Publications by authors named "Kanak L Dikshit"

34 Publications

Integration of VEK-30 peptide enhances fibrinolytic properties of staphylokinase.

Biotechnol Appl Biochem 2021 Apr 20;68(2):213-220. Epub 2020 May 20.

CSIR-Institute of Microbial Technology, Chandigarh, India.

Staphylokinase (SAK), a 136 amino acid bacterial protein with profibrinolytic properties, has emerged as an important thrombolytic agent because of its fibrin specificity and reduced inhibition by α-2 antiplasmin. In an attempt to enhance the clot dissolution ability of SAK, a 30 amino acid peptide (VEK-30) derived from a plasminogen (Pg) binding protein (PAM), was fused at the C-terminal end of SAK with a RGD (Arg-Gly-Asp) linker. The chimeric protein, SAKVEK, was expressed in E. coli and purified as a soluble protein. Pg activation by equimolar complexes of SAKVEK and SAK with plasmin revealed that the fusion of VEK-30 peptide has significantly enhanced the catalytic activity of SAK. The kinetic constant, k /K , of SAKVEK for the substrate Pg appeared 2.7 times higher than that of SAK and the time required for the fibrin and platelet rich clot lysis was shortened by 30% and 50%, respectively. The binary activator complex of SAKVEK with plasmin gets inhibited by α2- antiplasmin but remains protected in the presence of fibrin, very similar to SAK. Thus, the present study suggests that SAKVEK is more potent and effective as a thrombolytic agent due to its higher catalytic activity for Pg activation in a fibrin-specific manner and its ability to clear platelet-rich plasma clot faster than SAK.
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http://dx.doi.org/10.1002/bab.1912DOI Listing
April 2021

Truncated Hemoglobin O Carries an Autokinase Activity and Facilitates Adaptation of Under Hypoxia.

Antioxid Redox Signal 2020 02 24;32(6):351-362. Epub 2019 Jul 24.

CSIR-Institute of Microbial Technology, Chandigarh, India.

Although the human pathogen, (), is strictly aerobic and requires efficient supply of oxygen, it can survive long stretches of severe hypoxia. The mechanism responsible for this metabolic flexibility is unknown. We have investigated a novel mechanism by which hemoglobin O (HbO), operates and supports its host under oxygen stress. We discovered that the HbO exists in a phospho-bound state in and remains associated with the cell membrane under hypoxia. Deoxy-HbO carries an autokinase activity that disrupts its dimeric assembly into monomer and facilitates its association with the cell membrane, supporting survival and adaptation of under low oxygen conditions. Consistent with these observations, deletion of the O gene in bacillus Calmette-Guerin, which is identical to the O gene of , attenuated its survival under hypoxia and complementation of the O gene of rescued this inhibition, but phosphorylation-deficient mutant did not. These results demonstrated that autokinase activity of the HbO modulates its physiological function and plays a vital role in supporting the survival of its host under hypoxia. Our study demonstrates that the redox-dependent autokinase activity regulates oligomeric state and membrane association of HbO that generates a reservoir of oxygen in the proximity of respiratory membranes to sustain viability of under hypoxia. These results thus provide a novel insight into the physiological function of the HbO and demonstrate its pivotal role in supporting the survival and adaptation of under hypoxia.
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http://dx.doi.org/10.1089/ars.2018.7708DOI Listing
February 2020

Type II flavohemoglobin of Mycobacterium smegmatis oxidizes d-lactate and mediate electron transfer.

Int J Biol Macromol 2018 Jun 8;112:868-875. Epub 2018 Feb 8.

Department of Biotechnology, Panjab University, Sector 25, South Block, Chandigarh, India. Electronic address:

Two distantly related flavohemoglobins (FHbs), MsFHbI and MsFHbII, having crucial differences in their heme and reductase domains, co-exist in Mycobacterium smegmatis. Function of MsFHbI is associated with nitric-oxide detoxification but physiological relevance of MsFHbII remains unknown. This study unravels some unique spectral and functional characteristics of MsFHbII. Unlike conventional type I FHbs, MsFHbII lacks nitric-oxide dioxygenase and NADH oxidase activities but utilizes d-lactate as an electron donor to mediate electron transfer. MsFHbII carries a d-lactate dehydrogenase type FAD binding motif in its reductase domain and oxidizes d-lactate in a FAD dependent manner to reduce the heme iron, suggesting that the globin is acting as an electron acceptor. Importantly, expression of MsFHbII in Escherichia coli imparted protection under oxidative stress, suggesting its important role in stress management of its host. Since M. smegmatis lacks the gene encoding for d-lactate dehydrogenase and d-lactate is produced during aerobic metabolism and also as a by-product of lipid peroxidation, the ability of MsFHbII to metabolize d-lactate may provide it a unique ability to balance the oxidative stress generated due to accumulation of d-lactate in the cell and at the same time sequester electrons and pass it to the respiratory apparatus.
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http://dx.doi.org/10.1016/j.ijbiomac.2018.02.010DOI Listing
June 2018

Multidomain truncated hemoglobins: New members of the globin family exhibiting tandem repeats of globin units and domain fusion.

IUBMB Life 2017 07 10;69(7):479-488. Epub 2017 Apr 10.

Department of Biotechnology, Punjab University, Chandigarh, 160014, India.

Truncated hemoglobins (trHbs) are considered the most primitive members of globin superfamily and traditionally exist as a single domain heme protein in three distinct structural organizations, type I (trHb1_N), type II (trHb2_O) and type III (trHb3_P). Our search of microbial and lower eukaryotic genomes revealed a broad array of multidomain organization, representing multiunit and chimeric forms of trHbs, where multiple units of trHbs are joined together and/or integrated with distinct functional domains. Globin motifs of these multidomain trHbs were from all three groups of trHbs and unambiguously assigned to trHb1_N, trHb2_O and trHb3_P. Multiunit and chimeric forms of trHb1_N were identified exclusively in ciliated protozoan parasites, where multiple units of trHb are integrated in tandem and/or fused with another redox active or signalling domain, presenting an interesting example of gene duplication and fusion in lower eukaryotes. In contrast, trHb2_O and trHb3_P trHbs were found only in bacteria in two or multidomain organization, where amino or carboxy terminus of trHb unit is integrated with different redox-active or oxidoreductase domains. The identification of these new multiunit and chimeric trHbs and their specific phyletic distribution presents an interesting and challenging finding to explore and understand complex functionalities of these novel multidomain trHbs. © 2017 IUBMB Life, 69(7):479-488, 2017.
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http://dx.doi.org/10.1002/iub.1630DOI Listing
July 2017

BacHbpred: Support Vector Machine Methods for the Prediction of Bacterial Hemoglobin-Like Proteins.

Adv Bioinformatics 2016 29;2016:8150784. Epub 2016 Feb 29.

Centre for Chemistry and Biotechnology, Deakin University, Geelong, VIC 3217, Australia.

The recent upsurge in microbial genome data has revealed that hemoglobin-like (HbL) proteins may be widely distributed among bacteria and that some organisms may carry more than one HbL encoding gene. However, the discovery of HbL proteins has been limited to a small number of bacteria only. This study describes the prediction of HbL proteins and their domain classification using a machine learning approach. Support vector machine (SVM) models were developed for predicting HbL proteins based upon amino acid composition (AC), dipeptide composition (DC), hybrid method (AC + DC), and position specific scoring matrix (PSSM). In addition, we introduce for the first time a new prediction method based on max to min amino acid residue (MM) profiles. The average accuracy, standard deviation (SD), false positive rate (FPR), confusion matrix, and receiver operating characteristic (ROC) were analyzed. We also compared the performance of our proposed models in homology detection databases. The performance of the different approaches was estimated using fivefold cross-validation techniques. Prediction accuracy was further investigated through confusion matrix and ROC curve analysis. All experimental results indicate that the proposed BacHbpred can be a perspective predictor for determination of HbL related proteins. BacHbpred, a web tool, has been developed for HbL prediction.
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http://dx.doi.org/10.1155/2016/8150784DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4789356PMC
April 2016

Lipoprotein LprI of Mycobacterium tuberculosis Acts as a Lysozyme Inhibitor.

J Biol Chem 2016 Feb 20;291(6):2938-53. Epub 2015 Nov 20.

From the Council of Scientific and Industrial Research-Institute of Microbial Technology, Sector 39A, Chandigarh 160036, India

Mycobacterium tuberculosis executes numerous defense strategies for the successful establishment of infection under a diverse array of challenges inside the host. One such strategy that has been delineated in this study is the abrogation of lytic activity of lysozyme by a novel glycosylated and surface-localized lipoprotein, LprI, which is exclusively present in M. tuberculosis complex. The lprI gene co-transcribes with the glbN gene (encoding hemoglobin (HbN)) and both are synchronously up-regulated in M. tuberculosis during macrophage infection. Recombinant LprI, expressed in Escherichia coli, exhibited strong binding (Kd ≤ 2 nm) with lysozyme and abrogated its lytic activity completely, thereby conferring protection to fluorescein-labeled Micrococcus lysodeikticus from lysozyme-mediated hydrolysis. Expression of the lprI gene in Mycobacterium smegmatis (8-10-fold) protected its growth from lysozyme inhibition in vitro and enhanced its phagocytosis and survival during intracellular infection of peritoneal and monocyte-derived macrophages, known to secrete lysozyme, and in the presence of exogenously added lysozyme in secondary cell lines where lysozyme levels are low. In contrast, the presence of HbN enhanced phagocytosis and intracellular survival of M. smegmatis only in the absence of lysozyme but not under lysozyme stress. Interestingly, co-expression of the glbN-lprI gene pair elevated the invasion and survival of M. smegmatis 2-3-fold in secondary cell lines in the presence of lysozyme in comparison with isogenic cells expressing these genes individually. Thus, specific advantage against macrophage-generated lysozyme, conferred by the combination of LprI-HbN during invasion of M. tuberculosis, may have vital implications on the pathogenesis of tuberculosis.
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http://dx.doi.org/10.1074/jbc.M115.662593DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4742756PMC
February 2016

Bilobed shape of PadA reveals the connectivity from single to multi-domain bacterial plasminogen activators.

Int J Biol Macromol 2015 18;78:370-8. Epub 2015 Apr 18.

CSIR-Institute of Microbial Technology, Sector 39A, Chandigarh 160036, India. Electronic address:

The bacterial plasminogen activator, PadA activates bovine, ovine and caprine plasminogen but remains inert toward human plasminogen. It shows high sequence homology with human plasminogen activator, staphylokinase (SAK) but generates active-site in bovine plasminogen non-proteolytically, similar to streptokinase (SK). To examine the structural requirements for the function of this unique cofactor, attempts were made to visualize solution structure of the PadA using small-angle X-ray scattering (SAXS) data and compare its shape profile with structural models based on crystal structures of staphylokinase and streptokinase domains. The bilobal shape solved for the PadA matched closely with the structural model of α-domain of SK rather than its sequence homolog, SAK. The SAXS based solution structure of the PadA exhibited an extra volume and high mobility around Y(90)DKAEK(95) and P(104)ITES(108) loop regions that were found to play a crucial role in its cofactor function. Structure and sequence analysis of bacterial cofactors and mammalian plasminogens displayed evolutionary conservation of crucial complimentary amino acids required for making a functional binary activator complex between bacterial plasminogen activators and their cognate partner plasminogen. These studies highlighted the importance of structure-function related evolutionary strategies adopted by bacteria for exploiting mammalian plasminogen activation system and its understanding may help in designing and the development of new thrombolytic agents for clinical interventions.
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http://dx.doi.org/10.1016/j.ijbiomac.2015.04.019DOI Listing
February 2016

Recent applications of Vitreoscilla hemoglobin technology in bioproduct synthesis and bioremediation.

Appl Microbiol Biotechnol 2015 Feb 11;99(4):1627-36. Epub 2015 Jan 11.

Biology Division, Department of Biological and Chemical Sciences, Illinois Institute of Technology, Chicago, IL, 60616, USA,

Since its first use in 1990 to enhance production of α-amylase in E. coli, engineering of heterologous hosts to express the hemoglobin from the bacterium Vitreoscilla (VHb) has become a widely used strategy to enhance production of a variety of bioproducts, stimulate bioremediation, and increase growth and survival of engineered organisms. The hosts have included a variety of bacteria, yeast, fungi, higher plants, and even animals. The beneficial effects of VHb expression are presumably the result of one or more of its activities. The available evidence indicates that these include oxygen binding and delivery to the respiratory chain and oxygenases, protection against reactive oxygen species, and control of gene expression. In the past 4 to 5 years, the use of this "VHb technology" has continued in a variety of biotechnological applications in a wide range of organisms. These include enhancement of production of an ever wider array of bioproducts, new applications in bioremediation, a possible role in enhancing aerobic waste water treatment, and the potential to enhance growth and survival of both plants and animals of economic importance.
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http://dx.doi.org/10.1007/s00253-014-6350-yDOI Listing
February 2015

Mechanistic insight into the enzymatic reduction of truncated hemoglobin N of Mycobacterium tuberculosis: role of the CD loop and pre-A motif in electron cycling.

J Biol Chem 2014 Aug 13;289(31):21573-83. Epub 2014 Jun 13.

From the CSIR-Institute of Microbial Technology, Sector 39A, Chandigarh 160036, India,

Many pathogenic microorganisms have evolved hemoglobin-mediated nitric oxide (NO) detoxification mechanisms, where a globin domain in conjunction with a partner reductase catalyzes the conversion of toxic NO to innocuous nitrate. The truncated hemoglobin HbN of Mycobacterium tuberculosis displays a potent NO dioxygenase activity despite lacking a reductase domain. The mechanism by which HbN recycles itself during NO dioxygenation and the reductase that participates in this process are currently unknown. This study demonstrates that the NADH-ferredoxin/flavodoxin system is a fairly efficient partner for electron transfer to HbN with an observed reduction rate of 6.2 μM/min(-1), which is nearly 3- and 5-fold faster than reported for Vitreoscilla hemoglobin and myoglobin, respectively. Structural docking of the HbN with Escherichia coli NADH-flavodoxin reductase (FdR) together with site-directed mutagenesis revealed that the CD loop of the HbN forms contacts with the reductase, and that Gly(48) may have a vital role. The donor to acceptor electron coupling parameters calculated using the semiempirical pathway method amounts to an average of about 6.4 10(-5) eV, which is lower than the value obtained for E. coli flavoHb (8.0 10(-4) eV), but still supports the feasibility of an efficient electron transfer. The deletion of Pre-A abrogated the heme iron reduction by FdR in the HbN, thus signifying its involvement during intermolecular interactions of the HbN and FdR. The present study, thus, unravels a novel role of the CD loop and Pre-A motif in assisting the interactions of the HbN with the reductase and the electron cycling, which may be vital for its NO-scavenging function.
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http://dx.doi.org/10.1074/jbc.M114.578187DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4118117PMC
August 2014

Type I flavohemoglobin of mycobacterium smegmatis is a functional nitric oxide dioxygenase.

IUBMB Life 2014 Jun 26;66(6):396-404. Epub 2014 May 26.

CSIR-Institute of Microbial Technology, Sector 39 A, Chandigarh, India.

Two flavohemoglobins, type I and type II, displaying distinct structural features and cofactor binding sites coexist in Mycobacterium smegmatis; however, none of these flavohemeproteins are characterized so far. We have cloned and expressed type I flavohemoglobin (FHb1) of Mycobacterium smegmatis, encoded by MSMEG_1336, and characterized its spectral and functional properties. FHb1 exists as a monomer and displays spectral and functional characteristics similar to HMP of E. coli. Specific NO dioxygenase (NOD) activity of FHb1 was estimated to be 63.5 nmol heme(-1) sec(-1) , which was nearly eightfold higher than the HbN of M. tuberculosis and matched closely to the HMP of E. coli on the basis of cellular heme content. FHb1 preferred NADH for the NO dioxygenation and exhibited rapid reduction of flavin adenine dinucleotide and heme iron using NADH as electron donor. Level of FHb1 transcript increased significantly in M. smegmatis in the presence of acidified nitrite, and a nitric oxide-responsive transcriptional regulator of Rrf2 family exists together with the FHb1 under the same operon. These results suggested that FHb1 of M. smegmatis is a functional NOD and may be involved in the stress management of its host toward nitric oxide and nitrosative stress.
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http://dx.doi.org/10.1002/iub.1275DOI Listing
June 2014

Fibrin-targeted plasminogen activation by plasminogen activator, PadA, from Streptococcus dysgalactiae.

Protein Sci 2014 Jun 5;23(6):714-22. Epub 2014 Apr 5.

CSIR-Institute of Microbial Technology, Sector 39A, Chandigarh, 160036, India.

Bacterial plasminogen activators differ from each other in their mechanism of plasminogen activation besides their host specificity. Three-domain streptokinase (SK) and two-domain PauA generate nonproteolytic active site center in their cognate partner plasminogen but their binary activator complexes are resistant to α2-antiplasmin (a2AP) inhibition causing nonspecific plasminogen activation in plasma. In contrast, single-domain plasminogen activator, staphylokinase (SAK), requires proteolytic cleavage of human plasminogen into plasmin for the active site generation, and this activator complex is inhibited by a2AP. The single-domain plasminogen activator, PadA, from Streptococcus dysgalatiae, having close sequence and possible structure homology with SAK, was recently reported to activate bovine Pg in a nonproteolytic manner similar to SK. We report hereby that the binary activator complex of PadA with bovine plasminogen is inhibited by a2AP and PadA is recycled from this complex to catalyze the activation of plasminogen in the clot environment, where it is completely protected from a2AP inhibition. Catalytic efficiency of the activator complex formed by PadA and bovine plasminogen is amplified several folds in the presence of cyanogen bromide digested fibrinogen but not by intact fibrinogen indicating that PadA may be highly efficient at the fibrin surface. The present study, thus, demonstrates that PadA is a unique single-domain plasminogen activator that activates bovine plasminogen in a fibrin-targeted manner like SAK. The sequence optimization by PadA for acquiring the characteristics of both SK and SAK may be exploited for the development of efficient and fibrin-specific plasminogen activators for thrombolytic therapy.
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http://dx.doi.org/10.1002/pro.2455DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4093948PMC
June 2014

Haemoglobins of Mycobacteria: structural features and biological functions.

Adv Microb Physiol 2013 ;63:147-94

School of Life Sciences, Centre for Biomolecular Sciences, University of Nottingham, University Park, Nottingham, United Kingdom. Electronic address:

The genus Mycobacterium is comprised of Gram-positive bacteria occupying a wide range of natural habitats and includes species that range from severe intracellular pathogens to economically useful and harmless microbes. The recent upsurge in the availability of microbial genome data has shown that genes encoding haemoglobin-like proteins are ubiquitous among Mycobacteria and that multiple haemoglobins (Hbs) of different classes may be present in pathogenic and non-pathogenic species. The occurrence of truncated haemoglobins (trHbs) and flavohaemoglobins (flavoHbs) showing distinct haem active site structures and ligand-binding properties suggests that these Hbs may be playing diverse functions in the cellular metabolism of Mycobacteria. TrHbs and flavoHbs from some of the severe human pathogens such as Mycobacterium tuberculosis and Mycobacterium leprae have been studied recently and their roles in effective detoxification of reactive nitrogen and oxygen species, electron cycling, modulation of redox state of the cell and facilitation of aerobic respiration have been proposed. This multiplicity in the function of Hbs may aid these pathogens to cope with various environmental stresses and survive during their intracellular regime. This chapter provides recent updates on genomic, structural and functional aspects of Mycobacterial Hbs to address their role in Mycobacteria.
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http://dx.doi.org/10.1016/B978-0-12-407693-8.00005-4DOI Listing
April 2014

Truncated hemoglobin, HbN, is post-translationally modified in Mycobacterium tuberculosis and modulates host-pathogen interactions during intracellular infection.

J Biol Chem 2013 Oct 27;288(41):29987-99. Epub 2013 Aug 27.

From the Council of Scientific and Industrial Research (CSIR)-Institute of Microbial Technology, Sector 39A, Chandigarh 160036, India.

Mycobacterium tuberculosis (Mtb) is a phenomenally successful human pathogen having evolved mechanisms that allow it to survive within the hazardous environment of macrophages and establish long term, persistent infection in the host against the control of cell-mediated immunity. One such mechanism is mediated by the truncated hemoglobin, HbN, of Mtb that displays a potent O2-dependent nitric oxide dioxygenase activity and protects its host from the toxicity of macrophage-generated nitric oxide (NO). Here we demonstrate for the first time that HbN is post-translationally modified by glycosylation in Mtb and remains localized on the cell membrane and the cell wall. The glycan linkage in the HbN was identified as mannose. The elevated expression of HbN in Mtb and M. smegmatis facilitated their entry within the macrophages as compared with isogenic control cells, and mutation in the glycan linkage of HbN disrupted this effect. Additionally, HbN-expressing cells exhibited higher survival within the THP-1 and mouse peritoneal macrophages, simultaneously increasing the intracellular level of proinflammatory cytokines IL-6 and TNF-α and suppressing the expression of co-stimulatory surface markers CD80 and CD86. These results, thus, suggest the involvement of HbN in modulating the host-pathogen interactions and immune system of the host apart from protecting the bacilli from nitrosative stress inside the activated macrophages, consequently driving cells toward increased infectivity and intracellular survival.
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http://dx.doi.org/10.1074/jbc.M113.507301DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3795296PMC
October 2013

Role of PheE15 gate in ligand entry and nitric oxide detoxification function of mycobacterium tuberculosis truncated hemoglobin N.

PLoS One 2012 8;7(11):e49291. Epub 2012 Nov 8.

Department of Physical Chemistry and Institute of Biomedicine, IBUB, Faculty of Pharmacy, University of Barcelona - Recinte Torribera, Santa Coloma de Gramenet, Spain.

The truncated hemoglobin N, HbN, of Mycobacterium tuberculosis is endowed with a potent nitric oxide dioxygenase (NOD) activity that allows it to relieve nitrosative stress and enhance in vivo survival of its host. Despite its small size, the protein matrix of HbN hosts a two-branched tunnel, consisting of orthogonal short and long channels, that connects the heme active site to the protein surface. A novel dual-path mechanism has been suggested to drive migration of O(2) and NO to the distal heme cavity. While oxygen migrates mainly by the short path, a ligand-induced conformational change regulates opening of the long tunnel branch for NO, via a phenylalanine (PheE15) residue that acts as a gate. Site-directed mutagenesis and molecular simulations have been used to examine the gating role played by PheE15 in modulating the NOD function of HbN. Mutants carrying replacement of PheE15 with alanine, isoleucine, tyrosine and tryptophan have similar O(2)/CO association kinetics, but display significant reduction in their NOD function. Molecular simulations substantiated that mutation at the PheE15 gate confers significant changes in the long tunnel, and therefore may affect the migration of ligands. These results support the pivotal role of PheE15 gate in modulating the diffusion of NO via the long tunnel branch in the oxygenated protein, and hence the NOD function of HbN.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0049291PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3493545PMC
June 2013

An unconventional hexacoordinated flavohemoglobin from Mycobacterium tuberculosis.

J Biol Chem 2012 May 21;287(20):16435-46. Epub 2012 Mar 21.

Institute of Microbial Technology, Council of Scientific & Industrial Research, Sector 39 A, Chandigarh 160036, India.

Being an obligate aerobe, Mycobacterium tuberculosis faces a number of energetic challenges when it encounters hypoxia and environmental stress during intracellular infection. Consequently, it has evolved innovative strategies to cope with these unfavorable conditions. Here, we report a novel flavohemoglobin (MtbFHb) from M. tuberculosis that exhibits unique features within its heme and reductase domains distinct from conventional FHbs, including the absence of the characteristic hydrogen bonding interactions within the proximal heme pocket and mutations in the FAD and NADH binding regions of the reductase domain. In contrast to conventional FHbs, it has a hexacoordinate low-spin heme with a proximal histidine ligand lacking imidazolate character and a distal heme pocket with a relatively low electrostatic potential. Additionally, MtbFHb carries a new FAD binding site in its reductase domain similar to that of D-lactate dehydrogenase (D-LDH). When overexpressed in Escherichia coli or Mycobacterium smegmatis, MtbFHb remained associated with the cell membrane and exhibited D-lactate:phenazine methosulfate reductase activity and oxidized D-lactate into pyruvate by converting the heme iron from Fe(3+) to Fe(2+) in a FAD-dependent manner, indicating electron transfer from D-lactate to the heme via FAD cofactor. Under oxidative stress, MtbFHb-expressing cells exhibited growth advantage with reduced levels of lipid peroxidation. Given the fact that D-lactate is a byproduct of lipid peroxidation and that M. tuberculosis lacks the gene encoding D-LDH, we propose that the novel D-lactate metabolizing activity of MtbFHb uniquely equips M. tuberculosis to balance the stress level by protecting the cell membrane from oxidative damage via cycling between the Fe(3+)/Fe(2+) redox states.
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http://dx.doi.org/10.1074/jbc.M111.329920DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3351305PMC
May 2012

Pro42 and Val45 of staphylokinase modulate intermolecular interactions of His43-Tyr44 pair and specificity of staphylokinase-plasmin activator complex.

FEBS Lett 2012 Mar 2;586(6):653-8. Epub 2012 Feb 2.

Institute of Microbial Technology, CSIR, Chandigarh, India.

Staphylokinase (SAK) forms a 1:1 stoichiometric complex with plasmin (Pm) and changes its substrate specificity to create a plasminogen (Pg) activator complex. The His(43)-Tyr(44) pair of SAK resides within the active site cleft of the partner Pm and generates intermolecular contacts to confer Pg activator ability to the SAK-Pm bimolecular complex. Site-directed mutagenesis and molecular modeling studies unravelled that mutation at 42nd or 45th positions of SAK specifically disrupts cation-pi interaction of His(43) with Trp(215) of partner Pm within the active site, whereas pi-pi interaction of Tyr(44) with Trp(215) remain energetically favoured.
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http://dx.doi.org/10.1016/j.febslet.2012.01.046DOI Listing
March 2012

The Biochemistry of Vitreoscilla hemoglobin.

Comput Struct Biotechnol J 2012 29;3:e201210002. Epub 2012 Oct 29.

Department of Civil and Architectural Engineering, Illinois Institute of Technology, Chicago IL 60616, USA.

The hemoglobin (VHb) from Vitreoscilla was the first bacterial hemoglobin discovered. Its structure and function have been extensively investigated, and engineering of a wide variety of heterologous organisms to express VHb has been performed to increase their growth and productivity. This strategy has shown promise in applications as far-ranging as the production of antibiotics and petrochemical replacements by microorganisms to increasing stress tolerance in plants. These applications of "VHb technology" have generally been of the "black box" variety, wherein the endpoint studied is an increase in the levels of a certain product or improved growth and survival. Their eventual optimization, however, will require a thorough understanding of the various functions and activities of VHb, and how VHb expression ripples to affect metabolism more generally. Here we review the current knowledge of these topics. VHb's functions all involve oxygen binding (and often delivery) in one way or another. Several biochemical and structure-function studies have provided an insight into the molecular details of this binding and delivery. VHb activities are varied. They include supply of oxygen to oxygenases and the respiratory chain, particularly under low oxygen conditions; oxygen sensing and modulation of transcription factor activity; and detoxification of NO, and seem to require interactions of VHb with "partner proteins". VHb expression affects the levels of ATP and NADH, although not enormously. VHb expression may affect the level of many compounds of intermediary metabolism, and, apparently, alters the levels of expression of many genes. Thus, the metabolic changes in organisms engineered to express VHb are likely to be numerous and complicated.
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http://dx.doi.org/10.5936/csbj.201210002DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3962134PMC
June 2014

Recent advances in understanding the structure, function, and biotechnological usefulness of the hemoglobin from the bacterium Vitreoscilla.

Biotechnol Lett 2011 Sep 21;33(9):1705-14. Epub 2011 May 21.

Biology Division, Department of Biological, Chemical, and Physical Sciences, Illinois Institute of Technology, Chicago, IL, USA.

The hemoglobin from the bacterium Vitreoscilla (VHb) is the first microbial hemoglobin that was conclusively identified as such (in 1986). It has been extensively studied with respect to its ligand binding properties and mechanisms, structure, biochemical functions, and the mechanisms by which its expression is controlled. In addition, cloning of its gene (vgb) into a variety of heterologous hosts has proved that its expression results substantial increases in production of a variety of useful products and ability to degrade potentially harmful compounds. Recent studies (since 2005) have added significant knowledge to all of these areas and shown the broad range of biotechnological applications in which VHb can have a positive effect.
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http://dx.doi.org/10.1007/s10529-011-0621-9DOI Listing
September 2011

Intermolecular interactions in staphylokinase-plasmin(ogen) bimolecular complex: function of His43 and Tyr44.

FEBS Lett 2011 Jun 16;585(12):1814-20. Epub 2011 Apr 16.

Institute of Microbial Technology (CSIR), Chandigarh, India.

Staphylokinase (SAK) forms a 1:1 stoichiometric complex with human plasmin (Pm) and switches its substrate specificity to generate a plasminogen (Pg) activator complex. Site-directed mutagenesis of SAKHis43 and SAKTyr44 demonstrated the crucial requirement of a positively charged and an aromatic residue, respectively, at these positions for optimal functioning of SAK-Pm activator complex. Molecular modeling studies further revealed the role of these residues in making cation-pi and pi-pi interactions with Trp215 of Pm and thus establishing the crucial intermolecular contacts within the active site cleft of the activator complex for the cofactor activity of SAK.
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http://dx.doi.org/10.1016/j.febslet.2011.04.030DOI Listing
June 2011

Novel flavohemoglobins of mycobacteria.

IUBMB Life 2011 May 13;63(5):337-45. Epub 2011 Apr 13.

Institute of Microbial Technology, Chandigarh, India.

Flavohemoglobins (flavoHbs) constitute a distinct class of chimeric hemoglobins in which a globin domain is coupled with a ferredoxin reductase such as FAD- and NADH-binding modules. Structural features and active site of heme and reductase domains are highly conserved in various flavoHbs. A new class of flavoHbs, displaying crucial differences in functionally conserved regions of heme and reductase domains, have been identified in mycobacteria. Mining of microbial genome data indicated that the occurrence of such flavoHbs might be restricted to a small group of microbes unlike conventional flavoHbs that are widespread among prokaryotes and lower eukaryotes. One of the representative flavoHbs of this class, encoded by Rv0385 gene (MtbFHb) of Mycobacterium tuberculosis, has been cloned, expressed, and characterized. The ferric and deoxy spectra of MtbFHb displayed a hexacoordinate state indicating that its distal site may be occupied by an intrinsic amino acid or an external ligand and it may not be involved in nitric oxide detoxification. Phylogenetic analysis revealed that mycobacterial flavoHbs constitute a separate cluster distinct from conventional flavoHbs and may have novel function(s).
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http://dx.doi.org/10.1002/iub.460DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4533980PMC
May 2011

Redox-mediated interactions of VHb (Vitreoscilla haemoglobin) with OxyR: novel regulation of VHb biosynthesis under oxidative stress.

Biochem J 2010 Feb 24;426(3):271-80. Epub 2010 Feb 24.

Institute of Microbial Technology, Sector 39 A, Chandigarh 160036, India.

The bacterial haemoglobin from Vitreoscilla, VHb, displays several unusual properties that are unique among the globin family. When the gene encoding VHb, vgb, is expressed from its natural promoter in either Vitreoscilla or Escherichia coli, the level of VHb increases more than 50-fold under hypoxic conditions and decreases significantly during oxidative stress, suggesting similar functioning of the vgb promoter in both organisms. In the present study we show that expression of VHb in E. coli induced the antioxidant genes katG (catalase-peroxidase G) and sodA (superoxide dismutase A) and conferred significant protection from oxidative stress. In contrast, when vgb was expressed in an oxyR mutant of E. coli, VHb levels increased and the strain showed high sensitivity to oxidative stress without induction of antioxidant genes; this indicates the involvement of the oxidative stress regulator OxyR in mediating the protective effect of VHb under oxidative stress. A putative OxyR-binding site was identified within the vgb promoter and a gel-shift assay confirmed its interaction with oxidized OxyR, an interaction which was disrupted by the reduced form of the transcriptional activator Fnr (fumurate and nitrate reductase). This suggested that the redox state of OxyR and Fnr modulates their interaction with the vgb promoter. VHb associated with reduced OxyR in two-hybrid screen experiments and in vitro, converting it into an oxidized state in the presence of NADH, a condition where VHb is known to generate H2O2. These observations unveil a novel mechanism by which VHb may transmit signals to OxyR to autoregulate its own biosynthesis, simultaneously activating oxidative stress functions. The activation of OxyR via VHb, reported in the present paper for the first time, suggests the involvement of VHb in transcriptional control of many other genes as well.
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http://dx.doi.org/10.1042/BJ20091417DOI Listing
February 2010

Role of Pre-A motif in nitric oxide scavenging by truncated hemoglobin, HbN, of Mycobacterium tuberculosis.

J Biol Chem 2009 May 27;284(21):14457-68. Epub 2009 Mar 27.

Institute of Microbial Technology, Council of Scientific and Industrial Research, Chandigarh 160036, India.

Mycobacterium tuberculosis truncated hemoglobin, HbN, is endowed with a potent nitric-oxide dioxygenase activity and has been found to relieve nitrosative stress and enhance in vivo survival of a heterologous host, Salmonella enterica Typhimurium, within the macrophages. These findings implicate involvement of HbN in the defense of M. tuberculosis against nitrosative stress. The protein carries a tunnel system composed of a short and a long tunnel branch that has been proposed to facilitate diatomic ligand migration to the heme and an unusual Pre-A motif at the N terminus, which does not contribute significantly to the structural integrity of the protein, as it protrudes out of the compact globin fold. Strikingly, deletion of Pre-A region from the M. tuberculosis HbN drastically reduces its ability to scavenge nitric oxide (NO), whereas its insertion at the N terminus of Pre-A lacking HbN of Mycobacterium smegmatis improved its nitric-oxide dioxygenase activity. Titration of the oxygenated adduct of HbN and its mutants with NO indicated that the stoichiometric oxidation of protein is severalfold slower when the Pre-A region is deleted in HbN. Molecular dynamics simulations show that the excision of Pre-A motif results in distinct changes in the protein dynamics, which cause the gate of the tunnel long branch to be trapped into a closed conformation, thus impeding migration of diatomic ligands toward the heme active site. The present study, thus, unequivocally demonstrates vital function of Pre-A region in NO scavenging and unravels its unique role by which HbN might attain its efficient NO-detoxification ability.
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http://dx.doi.org/10.1074/jbc.M807436200DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2682894PMC
May 2009

Charged nylon membrane substrate for convenient and versatile high resolution microscopic analysis of Escherichia coli & mammalian cells in suspension culture.

Cytotechnology 2006 Jun 3;51(2):111-7. Epub 2006 Nov 3.

Institute of Microbial Technology, Sector 39 A, Chandigarh, 160036, India,

Preparation of isolated cells and microorganisms for ultrastructural examination always provides a challenge in terms of adequate immobilization of the cells and prevention of subsequent sample loss and damage during various steps of sample processing. Using a positively charged nylon membrane substrate we demonstrate that it is possible to easily immobilize and retain a sample of isolated cells in culture for a wide variety of microscopy-based techniques. Radiolabelled E. coli cells when immobilized on the charged membrane were seen to be highly resistant to detachment when subjected to the normal sample processing procedures associated with microscopy. In contrast cells on regular millipore membranes were rapidly lost during sample preparation. We demonstrate the utility of charged nylon membranes for a wide variety of microscopy based analysis including scanning and transmission electron microscopy (SEM and TEM), atomic force microscopy (AFM), TEM based immunogold labelling, laser confocal microscopy and SEM based elemental analysis.
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http://dx.doi.org/10.1007/s10616-006-9027-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3449682PMC
June 2006

Functional implications of the proximal site hydrogen bonding network in Vitreoscilla hemoglobin (VHb): role of Tyr95 (G5) and Tyr126 (H12).

FEBS Lett 2008 Oct 18;582(23-24):3494-500. Epub 2008 Sep 18.

Institute of Microbial Technology, Sector 39 A, Chandigarh 160036, India.

Although Vitreoscilla hemoglobin (VHb) carries a conventional globin fold, its proximal site geometry is unique in having a hydrogen-bonding network between proximal site residues, HisF8-TyrG5-GluH23 and TyrG5-TyrH12. TyrG5 and TyrH12 were mutated to study their relevance in VHb function. VHb G5 mutants (Tyr95Phe and Tyr95Leu showed no stable oxyform and nitric oxide dioxygenase activity, whereas, VHb H12 mutants (Tyr126Phe and Tyr126Leu) displayed little change in their oxygen affinity indicating a crucial role of Tyr95 in protein function. The VHb H12 mutant, Tyr126Leu, enhanced the intracellular pool of oxygen and cell growth better than VHb. Molecular modeling suggests that the replacement of tyrosine with leucine in Tyr126Leu creates an opening on the protein surface that may facilitate oxygen diffusion and accumulation.
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http://dx.doi.org/10.1016/j.febslet.2008.09.018DOI Listing
October 2008

Responses of Mycobacterium tuberculosis hemoglobin promoters to in vitro and in vivo growth conditions.

Appl Environ Microbiol 2008 Jun 4;74(11):3512-22. Epub 2008 Apr 4.

Institute of Microbial Technology, Sector 39 A, Chandigarh 160036, India.

The success of Mycobacterium tuberculosis as one of the dreaded human pathogens lies in its ability to utilize different defense mechanisms in response to the varied environmental challenges during the course of its intracellular infection, latency, and reactivation cycle. Truncated hemoglobins trHbN and trHbO are thought to play pivotal roles in the cellular metabolism of this organism during stress and hypoxia. To delineate the genetic regulation of the M. tuberculosis hemoglobins, transcriptional fusions of the promoters of the glbN and glbO genes with green fluorescent protein were constructed, and their responses were monitored in Mycobacterium smegmatis and M. tuberculosis H37Ra exposed to environmental stresses in vitro and in M. tuberculosis H37Ra after in vivo growth inside macrophages. The glbN promoter activity increased substantially during stationary phase and was nearly 3- to 3.5-fold higher than the activity of the glbO promoter, which remained more or less constant during different growth phases in M. smegmatis, as well as in M. tuberculosis H37Ra. In both mycobacterial hosts, the glbN promoter activity was induced 1.5- to 2-fold by the general nitrosative stress inducer, nitrite, as well as the NO releaser, sodium nitroprusside (SNP). The glbO promoter was more responsive to nitrite than to SNP, although the overall increase in its activity was much less than that of the glbN promoter. Additionally, the glbN promoter remained insensitive to the oxidative stress generated by H(2)O(2), but the glbO promoter activity increased nearly 1.5-fold under similar conditions, suggesting that the trHb gene promoters are regulated differently under nitrosative and oxidative stress conditions. In contrast, transition metal-induced hypoxia enhanced the activity of both the glbN and glbO promoters at all growth phases; the glbO promoter was induced approximately 2.3-fold, which was found to be the highest value for this promoter under all the conditions evaluated. Addition of iron along with nickel reversed the induction in both cases. Interestingly, a concentration-dependent decrease in the activity of both trHb gene promoters was observed when the levels of iron in the growth media were depleted by addition of an iron chelator. These results suggested that an iron/heme-containing oxygen sensor is involved in the modulation of the trHb gene promoter activities directly or indirectly in conjunction with other cellular factors. The modes of promoter regulation under different physiological conditions were found to be similar for the trHbs in both M. smegmatis and M. tuberculosis H37Ra, indicating that the promoters might be regulated by components that are common to the two systems. Confocal microscopy of THP-1 macrophages infected with M. tuberculosis carrying the trHb gene promoter fusions showed that there was a significant level of promoter activity during intracellular growth in macrophages. Time course evaluation of the promoter activity after various times up to 48 h by fluorescence-activated cell sorting analysis of the intracellular M. tuberculosis cells indicated that the glbN promoter was active at all time points assessed, whereas the activity of the glbO promoter remained at a steady-state level up to 24 h postinfection and increased approximately 2-fold after 48 h of infection. Thus, the overall regulation pattern of the M. tuberculosis trHb gene promoters correlates not only with the stresses that the tubercle bacillus is likely to encounter once it is in the macrophage environment but also with our current knowledge of their functions. The in vivo studies that demonstrated for the first time expression of trHbs during macrophage infection of M. tuberculosis strongly indicate that the hemoglobins are required, and thus important, during the intracellular phase of the bacterial cycle. The present study of transcriptional regulation of M. tuberculosis hemoglobins in vitro under various stress conditions and in vivo after macrophage infection supports the hypothesis that biosynthesis of both trHbs (trHbN and trHbO) in the native host is regulated via the environmental signals that the tubercle bacillus receives during macrophage infection and growth in its human host.
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http://dx.doi.org/10.1128/AEM.02663-07DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2423021PMC
June 2008

Cloning and characterization of promoter-active DNA sequences from Streptococcus equisimilis.

Curr Microbiol 2007 Jan 13;54(1):48-53. Epub 2006 Dec 13.

Department of Biotechnology, Panjab University, Chandigarh, India.

Genomic fragments of Streptococcus equisimilis exhibiting potent promoter activity in Escherichia coli were isolated by transcriptional fusion to chloramphenicol acetyl transferase (CAT) structural gene. Random S. equanimities DNA, cloned in E. coli, exhibited a higher frequency of strong promoter activity than did similarly cloned E. coli fragments. The determination of the relative promoter strength of randomly selected clones with CAT assay demonstrated the dominance of sequences acting as a strong promoter in E. coli. Removal of downstream terminator from the strong promoter containing plasmid resulted in a twofold to threefold increase in CAT expression in some cases. Structural characteristics of promoter sequences of some representative streptococcal genes clearly indicate that the streptococcal promoter regions are rich in secondary structures and might be one of the factors of their instability in E. coli.
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http://dx.doi.org/10.1007/s00284-006-0249-5DOI Listing
January 2007

Oxygen binding and NO scavenging properties of truncated hemoglobin, HbN, of Mycobacterium smegmatis.

FEBS Lett 2006 Jul 22;580(17):4031-41. Epub 2006 Jun 22.

Institute of Microbial Technology, Sector 39 A, Chandigarh 160036, India.

Unraveling of microbial genome data has indicated that two distantly related truncated hemoglobins (trHbs), HbN and HbO, might occur in many species of slow-growing pathogenic mycobacteria. Involvement of HbN in bacterial defense against NO toxicity and nitrosative stress has been proposed. A gene, encoding a putative HbN homolog with conserved features of typical trHbs, has been identified within the genome sequence of fast-growing mycobacterium, Mycobacterium smegmatis. Sequence analysis of M. smegmatis HbN indicated that it is relatively smaller in size and lacks N-terminal pre-A region, carrying 12-residue polar sequence motif that is present in HbN of M. tuberculosis. HbN encoding gene of M. smegmatis was expressed in E. coli as a 12.8kD homodimeric heme protein that binds oxygen reversibly with high affinity (P50 approximately 0.081 mm Hg) and autooxidizes faster than M. tuberculosis HbN. The circular dichroism spectra indicate that HbN of M. smegmatis and M. tuberculosis are structurally similar. Interestingly, an hmp mutant of E. coli, unable to metabolize nitric oxide, exhibited very low NO uptake activity in the presence of M. smegmatis HbN as compared to HbN of M. tuberculosis. On the basis of cellular heme content, specific nitric oxide dioxygenase (NOD) activity of M. smegmatis HbN was nearly one-third of that from M. tuberculosis. Additionally, the hmp mutant of E. coli, carrying M. smegmatis HbN, exhibited nearly 10-fold lower cell survival under nitrosative stress and nitrite derived reactive nitrogen species as compared to the isogenic strain harboring HbN of M. tuberculosis. Taken together, these results suggest that NO metabolizing activity and protection provided by M. smegmatis HbN against toxicity of NO and reactive nitrogen is significantly lower than HbN of M. tuberculosis. The lower efficiency of M. smegmatis HbN for NO detoxification as compared to M. tuberculosis HbN might be related to different level of NO exposure and nitrosative stress faced by these mycobacteria during their cellular metabolism.
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http://dx.doi.org/10.1016/j.febslet.2006.06.037DOI Listing
July 2006

Enhanced plasminogen activation by staphylokinase in the presence of streptokinase beta/betagamma domains: plasminogen kringles play a role.

FEBS Lett 2005 Mar;579(7):1565-72

Institute of Microbial Technology, Sector 39-A, Chandigarh 160036, India.

Presence of isolated beta or betagamma domains of streptokinase (SK) increased the catalytic activity of staphylokinase (SAK)-plasmin (Pm) complex up to 60%. In contrast, fusion of SK beta or betagamma domains with the C-terminal end of SAK drastically reduced the catalytic activity of the activator complex. The enhancement effect mediated by beta or betagamma domain on Pg activator activity of SAK-Pm complex was reduced greatly (45%) in the presence of isolated kringles of Pg, whereas, kringles did not change cofactor activity of SAK fusion proteins (carrying beta or betagamma domains) significantly. When catalytic activity of SAK-microPm (catalytic domain of Pm lacking kringle domains) complex was examined in the presence of isolated beta and betagamma domains, no enhancement effect on Pg activation was observed, whereas, enzyme complex formed between microplasmin and SAK fusion proteins (SAKbeta and SAKbetagamma) displayed 50-70% reduction in their catalytic activity. The present study, thus, suggests that the exogenously present beta and betagamma interact with Pg/Pm via kringle domains and elevate catalytic activity of SAK-Pm activator complex resulting in enhanced substrate Pg activation. Fusion of beta or betagamma domains with SAK might alter these intermolecular interactions resulting in attenuated functional activity of SAK.
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http://dx.doi.org/10.1016/j.febslet.2005.01.066DOI Listing
March 2005

Mutational study of the bacterial hemoglobin distal heme pocket.

Biochem Biophys Res Commun 2005 Jan;326(2):290-7

Biology Division, Department of Biological, Chemical, and Physical Sciences, Illinois Institute of Technology, Chicago, IL 60616, USA.

Ligand binding experiments on three mutants in the distal heme pocket of Vitreoscilla hemoglobin (GlnE7His, ProE8Ala, and GlnE7His,ProE8Ala) were used to probe the role of GlnE7 and ProE8 in the pocket's unusual structure. The oxygen dissociation constants for the wild type, E8Ala mutant, and E7His mutant proteins were 4.5, 4.7, and 1.7microM, respectively; the K(d) for the double mutant was not determinable by our technique. Visible-Soret spectra of the carbonyl and cyanyl forms and FT-IR of the carbonyl form of the E8 mutant were similar to those of the wild type; the opposite was true for the GlnE7His and GlnE7His,ProE8Ala mutants, which also differed from wild type in the visible-Soret spectra of their oxidized forms. Models of the effects of the mutations on distal pocket structure were consistent with the experimental findings, particularly the larger effects of the GlnE7His change.
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http://dx.doi.org/10.1016/j.bbrc.2004.11.025DOI Listing
January 2005

Nitric oxide scavenging and detoxification by the Mycobacterium tuberculosis haemoglobin, HbN in Escherichia coli.

Mol Microbiol 2002 Sep;45(5):1303-14

Institute of Microbial Technology, Sector 39A, Chandigarh, 160-036 India.

Nitric oxide (NO), generated in large amounts within the macrophages, controls and restricts the growth of internalized human pathogen, Mycobacterium tuberculosis H37Rv. The molecular mechanism by which tubercle bacilli survive within macrophages is currently of intense interest. In this work, we have demonstrated that dimeric haemoglobin, HbN, from M. tuberculosis exhibits distinct nitric oxide dioxygenase (NOD) activity and protects growth and cellular respiration of heterologous hosts, Escherichia coli and Mycobacterium smegmatis, from the toxic effect of exogenous NO and the NO-releasing compounds. A flavohaemoglobin (HMP)-deficient mutant of E. coli, unable to metabolize NO, acquired an oxygen-dependent NO consumption activity in the presence of HbN. On the basis of cellular haem content, the specific NOD activity of HbN was nearly 35-fold higher than the single-domain Vitreoscilla haemoglobin (VHb) but was sevenfold lower than the two-domain flavohaemoglobin. HbN-dependent NO consumption was sustained with repeated addition of NO, demonstrating that HbN is catalytically reduced within E. coli. Aerobic growth and respiration of a flavohaemoglobin (HMP) mutant of E. coli was inhibited in the presence of exogenous NO but remained insensitive to NO inhibition when these cells produced HbN, VHb or flavohaemoglobin. M. smegmatis, carrying a native HbN very similar to M. tuberculosis HbN, exhibited a 7.5-fold increase in NO uptake when exposed to gaseous NO, suggesting NO-induced NOD activity in these cells. In addition, expression of plasmid-encoded HbN of M. tuberculosis in M. smegmatis resulted in 100-fold higher NO consumption activity than the isogenic control cells. These results provide strong experimental evidence in support of NO scavenging and detoxification function for the M. tuberculosis HbN. The catalytic NO scavenging by HbN may be highly advantageous for the survival of tubercle bacilli during infection and pathogenesis.
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http://dx.doi.org/10.1046/j.1365-2958.2002.03095.xDOI Listing
September 2002