Publications by authors named "Ronald van Merkerk"

26 Publications

  • Page 1 of 1

Proteolysis Targeting Chimera (PROTAC) for Macrophage Migration Inhibitory Factor (MIF) Has Anti-Proliferative Activity in Lung Cancer Cells.

Angew Chem Int Ed Engl 2021 08 26;60(32):17514-17521. Epub 2021 Jun 26.

Department Chemical and Pharmaceutical Biology, Groningen Research Institute of Pharmacy (GRIP), University of Groningen, Antonius Deusinglaan 1, 9713, AV, Groningen, The Netherlands.

Macrophage migration inhibitory factor (MIF) is involved in protein-protein interactions that play key roles in inflammation and cancer. Current strategies to develop small molecule modulators of MIF functions are mainly restricted to the MIF tautomerase active site. Here, we use this site to develop proteolysis targeting chimera (PROTAC) in order to eliminate MIF from its protein-protein interaction network. We report the first potent MIF-directed PROTAC, denoted MD13, which induced almost complete MIF degradation at low micromolar concentrations with a DC around 100 nM in A549 cells. MD13 suppresses the proliferation of A549 cells, which can be explained by deactivation of the MAPK pathway and subsequent induction of cell cycle arrest at the G2/M phase. MD13 also exhibits antiproliferative effect in a 3D tumor spheroid model. In conclusion, we describe the first MIF-directed PROTAC (MD13) as a research tool, which also demonstrates the potential of PROTACs in cancer therapy.
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http://dx.doi.org/10.1002/anie.202101864DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8362126PMC
August 2021

Engineering of Multiple Modules to Improve Amorphadiene Production in Using CRISPR-Cas9.

J Agric Food Chem 2021 Apr 20;69(16):4785-4794. Epub 2021 Apr 20.

Department of Chemical and Pharmaceutical Biology, Groningen Research Institute of Pharmacy, University of Groningen, Antonius Deusinglaan 1, 9713 AV Groningen, The Netherlands.

Engineering strategies to improve terpenoids' production in mainly focus on 2-methyl-d-erythritol-4-phosphate (MEP) pathway overexpression. To systematically engineer the chassis strain for higher amorphadiene (precursor of artemisinin) production, a clustered regularly interspaced short palindromic repeat-Cas9 (CRISPR-Cas9) system was established in to facilitate precise and efficient genome editing. Then, this system was employed to engineer three more modules to improve amorphadiene production, including the terpene synthase module, the branch pathway module, and the central metabolic pathway module. Finally, our combination of all of the useful strategies within one strain significantly increased extracellular amorphadiene production from 81 to 116 mg/L after 48 h flask fermentation without medium optimization. For the first time, we attenuated the FPP-derived competing pathway to improve amorphadiene biosynthesis and investigated how the TCA cycle affects amorphadiene production in . Overall, this study provides a universal strategy for further increasing terpenoids' production in by comprehensive and systematic metabolic engineering.
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http://dx.doi.org/10.1021/acs.jafc.1c00498DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8154554PMC
April 2021

7-Hydroxycoumarins Are Affinity-Based Fluorescent Probes for Competitive Binding Studies of Macrophage Migration Inhibitory Factor.

J Med Chem 2020 10 13;63(20):11920-11933. Epub 2020 Oct 13.

Chemical and Pharmaceutical Biology, Groningen Research Institute of Pharmacy (GRIP), University of Groningen, Antonius Deusinglaan 1, 9713 AV Groningen, The Netherlands.

Macrophage migration inhibitory factor (MIF) is a cytokine with key roles in inflammation and cancer, which qualifies it as a potential drug target. Apart from its cytokine activity, MIF also harbors enzyme activity for keto-enol tautomerization. MIF enzymatic activity has been used for identification of MIF binding molecules that also interfere with its biological activity. However, MIF tautomerase activity assays are troubled by irregularities, thus creating a need for alternative methods. In this study, we identified a 7-hydroxycoumarin fluorophore with high affinity for the MIF tautomerase active site ( = 18 ± 1 nM) that binds with concomitant quenching of its fluorescence. This property enabled development of a novel competition-based assay format to quantify MIF binding. We also demonstrated that the 7-hydroxycoumarin fluorophore interfered with the MIF-CD74 interaction and inhibited proliferation of A549 cells. Thus, we provide a high-affinity MIF binder as a novel tool to advance MIF-oriented research.
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http://dx.doi.org/10.1021/acs.jmedchem.0c01160DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7586407PMC
October 2020

Engineering the specificity of Streptococcus pyogenes sortase A by loop grafting.

Proteins 2020 11 21;88(11):1394-1400. Epub 2020 Jun 21.

Department of Chemical and Pharmaceutical Biology, Groningen Research Institute of Pharmacy, University of Groningen, Groningen, The Netherlands.

Sortases are a group of enzymes displayed on the cell-wall of Gram-positive bacteria. They are responsible for the attachment of virulence factors onto the peptidoglycan in a transpeptidation reaction through recognition of a pentapeptide substrate. Most housekeeping sortases recognize one specific pentapeptide motif; however, Streptococcus pyogenes sortase A (SpSrtA WT) recognizes LPETG, LPETA and LPKLG motifs. Here, we examined SpSrtA's flexible substrate specificity by investigating the role of the β7/β8 loop in determining substrate specificity. We exchanged the β7/β8 loop in SpSrtA with corresponding β7/β8 loops from Staphylococcus aureus (SaSrtA WT) and Bacillus anthracis (BaSrtA WT). While the BaSrtA-derived variant showed no enzymatic activity toward either LPETG or LPETA substrates, the activity of the SaSrtA-derived mutant toward the LPETA substrate was completely abolished. Instead, the mutant had an improved activity toward LPETG, the preferred substrate of SaSrtA WT.
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http://dx.doi.org/10.1002/prot.25958DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7586933PMC
November 2020

Production of Squalene in by Squalene Synthase Screening and Metabolic Engineering.

J Agric Food Chem 2020 Apr 3;68(15):4447-4455. Epub 2020 Apr 3.

Department of Chemical and Pharmaceutical Biology, Groningen Research Institute of Pharmacy, University of Groningen, Antonius Deusinglaan 1, 9713 AV Groningen, The Netherlands.

Squalene synthase (SQS) catalyzes the conversion of two farnesyl pyrophosphates to squalene, an important intermediate in between isoprene and valuable triterpenoids. In this study, we have constructed a novel biosynthesis pathway for squalene in and performed metabolic engineering aiming at facilitating further exploitation and production of squalene-derived triterpenoids. Therefore, systematic studies and analysis were performed including selection of multiple SQS candidates from various organisms, comparison of expression vectors, optimization of cultivation temperatures, and examination of rate-limiting factors within the synthetic pathway. We were, for the first time, able to obtain squalene synthesis in . Furthermore, we achieved a 29-fold increase of squalene yield (0.26-7.5 mg/L) by expressing SQS from and eliminating bottlenecks within the upstream methylerythritol-phosphate pathway. Moreover, our findings showed that also could positively affect the production of squalene.
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http://dx.doi.org/10.1021/acs.jafc.0c00375DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7168599PMC
April 2020

A regulated synthetic operon facilitates stable overexpression of multigene terpenoid pathway in Bacillus subtilis.

J Ind Microbiol Biotechnol 2020 Feb 1;47(2):243-249. Epub 2020 Jan 1.

Department of Chemical and Pharmaceutical Biology, Groningen Research Institute of Pharmacy, University of Groningen, Antonius Deusinglaan 1, 9713 AV, Groningen, The Netherlands.

The creation of microbial cell factories for sustainable production of natural products is important for medical and industrial applications. This requires stable expression of biosynthetic pathways in a host organism with favorable fermentation properties such as Bacillus subtilis. The aim of this study is to construct B. subtilis strains that produce valuable terpenoid compounds by overexpressing the innate methylerythritol phosphate (MEP) pathway. A synthetic operon allowing the concerted and regulated expression of multiple genes was developed. Up to 8 genes have been combined in this operon and a stably inherited plasmid-based vector was constructed resulting in a high production of C carotenoids. For this, two vectors were examined, one with rolling circle replication and another with theta replication. Theta-replication constructs were clearly superior in structural and segregational stability compared to rolling circle constructs. A strain overexpressing all eight genes of the MEP pathway on a theta-replicating plasmid clearly produced the highest level of carotenoids. The level of transcription for each gene in the operon was similar as RT-qPCR analysis indicated. Hence, that corresponding strain can be used as a stable cell factory for production of terpenoids. This is the first report of merging and stably expressing this large-size operon (eight genes) from a plasmid-based system in B. subtilis enabling high C carotenoid production.
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http://dx.doi.org/10.1007/s10295-019-02257-4DOI Listing
February 2020

Tuning Enzyme Activity for Nonaqueous Solvents: Engineering an Enantioselective "Michaelase" for Catalysis in High Concentrations of Ethanol.

Chembiochem 2020 05 18;21(10):1499-1504. Epub 2020 Feb 18.

Department of Chemical and Pharmaceutical Biology, Groningen Research Institute of Pharmacy, University of Groningen, Antonius Deusinglaan 1, 9713 AV, Groningen, The Netherlands.

Enzymes have evolved to function under aqueous conditions and may not exhibit features essential for biocatalytic application, such as the ability to function in high concentrations of an organic solvent. Consequently, protein engineering is often required to tune an enzyme for catalysis in non-aqueous solvents. In this study, we have used a collection of nearly all single mutants of 4-oxalocrotonate tautomerase, which promiscuously catalyzes synthetically useful Michael-type additions of acetaldehyde to various nitroolefins, to investigate the effect of each mutation on the ability of this enzyme to retain its "Michaelase" activity in elevated concentrations of ethanol. Examination of this mutability landscape allowed the identification of two hotspot positions, Ser30 and Ala33, at which mutations are beneficial for catalysis in high ethanol concentrations. The "hotspot" position Ala33 was then randomized in a highly enantioselective, but ethanol-sensitive 4-OT variant (L8F/M45Y/F50A) to generate an improved enzyme variant (L8F/A33I/M45Y/F50A) that showed great ethanol stability and efficiently catalyzes the enantioselective addition of acetaldehyde to nitrostyrene in 40 % ethanol (permitting high substrate loading) to give the desired γ-nitroaldehyde product in excellent isolated yield (89 %) and enantiopurity (ee=98 %). The presented work demonstrates the power of mutability-landscape-guided enzyme engineering for efficient biocatalysis in non-aqueous solvents.
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http://dx.doi.org/10.1002/cbic.201900721DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7317446PMC
May 2020

Structure-activity relationships for binding of 4-substituted triazole-phenols to macrophage migration inhibitory factor (MIF).

Eur J Med Chem 2020 Jan 11;186:111849. Epub 2019 Nov 11.

Chemical and Pharmaceutical Biology, Groningen Research Institute of Pharmacy (GRIP), University of Groningen, Groningen, the Netherlands. Electronic address:

Macrophage migration inhibitory factor (MIF) is a versatile protein that plays a role in inflammation, autoimmune diseases and cancers. Development of novel inhibitors will enable further exploration of MIF as a drug target. In this study, we investigated structure-activity relationships of MIF inhibitors using a MIF tautomerase activity assay to measure binding. Importantly, we notified that transition metals such as copper (II) and zinc (II) interfere with the MIF tautomerase activity under the assay conditions applied. EDTA was added to the assay buffer to avoid interference of residual heavy metals with tautomerase activity measurements. Using these assay conditions the structure-activity relationships for MIF binding of a series of triazole-phenols was explored. The most potent inhibitors in this series provided activities in the low micromolar range. Enzyme kinetic analysis indicates competitive binding that proved reversible. Binding to the enzyme was confirmed using a microscale thermophoresis (MST) assay. Molecular modelling was used to rationalize the observed structure-activity relationships. The most potent inhibitor 2d inhibited proliferation of A549 cells in a clonogenic assay. In addition, 2d attenuated MIF induced ERK phosphorylation in A549 cells. Altogether, this study provides insights in the structure-activity relationships for MIF binding of triazole-phenols and further validates this class of compounds as MIF binding agents in cell-based studies.
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http://dx.doi.org/10.1016/j.ejmech.2019.111849DOI Listing
January 2020

Inhibitory selectivity among class I HDACs has a major impact on inflammatory gene expression in macrophages.

Eur J Med Chem 2019 Sep 18;177:457-466. Epub 2019 May 18.

Chemical and Pharmaceutical Biology, Groningen Research Institute of Pharmacy (GRIP), University of Groningen, Groningen, 9713 AV, the Netherlands. Electronic address:

Histone deacetylases (HDACs) play an important role in cancer, degenerative diseases and inflammation. The currently applied HDAC inhibitors in the clinic lack selectivity among HDAC isoforms, which limits their application for novel indications such as inflammatory diseases. Recent, literature indicates that HDAC 3 plays an important role among class I HDACs in gene expression in inflammation. In this perspective, the development and understanding of inhibitory selectivity among HDACs 1, 2 and 3 and their respective influence on gene expression need to be characterized to facilitate drug discovery. Towards this aim, we synthesized nine structural analogues of the class I HDAC inhibitor Entinostat and investigated their selectivity profile among HDACs 1, 2 and 3. We found that we can explain the observed structure activity relationships by small structural and conformational differences between HDAC 1 and HDAC 3 in the 'lid' interacting region. Cell-based studies indicated, however, that application of inhibitors with improved HDAC 3 selectivity did not provide an anti-inflammatory response in contrast to expectations from biochemical evidence in literature. Altogether, in this study, we identified structure activity relationships among class I HDACs and we connected isoform selectivity among class I HDACs with pro- and anti-inflammatory gene transcription in macrophages.
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http://dx.doi.org/10.1016/j.ejmech.2019.05.038DOI Listing
September 2019

Creation of RANKL mutants with low affinity for decoy receptor OPG and their potential anti-fibrosis activity.

FEBS J 2019 09 25;286(18):3582-3593. Epub 2019 May 25.

Department of Chemical and Pharmaceutical Biology, Groningen Research Institute of Pharmacy, University of Groningen, The Netherlands.

Fibrosis is characterized by the progressive alteration of the tissue structure due to the excessive production of extracellular matrix (ECM). The signaling system encompassing Receptor Activator of Nuclear factor NF-κB Ligand (RANKL)/RANK/Osteoprotegerin (OPG) was discovered to play an important role in the regulation of ECM formation and degradation in bone tissue. However, whether and how this signaling pathway plays a role in liver or pulmonary ECM degradation is unclear up to now. Interestingly, increased decoy receptor OPG levels are found in fibrotic tissues. We hypothesize that RANKL can stimulate RANK on macrophages and initiate the process of ECM degradation. This process may be inhibited by highly expressed OPG in fibrotic conditions. In this case, RANKL mutants that can bind to RANK without binding to OPG might become promising therapeutic candidates. In this study, we built a structure-based library containing 44 RANKL mutants and found that the Q236 residue of RANKL is important for OPG binding. We show that RANKL_Q236D can activate RAW cells to initiate the process of ECM degradation and is able to escape from the obstruction by exogenous OPG. We propose that the generation of RANKL mutants with reduced affinity for OPG is a promising strategy for the exploration of new therapeutics against fibrosis.
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http://dx.doi.org/10.1111/febs.14925DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6852375PMC
September 2019

Novel 15-Lipoxygenase-1 Inhibitor Protects Macrophages from Lipopolysaccharide-Induced Cytotoxicity.

J Med Chem 2019 05 19;62(9):4624-4637. Epub 2019 Apr 19.

Department of Chemical Biology 2, Stratingh Institute for Chemistry , University of Groningen , Nijenborgh 7 , 9747 AG Groningen , The Netherlands.

Various mechanisms for regulated cell death include the formation of oxidative mediators such as lipid peroxides and nitric oxide (NO). In this respect, 15-lipoxygenase-1 (15-LOX-1) is a key enzyme that catalyzes the formation of lipid peroxides. The actions of these peroxides are interconnected with nuclear factor-κB signaling and NO production. Inhibition of 15-LOX-1 holds promise to interfere with regulated cell death in inflammatory conditions. In this study, a novel potent 15-LOX-1 inhibitor, 9c (i472), was developed and structure-activity relationships were explored. In vitro, this inhibitor protected cells from lipopolysaccharide-induced cell death, inhibiting NO formation and lipid peroxidation. Thus, we provide a novel 15-LOX-1 inhibitor that inhibits cellular NO production and lipid peroxidation, which set the stage for further exploration of these mechanisms.
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http://dx.doi.org/10.1021/acs.jmedchem.9b00212DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6511944PMC
May 2019

Metabolic Engineering of Toward Taxadiene Biosynthesis as the First Committed Step for Taxol Production.

Front Microbiol 2019 20;10:218. Epub 2019 Feb 20.

Department of Chemical and Pharmaceutical Biology, Groningen Research Institute of Pharmacy, University of Groningen, Groningen, Netherlands.

Terpenoids are natural products known for their medicinal and commercial applications. Metabolic engineering of microbial hosts for the production of valuable compounds, such as artemisinin and Taxol, has gained vast interest in the last few decades. The Generally Regarded As Safe (GRAS) 168 with its broad metabolic potential is considered one of these interesting microbial hosts. In the effort toward engineering as a cell factory for the production of the chemotherapeutic Taxol, we expressed the plant-derived taxadiene synthase (TXS) enzyme. TXS is responsible for the conversion of the precursor geranylgeranyl pyrophosphate (GGPP) to taxa-4,11-diene, which is the first committed intermediate in Taxol biosynthesis. Furthermore, overexpression of eight enzymes in the biosynthesis pathway was performed to increase the flux of the GGPP precursor. This was achieved by creating a synthetic operon harboring the genes encoding the 2-C-methyl-D-erythritol-4-phosphate (MEP) pathway (, , , , , , ) together with (encoding geranyl and farnesyl pyrophosphate synthases) responsible for providing farnesyl pyrophosphate (FPP). In addition, a vector harboring the gene (encoding geranylgeranyl pyrophosphate synthase, GGPPS, of ) to increase the supply of GGPP was introduced. The overexpression of the MEP pathway enzymes along with IspA and GGPPS caused an 83-fold increase in the amount of taxadiene produced compared to the strain only expressing TXS and relying on the innate pathway of . The total amount of taxadiene produced by that strain was 17.8 mg/l. This is the first account of the successful expression of taxadiene synthase in . We determined that the expression of GGPPS through the gene is essential for the formation of sufficient precursor, GGPP, in as its innate metabolism is not efficient in producing it. Finally, the extracellular localization of taxadiene production by overexpressing the complete MEP pathway along with IspA and GGPPS presents the prospect for further engineering aiming for semisynthesis of Taxol.
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http://dx.doi.org/10.3389/fmicb.2019.00218DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6391936PMC
February 2019

Enantioselective Synthesis of Pharmaceutically Active γ-Aminobutyric Acids Using a Tailor-Made Artificial Michaelase in One-Pot Cascade Reactions.

ACS Catal 2019 Feb 7;9(2):1503-1513. Epub 2019 Jan 7.

Department of Chemical and Pharmaceutical Biology, Groningen Research Institute of Pharmacy, University of Groningen, Antonius Deusinglaan 1, 9713 AV Groningen, The Netherlands.

Chiral γ-aminobutyric acid (GABA) analogues represent abundantly prescribed drugs, which are broadly applied as anticonvulsants, as antidepressants, and for the treatment of neuropathic pain. Here we report a one-pot two-step biocatalytic cascade route for synthesis of the pharmaceutically relevant enantiomers of γ-nitrobutyric acids, starting from simple precursors (acetaldehyde and nitroalkenes), using a tailor-made highly enantioselective artificial "Michaelase" (4-oxalocrotonate tautomerase mutant L8Y/M45Y/F50A), an aldehyde dehydrogenase with a broad non-natural substrate scope, and a cofactor recycling system. We also report a three-step chemoenzymatic cascade route for the efficient chemical reduction of enzymatically prepared γ-nitrobutyric acids into GABA analogues in one pot, achieving high enantiopurity (e.r. up to 99:1) and high overall yields (up to 70%). This chemoenzymatic methodology offers a step-economic alternative route to important pharmaceutically active GABA analogues, and highlights the exciting opportunities available for combining chemocatalysts, natural enzymes, and designed artificial biocatalysts in multistep syntheses.
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http://dx.doi.org/10.1021/acscatal.8b04299DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6366683PMC
February 2019

Catalysis of amorpha-4,11-diene synthase unraveled and improved by mutability landscape guided engineering.

Sci Rep 2018 07 2;8(1):9961. Epub 2018 Jul 2.

Department of Chemical and Pharmaceutical Biology, Groningen Research Institute of Pharmacy, University of Groningen, 9713 AV, Groningen, The Netherlands.

Amorpha-4,11-diene synthase (ADS) cyclizes the substrate farnesyl pyrophosphate to produce amorpha-4,11-diene as a major product. This is considered the first committed and rate-limiting step in the biosynthesis of the antimalarial artemisinin. Here, we utilize a reported 3D model of ADS to perform mutability landscape guided enzyme engineering. A mutant library of 258 variants along sixteen active site residues was created then screened for catalytic activity and product profile. This allowed for identification of the role of some of these residues in the mechanism. R262 constrains the released pyrophosphate group along with magnesium ions. The aromatic residues (W271, Y519 and F525) stabilize the intermediate carbocations while T296, G400, G439 and L515 help with the 1,6- and 1,10-ring closures. Finally, W271 is suggested to act as active site base along with T399, which ensures regioselective deprotonation. The mutability landscape also helped determine variants with improved catalytic activity. H448A showed ~4 fold increase in catalytic efficiency and the double mutation T399S/H448A improved k by 5 times. This variant can be used to enhance amorphadiene production and in turn artemisinin biosynthesis. Our findings provide the basis for the first step in improving industrial production of artemisinin and they open up possibilities for further engineering and understanding of ADS.
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http://dx.doi.org/10.1038/s41598-018-28177-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6028579PMC
July 2018

Novel RANKL DE-loop mutants antagonize RANK-mediated osteoclastogenesis.

FEBS J 2017 08 7;284(15):2501-2512. Epub 2017 Jul 7.

Department of Chemical and Pharmaceutical Biology, Groningen Research Institute of Pharmacy, University of Groningen, The Netherlands.

Bone is a dynamic tissue that is maintained by continuous renewal. An imbalance in bone resorption and bone formation can lead to a range of disorders, such as osteoporosis. The receptor activator of NF-κB (RANK)-RANK-ligand (RANKL) pathway plays a major role in bone remodeling. Here, we investigated the effect of mutations at position I248 in the DE-loop of murine RANKL on the interaction of RANKL with RANK, and subsequent activation of osteoclastogenesis. Two single mutants, RANKL I248Y and I248K, were found to maintain binding and have the ability to reduce wild-type RANKL-induced osteoclastogenesis. The generation of RANK-antagonists is a promising strategy for the exploration of new therapeutics against osteoporosis.
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http://dx.doi.org/10.1111/febs.14142DOI Listing
August 2017

Penicillin V acylases from gram-negative bacteria degrade N-acylhomoserine lactones and attenuate virulence in Pseudomonas aeruginosa.

Appl Microbiol Biotechnol 2017 Mar 8;101(6):2383-2395. Epub 2016 Dec 8.

Division of Biochemical Sciences, CSIR-National Chemical Laboratory, Dr. Homi Bhabha Road, Pune, 411008, India.

Virulence pathways in gram-negative pathogenic bacteria are regulated by quorum sensing mechanisms, through the production and sensing of N-acylhomoserine lactone (AHL) signal molecules. Enzymatic degradation of AHLs leading to attenuation of virulence (quorum quenching) could pave the way for the development of new antibacterials. Penicillin V acylases (PVAs) belong to the Ntn hydrolase superfamily, together with AHL acylases. PVAs are exploited widely in the pharmaceutical industry, but their role in the natural physiology of their native microbes is not clearly understood. This report details the characterization of AHL degradation activity by homotetrameric PVAs from two gram-negative plant pathogenic bacteria, Pectobacterium atrosepticum (PaPVA) and Agrobacterium tumefaciens (AtPVA). Both the PVAs exhibited substrate specificity for degrading long-chain AHLs. Exogenous addition of these enzymes into Pseudomonas aeruginosa greatly diminished the production of elastase and pyocyanin and biofilm formation and increased the survival rate in an insect model of acute infection. Subtle structural differences in the PVA active site that regulate specificity for acyl chain length have been characterized, which could reflect the evolution of AHL-degrading acylases in relation to the environment of the bacteria that produce them and also provide strategies for enzyme engineering. The potential for using these enzymes as therapeutic agents in clinical applications and a few ideas about their possible significance in microbial physiology have also been discussed.
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http://dx.doi.org/10.1007/s00253-016-8031-5DOI Listing
March 2017

Insights into the Three-Dimensional Structure of Amorpha-4,11-diene Synthase and Probing of Plasticity Residues.

J Nat Prod 2016 10 27;79(10):2455-2463. Epub 2016 Sep 27.

Department of Chemical and Pharmaceutical Biology, Groningen Research Institute of Pharmacy, University of Groningen , 9713 AV, Groningen, The Netherlands.

Amorphadiene synthase (ADS) is known for its vital role as a key enzyme in the biosynthesis of the antimalarial drug artemisinin. Despite the vast research targeting this enzyme, an X-ray crystal structure of the enzyme has not yet been reported. In spite of the remarkable difference in product profile among various sesquiterpene synthases, they all share a common α-helical fold with many highly conserved regions especially the bivalent metal ion binding motifs. Hence, to better understand the structural basis of the mechanism of ADS, a reliable 3D homology model representing the conformation of the ADS enzyme and the position of its substrate, farnesyl diphosphate, in the active site was constructed. The model was generated using the reported crystal structure of α-bisabolol synthase mutant, an enzyme with high sequence identity with ADS, as a template. Site-directed mutagenesis was used to probe the active site residues. Seven residues were probed showing their vital role in the ADS mechanism and/or their effect on product profile. The generated variants confirmed the validity of the ADS model. This model will serve as a basis for exploring structure-function relationships of all residues in the active site to obtain further insight into the ADS mechanism.
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http://dx.doi.org/10.1021/acs.jnatprod.6b00236DOI Listing
October 2016

Using mutability landscapes of a promiscuous tautomerase to guide the engineering of enantioselective Michaelases.

Nat Commun 2016 Mar 8;7:10911. Epub 2016 Mar 8.

Department of Pharmaceutical Biology, Groningen Research Institute of Pharmacy, University of Groningen, Antonius Deusinglaan 1, Groningen 9713 AV, The Netherlands.

The Michael-type addition reaction is widely used in organic synthesis for carbon-carbon bond formation. However, biocatalytic methodologies for this type of reaction are scarce, which is related to the fact that enzymes naturally catalysing carbon-carbon bond-forming Michael-type additions are rare. A promising template to develop new biocatalysts for carbon-carbon bond formation is the enzyme 4-oxalocrotonate tautomerase, which exhibits promiscuous Michael-type addition activity. Here we present mutability landscapes for the expression, tautomerase and Michael-type addition activities, and enantioselectivity of 4-oxalocrotonate tautomerase. These maps of neutral, beneficial and detrimental amino acids for each residue position and enzyme property provide detailed insight into sequence-function relationships. This offers exciting opportunities for enzyme engineering, which is illustrated by the redesign of 4-oxalocrotonate tautomerase into two enantiocomplementary 'Michaelases'. These 'Michaelases' catalyse the asymmetric addition of acetaldehyde to various nitroolefins, providing access to both enantiomers of γ-nitroaldehydes, which are important precursors for pharmaceutically active γ-aminobutyric acid derivatives.
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http://dx.doi.org/10.1038/ncomms10911DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4786785PMC
March 2016

Development of a dry, stable and inhalable acyl-homoserine-lactone-acylase powder formulation for the treatment of pulmonary Pseudomonas aeruginosa infections.

Eur J Pharm Sci 2013 Mar 28;48(4-5):637-43. Epub 2012 Dec 28.

Department of Pharmaceutical Biology, University of Groningen, Antonius Deusiglaan 1, 9713 AV Groningen, The Netherlands.

In the lungs of cystic fibrosis (CF) patients, Pseudomonas aeruginosa commonly causes chronic infections. It has been shown that the P. aeruginosa quorum sensing (QS) system controls the expression of virulence factors during invasion and infection to host cells. PvdQ is an acyl-homoserine lactone (AHL) acylase able to degrade the signal molecule of P. aeruginosa QS. The role of PvdQ in inhibiting the QS and its successive virulence determinants has been established in in vitro as well as in in vivo, the latter in a Caenorabdhitis elegans model. For the treatment of pulmonary P. aeruginosa infections, we propose that PvdQ can be best administered directly to the lungs of the patients as a dry powder because this is expected to give specific advantages in delivery as compared to nebulizing. Therefore in this study we investigated the production of a PvdQ powder by spray-freeze drying using mannitol, trehalose and inulin as excipient. The activity of PvdQ in the powder was determined immediately after production and after subsequent storage during 4 weeks at 20°C and 55°C. We found that the enzymatic activity of PvdQ is fully maintained during spray-freeze drying using mannitol, trehalose or inulin as excipient. However, mannitol was not able to stabilize the protein during storage, while PvdQ incorporated in trehalose or inulin was fully stabilized even during storage at 55°C for at least 4 weeks. The poor stabilizing capacities of mannitol during storage could be related to its crystalline nature while the excellent stabilizing capacities of trehalose and inulin during storage could be related to their amorphous nature. The trehalose and inulin-based particles consisted of porous spheres with a volume average aerodynamical diameter of ∼1.8 μm implying that they are suitable for pulmonary delivery. This is the first study in which an AHL-degrading enzyme is processed into spray-freeze-dried powder suitable for inhalation.
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http://dx.doi.org/10.1016/j.ejps.2012.12.015DOI Listing
March 2013

Lipase A gene transcription in Pseudomonas alcaligenes is under control of RNA polymerase σ54 and response regulator LipR.

FEMS Microbiol Lett 2012 Apr 24;329(2):146-53. Epub 2012 Feb 24.

Department of Pharmaceutical Biology, University Center of Pharmacy, Groningen University Institute for Drug Exploration, Groningen, The Netherlands.

Initial analysis has shown that the transcription of the Pseudomonas alcaligenes lipA gene, which encodes an extracellular lipase, is governed by the LipQR two-component system consisting of sensor kinase LipQ and DNA-binding regulator LipR. This study further analyzes lipA gene expression and demonstrates that the RNA polymerase σ54 is involved in the transcription. Purified LipR has an ATPase activity that is stimulated by the presence of lipA promoter DNA. Surface plasmon resonance measurements with purified and in vitro phosphorylated LipR reveal that phosphorylation of LipR is required for specific binding to the upstream activating sequence of the lipA promoter. Furthermore, mass spectrometric analysis combined with mutagenesis demonstrates that Asp52 is the phosphorylated aspartate. This analysis exposes LipR as a prominent member of the growing family of bacterial enhancer-binding proteins.
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http://dx.doi.org/10.1111/j.1574-6968.2012.02516.xDOI Listing
April 2012

PA0305 of Pseudomonas aeruginosa is a quorum quenching acylhomoserine lactone acylase belonging to the Ntn hydrolase superfamily.

Microbiology (Reading) 2011 Jul 3;157(Pt 7):2042-2055. Epub 2011 Mar 3.

Department of Pharmaceutical Biology, University of Groningen, 9713AV Groningen, The Netherlands.

The Pseudomonas aeruginosa PAO1 genome has at least two genes, pvdQ and quiP, encoding acylhomoserine lactone (AHL) acylases. Two additional genes, pa1893 and pa0305, have been predicted to encode penicillin acylase proteins, but have not been characterized. Initial studies on a pa0305 transposon insertion mutant suggested that the gene is not related to the AHL growth phenotype of P. aeruginosa. The close similarity (67 %) of pa0305 to HacB, an AHL acylase of Pseudomonas syringae, prompted us to investigate whether the PA0305 protein might also function as an AHL acylase. The pa0305 gene has been cloned and the protein (PA0305) has been overproduced, purified and subjected to functional characterization. Analysis of the purified protein showed that, like β-lactam acylases, PA0305 undergoes post-translational processing resulting in α- and β-subunits, with the catalytic serine as the first amino acid of the β-subunit, strongly suggesting that PA0305 is a member of the N-terminal nucleophile hydrolase superfamily. Using a biosensor assay, PA0305his was shown to degrade AHLs with acyl side chains ranging in length from 6 to 14 carbons. Kinetics studies using N-octanoyl-L-homoserine lactone (C(8)-HSL) and N-(3-oxo-dodecanoyl)-L-homoserine lactone (3-oxo-C(12)-HSL) as substrates showed that the enzyme has a robust activity towards these two AHLs, with apparent K(cat)/K(m) values of 0.14 × 10(4) M(-1) s(-1) towards C(8)-HSL and 7.8 × 10(4) M(-1 )s(-1) towards 3-oxo-C(12)-HSL. Overexpression of the pa0305 gene in P. aeruginosa showed significant reductions in both accumulation of 3-oxo-C(12)-HSL and expression of virulence factors. A mutant P. aeruginosa strain with a deleted pa0305 gene showed a slightly increased capacity to kill Caenorhabditis elegans compared with the P. aeruginosa PAO1 wild-type strain and the PAO1 strain carrying a plasmid overexpressing pa0305. The harmful effects of the Δpa0305 strain on the animals were most visible at 5 days post-exposure and the mortality rate of the animals fed on the Δpa0305 strain was faster than for the animals fed on either the wild-type strain or the strain overexpressing pa0305. In conclusion, the pa0305 gene encodes an efficient acylase with activity towards long-chain homoserine lactones, including 3-oxo-C(12)-HSL, the natural quorum sensing signal molecule in P. aeruginosa, and we propose to name this acylase HacB.
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http://dx.doi.org/10.1099/mic.0.043935-0DOI Listing
July 2011

Lipase expression in Pseudomonas alcaligenes is under the control of a two-component regulatory system.

Appl Environ Microbiol 2008 Mar 11;74(5):1402-11. Epub 2008 Jan 11.

Pharmaceutical Biology, University of Groningen, Antonius Deusinglaan 1, 9713 AV Groningen, The Netherlands.

Preliminary observations in a large-scale fermentation process suggested that the lipase expression of Pseudomonas alcaligenes can be switched on by the addition of certain medium components, such as soybean oil. In an attempt to elucidate the mechanism of induction of lipase expression, we have set up a search method for genes controlling lipase expression by use of a cosmid library containing fragments of P. alcaligenes genomic DNA. A screen for lipase hyperproduction resulted in the selection of multiple transformants, of which the best-producing strains comprised cosmids that shared an overlapping genomic fragment. Within this fragment, two previously unidentified genes were found and named lipQ and lipR. Their encoded proteins belong to the NtrBC family of regulators that regulate gene expression via binding to a specific upstream activator sequence (UAS). Such an NtrC-like UAS was identified in a previous study in the P. alcaligenes lipase promoter, strongly suggesting that LipR acts as a positive regulator of lipase expression. The regulating role could be confirmed by down-regulated lipase expression in a strain with an inactivated lipR gene and a threefold increase in lipase yield in a large-scale fermentation when expressing the lipQR operon from the multicopy plasmid pLAFR3. Finally, cell extracts of a LipR-overexpressing strain caused a retardation of the lipase promoter fragment in a band shift assay. Our results indicate that lipase expression in Pseudomonas alcaligenes is under the control of the LipQR two-component system.
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http://dx.doi.org/10.1128/AEM.01632-07DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2258621PMC
March 2008

LmrCD is a major multidrug resistance transporter in Lactococcus lactis.

Mol Microbiol 2006 Aug;61(3):771-81

Department of Molecular Microbiology, Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, Kerklaan 30, NL-9751 Haren, the Netherlands.

When Lactococcus lactis is challenged with drugs it displays a multidrug resistance (MDR) phenotype. In silico analysis of the genome of L. lactis indicates the presence of at least 40 putative MDR transporters, of which only four, i.e. the ABC transporters LmrA, LmrC and LmrD, and the major facilitator LmrP, have been experimentally associated with the MDR. To understand the molecular basis of the MDR phenotype in L. lactis, we have performed a global transcriptome analysis comparing four independently isolated drug-resistant strains of L. lactis with the wild-type strain. The results show a strong and consistent upregulation of the lmrC and lmrD genes in all four strains, while the mRNA levels of other putative MDR transporters were not significantly altered. Deletion of lmrCD renders L. lactis sensitive to several toxic compounds, and this phenotype is associated with a reduced ability to secrete these compounds. Another gene, which is strongly upregulated in all mutant strains, specifies LmrR (YdaF), a local transcriptional repressor of lmrCD that belongs to the PadR family of transcriptional regulators and that binds to the promoter region of lmrCD. These results demonstrate that the heterodimeric MDR ABC transporter LmrCD is a major determinant of both acquired and intrinsic drug resistance of L. lactis.
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http://dx.doi.org/10.1111/j.1365-2958.2006.05267.xDOI Listing
August 2006

Nucleotide-binding sites of the heterodimeric LmrCD ABC-multidrug transporter of Lactococcus lactis are asymmetric.

Biochemistry 2006 Jan;45(2):648-56

Department of Microbiology, Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, Kerklaan 30, NL-9751 Haren, The Netherlands.

LmrCD is a lactococcal, heterodimeric multidrug transporter, which belongs to the ABC superfamily. It consists of two half-transporters, LmrC and LmrD, that are necessary and sufficient for drug extrusion and ATP hydrolysis. LmrCD is asymmetric in terms of the conservation of the functional motifs of the nucleotide-binding domains (NBDs). Important residues of the nucleotide-binding site of LmrC and the C loop of LmrD are not conserved. To investigate the functional importance of the LmrC and LmrD subunits, the putative catalytic base residue adjacent to the Walker B motif of both NBDs were substituted for the respective carboxamides. Our data demonstrate that Glu587 of LmrD is essential for both drug transport and ATPase activity of the LmrCD heterodimer, whereas mutation of Asp495 of LmrC has a less severe effect on the activity of the complex. Structural and/or functional asymmetry is further demonstrated by differential labeling of both subunits by 8-azido-[alpha-32P]ATP, which, at 4 degrees C, occurs predominantly at LmrC, while aluminiumfluoride (AlF(x))-induced trapping of the hydrolyzed nucleotide at 30 degrees C results in an almost exclusive labeling of LmrD. It is concluded that the LmrCD heterodimer contains two structurally and functionally distinct NBDs.
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http://dx.doi.org/10.1021/bi051276sDOI Listing
January 2006

Proton motive force-dependent Hoechst 33342 transport by the ABC transporter LmrA of Lactococcus lactis.

Biochemistry 2005 Dec;44(51):16931-8

Department of Microbiology, Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, Kerklaan 30, NL-9751 NN Haren, The Netherlands.

The fluorescent compound Hoechst 33342 is a substrate for many multidrug resistance (MDR) transporters and is widely used to characterize their transport activity. We have constructed mutants of the adenosine triphosphate (ATP) binding cassette (ABC)-type MDR transporter LmrA of Lactococcus lactis that are defective in ATP hydrolysis. These mutants and wild-type LmrA exhibited an atypical behavior in the Hoechst 33342 transport assay. In membrane vesicles, Hoechst 33342 transport was shown to be independent of the ATPase activity of LmrA, and it was not inhibited by orthovanadate but sensitive to uncouplers that collapse the proton gradient and to N,N'-dicyclohexylcarbodiimide, an inhibitor of the F0F1-ATPase. In contrast, transport of Hoechst 33342 by the homologous, heterodimeric MDR transporter LmrCD showed a normal ATP dependence and was insensitive to uncouplers of the proton gradient. With intact cells, expression of LmrA resulted in an increased rate of Hoechst 33342 influx while LmrCD caused a decrease in the rate of Hoechst 33342 influx. Cellular toxicity assays using a triple knockout strain, i.e., L. lactis delta lmrA delta lmrCD, demonstrate that expression of LmrCD protects cells against the growth inhibitory effects of Hoechst 33342, while in the presence of LmrA, cells are more susceptible to Hoechst 33342. Our data demonstrate that the LmrA-mediated Hoechst 33342 transport in membrane vesicles is influenced by the transmembrane pH gradient due to a pH-dependent partitioning of Hoechst 33342 into the membrane.
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http://dx.doi.org/10.1021/bi051497yDOI Listing
December 2005

ydaG and ydbA of Lactococcus lactis encode a heterodimeric ATP-binding cassette-type multidrug transporter.

J Biol Chem 2004 Aug 10;279(33):34449-55. Epub 2004 Jun 10.

Department of Microbiology, Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, Kerklaan 30, NL-9751 NN Haren, The Netherlands.

Multidrug resistance (MDR)-type transporters mediate the active extrusion of structurally and functionally dissimilar compounds from the cells, thereby rendering cells resistant to a range of drugs. The ydaG and ydbA genes of Lactococcus lactis encode two ATP-binding cassette half-transporters, which both share homology with MDR proteins such as LmrA from L. lactis or the mammalian P-glycoprotein. The ydaG/ydbA genes were cloned and expressed separately and jointly in L. lactis using the nisin-inducible system. When both proteins are co-expressed, several structurally dissimilar drugs such as ethidium, daunomycin, and BCECF-AM are extruded from the cell. YdaG and YdbA could be co-purified as a stable heterodimer. ATPase activity was found to be associated with the YdaG/YdbA heterodimer only and not with the individual subunits. Both the ydaG and ydbA genes are up-regulated in multidrug-resistant L. lactis strains selected for growth in the presence of a variety of toxic compounds. This is the first demonstration of a functional heterodimeric ATP-binding cassette-type MDR transporter.
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http://dx.doi.org/10.1074/jbc.M404072200DOI Listing
August 2004
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