Publications by authors named "Amanda A Brindley"

31 Publications

The active site of magnesium chelatase.

Nat Plants 2020 12 30;6(12):1491-1502. Epub 2020 Nov 30.

Department of Molecular Biology and Biotechnology, The University of Sheffield, Sheffield, UK.

The insertion of magnesium into protoporphyrin initiates the biosynthesis of chlorophyll, the pigment that underpins photosynthesis. This reaction, catalysed by the magnesium chelatase complex, couples ATP hydrolysis by a ChlID motor complex to chelation within the ChlH subunit. We probed the structure and catalytic function of ChlH using a combination of X-ray crystallography, computational modelling, mutagenesis and enzymology. Two linked domains of ChlH in an initially open conformation of ChlH bind protoporphyrin IX, and the rearrangement of several loops envelops this substrate, forming an active site cavity. This induced fit brings an essential glutamate (E660), proposed to be the key catalytic residue for magnesium insertion, into proximity with the porphyrin. A buried solvent channel adjacent to E660 connects the exterior bulk solvent to the active site, forming a possible conduit for the delivery of magnesium or abstraction of protons.
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http://dx.doi.org/10.1038/s41477-020-00806-9DOI Listing
December 2020

Dissecting the cytochrome -reaction centre interaction in bacterial photosynthesis using single molecule force spectroscopy.

Biochem J 2019 08 9;476(15):2173-2190. Epub 2019 Aug 9.

Department of Molecular Biology and Biotechnology, University of Sheffield, Sheffield S10 2TN, U.K.

The reversible docking of small, diffusible redox proteins onto a membrane protein complex is a common feature of bacterial, mitochondrial and photosynthetic electron transfer (ET) chains. Spectroscopic studies of ensembles of such redox partners have been used to determine ET rates and dissociation constants. Here, we report a single-molecule analysis of the forces that stabilise transient ET complexes. We examined the interaction of two components of bacterial photosynthesis, cytochrome and the reaction centre (RC) complex, using dynamic force spectroscopy and PeakForce quantitative nanomechanical imaging. RC-LH1-PufX complexes, attached to silicon nitride AFM probes and maintained in a photo-oxidised state, were lowered onto a silicon oxide substrate bearing dispersed, immobilised and reduced cytochrome molecules. Microscale patterns of cytochrome and the cyan fluorescent protein were used to validate the specificity of recognition between tip-attached RCs and surface-tethered cytochrome Following the transient association of photo-oxidised RC and reduced cytochrome molecules, retraction of the RC-functionalised probe met with resistance, and forces between 112 and 887 pN were required to disrupt the post-ET RC- complex, depending on the retraction velocities used. If tip-attached RCs were reduced instead, the probability of interaction with reduced cytochrome molecules decreased 5-fold. Thus, the redox states of the cytochrome haem cofactor and RC 'special pair' bacteriochlorophyll dimer are important for establishing a productive ET complex. The millisecond persistence of the post-ET cytochrome [oxidised]-RC[reduced] 'product' state is compatible with rates of cyclic photosynthetic ET, at physiologically relevant light intensities.
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http://dx.doi.org/10.1042/BCJ20170519DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6688529PMC
August 2019

The ChlD subunit links the motor and porphyrin binding subunits of magnesium chelatase.

Biochem J 2019 07 2;476(13):1875-1887. Epub 2019 Jul 2.

Department of Molecular Biology and Biotechnology, The University of Sheffield, Sheffield S10 2TN, U.K.

Magnesium chelatase initiates chlorophyll biosynthesis, catalysing the MgATP-dependent insertion of a Mg ion into protoporphyrin IX. The catalytic core of this large enzyme complex consists of three subunits: Bch/ChlI, Bch/ChlD and Bch/ChlH (in bacteriochlorophyll and chlorophyll producing species, respectively). The D and I subunits are members of the AAA (ATPases associated with various cellular activities) superfamily of enzymes, and they form a complex that binds to H, the site of metal ion insertion. In order to investigate the physical coupling between ChlID and ChlH and , ChlD was FLAG-tagged in the cyanobacterium sp. PCC 6803 and co-immunoprecipitation experiments showed interactions with both ChlI and ChlH. Co-production of recombinant ChlD and ChlH in yielded a ChlDH complex. Quantitative analysis using microscale thermophoresis showed magnesium-dependent binding ( 331 ± 58 nM) between ChlD and H. The physical basis for a ChlD-H interaction was investigated using chemical cross-linking coupled with mass spectrometry (XL-MS), together with modifications that either truncate ChlD or modify single residues. We found that the C-terminal integrin I domain of ChlD governs association with ChlH, the Mg dependence of which also mediates the cooperative response of the chelatase to magnesium. The interaction site between the AAA motor and the chelatase domain of magnesium chelatase will be essential for understanding how free energy from the hydrolysis of ATP on the AAA ChlI subunit is transmitted via the bridging subunit ChlD to the active site on ChlH.
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http://dx.doi.org/10.1042/BCJ20190095DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6604950PMC
July 2019

Probing the quality control mechanism of the twin-arginine translocase with folding variants of a -designed heme protein.

J Biol Chem 2018 05 20;293(18):6672-6681. Epub 2018 Mar 20.

From the Department of Molecular Biology and Biotechnology, University of Sheffield, Sheffield S10 2TN, United Kingdom,

Protein transport across the cytoplasmic membrane of bacterial cells is mediated by either the general secretion (Sec) system or the twin-arginine translocase (Tat). The Tat machinery exports folded and cofactor-containing proteins from the cytoplasm to the periplasm by using the transmembrane proton motive force as a source of energy. The Tat apparatus apparently senses the folded state of its protein substrates, a quality-control mechanism that prevents premature export of nascent unfolded or misfolded polypeptides, but its mechanistic basis has not yet been determined. Here, we investigated the innate ability of the model Tat system to recognize and translocate -designed protein substrates with experimentally determined differences in the extent of folding. Water-soluble, four-helix bundle maquette proteins were engineered to bind two, one, or no heme cofactors, resulting in a concomitant reduction in the extent of their folding, assessed with temperature-dependent CD spectroscopy and one-dimensional H NMR spectroscopy. Fusion of the archetypal N-terminal Tat signal peptide of the trimethylamine--oxide (TMAO) reductase (TorA) to the N terminus of the protein maquettes was sufficient for the Tat system to recognize them as substrates. The clear correlation between the level of Tat-dependent export and the degree of heme -induced folding of the maquette protein suggested that the membrane-bound Tat machinery can sense the extent of folding and conformational flexibility of its substrates. We propose that these artificial proteins are ideal substrates for future investigations of the Tat system's quality-control mechanism.
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http://dx.doi.org/10.1074/jbc.RA117.000880DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5936819PMC
May 2018

Complete enzyme set for chlorophyll biosynthesis in .

Sci Adv 2018 01 26;4(1):eaaq1407. Epub 2018 Jan 26.

Department of Molecular Biology and Biotechnology, University of Sheffield, Firth Court, Western Bank, Sheffield S10 2TN, UK.

Chlorophylls are essential cofactors for photosynthesis, which sustains global food chains and oxygen production. Billions of tons of chlorophylls are synthesized annually, yet full understanding of chlorophyll biosynthesis has been hindered by the lack of characterization of the Mg-protoporphyrin IX monomethyl ester oxidative cyclase step, which confers the distinctive green color of these pigments. We demonstrate cyclase activity using heterologously expressed enzyme. Next, we assemble a genetic module that encodes the complete chlorophyll biosynthetic pathway and show that it functions in . Expression of 12 genes converts endogenous protoporphyrin IX into chlorophyll a, turning cells green. Our results delineate a minimum set of enzymes required to make chlorophyll and establish a platform for engineering photosynthesis in a heterotrophic model organism.
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http://dx.doi.org/10.1126/sciadv.aaq1407DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5787379PMC
January 2018

Repurposing a photosynthetic antenna protein as a super-resolution microscopy label.

Sci Rep 2017 12 1;7(1):16807. Epub 2017 Dec 1.

Department of Molecular Biology and Biotechnology, Firth Court, Western Bank, Sheffield, S10 2TN, UK.

Techniques such as Stochastic Optical Reconstruction Microscopy (STORM) and Structured Illumination Microscopy (SIM) have increased the achievable resolution of optical imaging, but few fluorescent proteins are suitable for super-resolution microscopy, particularly in the far-red and near-infrared emission range. Here we demonstrate the applicability of CpcA, a subunit of the photosynthetic antenna complex in cyanobacteria, for STORM and SIM imaging. The periodicity and width of fabricated nanoarrays of CpcA, with a covalently attached phycoerythrobilin (PEB) or phycocyanobilin (PCB) chromophore, matched the lines in reconstructed STORM images. SIM and STORM reconstructions of Escherichia coli cells harbouring CpcA-labelled cytochrome bd ubiquinol oxidase in the cytoplasmic membrane show that CpcA-PEB and CpcA-PCB are suitable for super-resolution imaging in vivo. The stability, ease of production, small size and brightness of CpcA-PEB and CpcA-PCB demonstrate the potential of this largely unexplored protein family as novel probes for super-resolution microscopy.
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http://dx.doi.org/10.1038/s41598-017-16834-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5711914PMC
December 2017

The molecular basis of phosphite and hypophosphite recognition by ABC-transporters.

Nat Commun 2017 11 23;8(1):1746. Epub 2017 Nov 23.

Department of Molecular Biology and Biotechnology, University of Sheffield, Firth Court, Western Bank, Sheffield, S10 2TN, UK.

Inorganic phosphate is the major bioavailable form of the essential nutrient phosphorus. However, the concentration of phosphate in most natural habitats is low enough to limit microbial growth. Under phosphate-depleted conditions some bacteria utilise phosphite and hypophosphite as alternative sources of phosphorus, but the molecular basis of reduced phosphorus acquisition from the environment is not fully understood. Here, we present crystal structures and ligand binding affinities of periplasmic binding proteins from bacterial phosphite and hypophosphite ATP-binding cassette transporters. We reveal that phosphite and hypophosphite specificity results from a combination of steric selection and the presence of a P-H…π interaction between the ligand and a conserved aromatic residue in the ligand-binding pocket. The characterisation of high affinity and specific transporters has implications for the marine phosphorus redox cycle, and might aid the use of phosphite as an alternative phosphorus source in biotechnological, industrial and agricultural applications.
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http://dx.doi.org/10.1038/s41467-017-01226-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5700983PMC
November 2017

The catalytic power of magnesium chelatase: a benchmark for the AAA(+) ATPases.

FEBS Lett 2016 06 2;590(12):1687-93. Epub 2016 Jun 2.

Department of Chemistry, University of Sheffield, UK.

In the first committed reaction of chlorophyll biosynthesis, magnesium chelatase couples ATP hydrolysis to the thermodynamically unfavorable Mg(2+) insertion into protoporphyrin IX (ΔG°' of circa 25-33 kJ·mol(-1) ). We explored the thermodynamic constraints on magnesium chelatase and demonstrate the effect of nucleotide hydrolysis on both the reaction kinetics and thermodynamics. The enzyme produces a significant rate enhancement (kcat /kuncat of 400 × 10(6) m) and a catalytic rate enhancement, kcat/KmDIXK0.5Mgkuncat, of 30 × 10(15) m(-1) , increasing to 300 × 10(15) m(-1) with the activator protein Gun4. This is the first demonstration of the thermodynamic benefit of ATP hydrolysis in the AAA(+) family.
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http://dx.doi.org/10.1002/1873-3468.12214DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4982103PMC
June 2016

Nanomechanical and Thermophoretic Analyses of the Nucleotide-Dependent Interactions between the AAA(+) Subunits of Magnesium Chelatase.

J Am Chem Soc 2016 05 12;138(20):6591-7. Epub 2016 May 12.

Department of Molecular Biology and Biotechnology, The University of Sheffield , Sheffield S10 2TN, United Kingdom.

In chlorophyll biosynthesis, the magnesium chelatase enzyme complex catalyzes the insertion of a Mg(2+) ion into protoporphyrin IX. Prior to this event, two of the three subunits, the AAA(+) proteins ChlI and ChlD, form a ChlID-MgATP complex. We used microscale thermophoresis to directly determine dissociation constants for the I-D subunits from Synechocystis, and to show that the formation of a ChlID-MgADP complex, mediated by the arginine finger and the sensor II domain on ChlD, is necessary for the assembly of the catalytically active ChlHID-MgATP complex. The N-terminal AAA(+) domain of ChlD is essential for complex formation, but some stability is preserved in the absence of the C-terminal integrin domain of ChlD, particularly if the intervening polyproline linker region is retained. Single molecule force spectroscopy (SMFS) was used to determine the factors that stabilize formation of the ChlID-MgADP complex at the single molecule level; ChlD was attached to an atomic force microscope (AFM) probe in two different orientations, and the ChlI subunits were tethered to a silica surface; the probability of subunits interacting more than doubled in the presence of MgADP, and we show that the N-terminal AAA(+) domain of ChlD mediates this process, in agreement with the microscale thermophoresis data. Analysis of the unbinding data revealed a most probable interaction force of around 109 pN for formation of single ChlID-MgADP complexes. These experiments provide a quantitative basis for understanding the assembly and function of the Mg chelatase complex.
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http://dx.doi.org/10.1021/jacs.6b02827DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4882731PMC
May 2016

Five glutamic acid residues in the C-terminal domain of the ChlD subunit play a major role in conferring Mg(2+) cooperativity upon magnesium chelatase.

Biochemistry 2015 Nov 2;54(44):6659-62. Epub 2015 Nov 2.

Department of Chemistry, The University of Sheffield , Sheffield S3 7HF, U.K.

Magnesium chelatase catalyzes the first committed step in chlorophyll biosynthesis by inserting a Mg(2+) ion into protoporphyrin IX in an ATP-dependent manner. The cyanobacterial (Synechocystis) and higher-plant chelatases exhibit a complex cooperative response to free magnesium, while the chelatases from Thermosynechococcus elongatus and photosynthetic bacteria do not. To investigate the basis for this cooperativity, we constructed a series of chimeric ChlD proteins using N-terminal, central, and C-terminal domains from Synechocystis and Thermosynechococcus. We show that five glutamic acid residues in the C-terminal domain play a major role in this process.
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http://dx.doi.org/10.1021/acs.biochem.5b01080DOI Listing
November 2015

Porphyrin Binding to Gun4 Protein, Facilitated by a Flexible Loop, Controls Metabolite Flow through the Chlorophyll Biosynthetic Pathway.

J Biol Chem 2015 Nov 7;290(47):28477-28488. Epub 2015 Oct 7.

Institute of Microbiology, Academy of Sciences, 37981 Třeboň, Czech Republic; Faculty of Science, University of South Bohemia, 370 05 České Budějovice, Czech Republic. Electronic address:

In oxygenic phototrophs, chlorophylls, hemes, and bilins are synthesized by a common branched pathway. Given the phototoxic nature of tetrapyrroles, this pathway must be tightly regulated, and an important regulatory role is attributed to magnesium chelatase enzyme at the branching between the heme and chlorophyll pathway. Gun4 is a porphyrin-binding protein known to stimulate in vitro the magnesium chelatase activity, but how the Gun4-porphyrin complex acts in the cell was unknown. To address this issue, we first performed simulations to determine the porphyrin-docking mechanism to the cyanobacterial Gun4 structure. After correcting crystallographic loop contacts, we determined the binding site for magnesium protoporphyrin IX. Molecular modeling revealed that the orientation of α6/α7 loop is critical for the binding, and the magnesium ion held within the porphyrin is coordinated by Asn-211 residue. We also identified the basis for stronger binding in the Gun4-1 variant and for weaker binding in the W192A mutant. The W192A-Gun4 was further characterized in magnesium chelatase assay showing that tight porphyrin binding in Gun4 facilitates its interaction with the magnesium chelatase ChlH subunit. Finally, we introduced the W192A mutation into cells and show that the Gun4-porphyrin complex is important for the accumulation of ChlH and for channeling metabolites into the chlorophyll biosynthetic pathway.
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http://dx.doi.org/10.1074/jbc.M115.664987DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4653704PMC
November 2015

PucC and LhaA direct efficient assembly of the light-harvesting complexes in Rhodobacter sphaeroides.

Mol Microbiol 2016 Jan 5;99(2):307-27. Epub 2015 Nov 5.

Department of Molecular Biology and Biotechnology, University of Sheffield, Firth Court, Western Bank, Sheffield, S10 2TN, UK.

The mature architecture of the photosynthetic membrane of the purple phototroph Rhodobacter sphaeroides has been characterised to a level where an atomic-level membrane model is available, but the roles of the putative assembly proteins LhaA and PucC in establishing this architecture are unknown. Here we investigate the assembly of light-harvesting LH2 and reaction centre-light-harvesting1-PufX (RC-LH1-PufX) photosystem complexes using spectroscopy, pull-downs, native gel electrophoresis, quantitative mass spectrometry and fluorescence lifetime microscopy to characterise a series of lhaA and pucC mutants. LhaA and PucC are important for specific assembly of LH1 or LH2 complexes, respectively, but they are not essential; the few LH1 subunits found in ΔlhaA mutants assemble to form normal RC-LH1-PufX core complexes showing that, once initiated, LH1 assembly round the RC is cooperative and proceeds to completion. LhaA and PucC form oligomers at sites of initiation of membrane invagination; LhaA associates with RCs, bacteriochlorophyll synthase (BchG), the protein translocase subunit YajC and the YidC membrane protein insertase. These associations within membrane nanodomains likely maximise interactions between pigments newly arriving from BchG and nascent proteins within the SecYEG-SecDF-YajC-YidC assembly machinery, thereby co-ordinating pigment delivery, the co-translational insertion of LH polypeptides and their folding and assembly to form photosynthetic complexes.
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http://dx.doi.org/10.1111/mmi.13235DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4949548PMC
January 2016

Structural and functional consequences of removing the N-terminal domain from the magnesium chelatase ChlH subunit of Thermosynechococcus elongatus.

Biochem J 2014 Dec;464(3):315-22

*Department of Molecular Biology and Biotechnology, University of Sheffield, Sheffield S10 2TN, U.K.

Magnesium chelatase (MgCH) initiates chlorophyll biosynthesis by catalysing the ATP-dependent insertion of Mg2+ into protoporphyrin. This large enzyme complex comprises ChlH, I and D subunits, with I and D involved in ATP hydrolysis, and H the protein that handles the substrate and product. The 148 kDa ChlH subunit has a globular N-terminal domain attached by a narrow linker to a hollow cage-like structure. Following deletion of this ~18 kDa domain from the Thermosynechoccus elongatus ChlH, we used single particle reconstruction to show that the apo- and porphyrin-bound forms of the mutant subunit consist of a hollow globular protein with three connected lobes; superposition of the mutant and native ChlH structures shows that, despite the clear absence of the N-terminal 'head' region, the rest of the protein appears to be correctly folded. Analyses of dissociation constants shows that the ΔN159ChlH mutant retains the ability to bind protoporphyrin and the Gun4 enhancer protein, although the addition of I and D subunits yields an extremely impaired active enzyme complex. Addition of the Gun4 enhancer protein, which stimulates MgCH activity significantly especially at low Mg2+ concentrations, partially reactivates the ΔN159ChlH-I-D mutant enzyme complex, suggesting that the binding site or sites for Gun4 on H do not wholly depend on the N-terminal domain.
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http://dx.doi.org/10.1042/BJ20140463DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4255732PMC
December 2014

Integration of energy and electron transfer processes in the photosynthetic membrane of Rhodobacter sphaeroides.

Biochim Biophys Acta 2014 Oct 13;1837(10):1769-80. Epub 2014 Feb 13.

Department of Molecular Biology and Biotechnology, University of Sheffield, Sheffield S10 2TN, UK. Electronic address:

Photosynthesis converts absorbed solar energy to a protonmotive force, which drives ATP synthesis. The membrane network of chlorophyll-protein complexes responsible for light absorption, photochemistry and quinol (QH2) production has been mapped in the purple phototrophic bacterium Rhodobacter (Rba.) sphaeroides using atomic force microscopy (AFM), but the membrane location of the cytochrome bc1 (cytbc1) complexes that oxidise QH2 to quinone (Q) to generate a protonmotive force is unknown. We labelled cytbc1 complexes with gold nanobeads, each attached by a Histidine10 (His10)-tag to the C-terminus of cytc1. Electron microscopy (EM) of negatively stained chromatophore vesicles showed that the majority of the cytbc1 complexes occur as dimers in the membrane. The cytbc1 complexes appeared to be adjacent to reaction centre light-harvesting 1-PufX (RC-LH1-PufX) complexes, consistent with AFM topographs of a gold-labelled membrane. His-tagged cytbc1 complexes were retrieved from chromatophores partially solubilised by detergent; RC-LH1-PufX complexes tended to co-purify with cytbc1 whereas LH2 complexes became detached, consistent with clusters of cytbc1 complexes close to RC-LH1-PufX arrays, but not with a fixed, stoichiometric cytbc1-RC-LH1-PufX supercomplex. This information was combined with a quantitative mass spectrometry (MS) analysis of the RC, cytbc1, ATP synthase, cytaa3 and cytcbb3 membrane protein complexes, to construct an atomic-level model of a chromatophore vesicle comprising 67 LH2 complexes, 11 LH1-RC-PufX dimers & 2 RC-LH1-PufX monomers, 4 cytbc1 dimers and 2 ATP synthases. Simulation of the interconnected energy, electron and proton transfer processes showed a half-maximal ATP turnover rate for a light intensity equivalent to only 1% of bright sunlight. Thus, the photosystem architecture of the chromatophore is optimised for growth at low light intensities.
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http://dx.doi.org/10.1016/j.bbabio.2014.02.003DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4143486PMC
October 2014

Characterization of the magnesium chelatase from Thermosynechococcus elongatus.

Biochem J 2014 Jan;457(1):163-70

*Department of Molecular Biology and Biotechnology, The University of Sheffield, Sheffield S10 2TN, U.K.

The first committed step in chlorophyll biosynthesis is catalysed by magnesium chelatase (E.C. 6.6.1.1), which uses the free energy of ATP hydrolysis to insert an Mg(2+) ion into the ring of protoporphyrin IX. We have characterized magnesium chelatase from the thermophilic cyanobacterium Thermosynechococcus elongatus. This chelatase is thermostable, with subunit melting temperatures between 55 and 63°C and optimal activity at 50°C. The T. elongatus chelatase (kcat of 0.16 μM/min) shows a Michaelis-Menten-type response to both Mg(2+) (Km of 2.3 mM) and MgATP(2-) (Km of 0.8 mM). The response to porphyrin is more complex; porphyrin inhibits at high concentrations of ChlH, but when the concentration of ChlH is comparable with the other two subunits the response is of a Michaelis-Menten type (at 0.4 μM ChlH, Km is 0.2 μM). Hybrid magnesium chelatases containing a mixture of subunits from the mesophilic Synechocystis and Thermosynechococcus enzymes are active. We generated all six possible hybrid magnesium chelatases; the hybrid chelatase containing Thermosynechococcus ChlD and Synechocystis ChlI and ChlH is not co-operative towards Mg(2+), in contrast with the Synechocystis magnesium chelatase. This loss of co-operativity reveals the significant regulatory role of Synechocystis ChlD.
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http://dx.doi.org/10.1042/BJ20130834DOI Listing
January 2014

Three-dimensional structure of the Rhodobacter sphaeroides RC-LH1-PufX complex: dimerization and quinone channels promoted by PufX.

Biochemistry 2013 Oct 16;52(43):7575-85. Epub 2013 Oct 16.

Department of Molecular Biology and Biotechnology, University of Sheffield , Western Bank, Firth Court, Sheffield S10 2TN, United Kingdom.

Reaction center-light harvesting 1 (RC-LH1) complexes are the fundamental units of bacterial photosynthesis, which use solar energy to power the reduction of quinone to quinol prior to the formation of the proton gradient that drives ATP synthesis. The dimeric RC-LH1-PufX complex of Rhodobacter sphaeroides is composed of 64 polypeptides and 128 cofactors, including 56 LH1 bacteriochlorophyll a (BChl a) molecules that surround and donate energy to the two RCs. The 3D structure was determined to 8 Å by X-ray crystallography, and a model was built with constraints provided by electron microscopy (EM), nuclear magnetic resonance (NMR), mass spectrometry (MS), and site-directed mutagenesis. Each half of the dimer complex consists of a RC surrounded by an array of 14 LH1 αβ subunits, with two BChls sandwiched between each αβ pair of transmembrane helices. The N- and C-terminal extrinsic domains of PufX promote dimerization by interacting with the corresponding domains of an LH1 β polypeptide from the other half of the RC-LH1-PufX complex. Close contacts between PufX, an LH1 αβ subunit, and the cytoplasmic domain of the RC-H subunit prevent the LH1 complex from encircling the RC and create a channel connecting the RC QB site to an opening in the LH1 ring, allowing Q/QH₂ exchange with the external quinone pool. We also identified a channel that connects the two halves of the dimer, potentially forming a long-range pathway for quinone migration along rows of RC-LH1-PufX complexes in the membrane. The structure of the RC-LH1-PufX complex explains the crucial role played by PufX in dimer formation, and it shows how quinone traffic traverses the LH1 complex as it shuttles between the RC and the cytochrome bc₁ complex.
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http://dx.doi.org/10.1021/bi4011946DOI Listing
October 2013

Nano-mechanical mapping of the interactions between surface-bound RC-LH1-PufX core complexes and cytochrome c 2 attached to an AFM probe.

Photosynth Res 2014 May 29;120(1-2):169-80. Epub 2013 Mar 29.

Department of Molecular Biology and Biotechnology, University of Sheffield, Sheffield, S10 2TN, UK,

Electron transfer pathways in photosynthesis involve interactions between membrane-bound complexes such as reaction centres with an extrinsic partner. In this study, the biological specificity of electron transfer between the reaction centre-light-harvesting 1-PufX complex and its extrinsic electron donor, cytochrome c 2, formed the basis for mapping the location of surface-attached RC-LH1-PufX complexes using atomic force microscopy (AFM). This nano-mechanical mapping method used an AFM probe functionalised with cyt c 2 molecules to quantify the interaction forces involved, at the single-molecule level under native conditions. With surface-bound RC-His12-LH1-PufX complexes in the photo-oxidised state, the mean interaction force with cyt c 2 is approximately 480 pN with an interaction frequency of around 66 %. The latter value lowered 5.5-fold when chemically reduced RC-His12-LH1-PufX complexes are imaged in the dark to abolish electron transfer from cyt c 2 to the RC. The correspondence between topographic and adhesion images recorded over the same area of the sample shows that affinity-based AFM methods are a useful tool when topology alone is insufficient for spatially locating proteins at the surface of photosynthetic membranes.
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http://dx.doi.org/10.1007/s11120-013-9812-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4104003PMC
May 2014

Structure of the cyanobacterial Magnesium Chelatase H subunit determined by single particle reconstruction and small-angle X-ray scattering.

J Biol Chem 2012 Feb 15;287(7):4946-56. Epub 2011 Dec 15.

Department of Molecular Biology and Biotechnology, University of Sheffield, Sheffield S10 2TN, United Kingdom.

The biosynthesis of chlorophyll, an essential cofactor for photosynthesis, requires the ATP-dependent insertion of Mg(2+) into protoporphyrin IX catalyzed by the multisubunit enzyme magnesium chelatase. This enzyme complex consists of the I subunit, an ATPase that forms a complex with the D subunit, and an H subunit that binds both the protoporphyrin substrate and the magnesium protoporphyrin product. In this study we used electron microscopy and small-angle x-ray scattering to investigate the structure of the magnesium chelatase H subunit, ChlH, from the thermophilic cyanobacterium Thermosynechococcus elongatus. Single particle reconstruction of negatively stained apo-ChlH and Chl-porphyrin proteins was used to reconstitute three-dimensional structures to a resolution of ∼30 Å. ChlH is a large, 148-kDa protein of 1326 residues, forming a cage-like assembly comprising the majority of the structure, attached to a globular N-terminal domain of ∼16 kDa by a narrow linker region. This N-terminal domain is adjacent to a 5 nm-diameter opening in the structure that allows access to a cavity. Small-angle x-ray scattering analysis of ChlH, performed on soluble, catalytically active ChlH, verifies the presence of two domains and their relative sizes. Our results provide a basis for the multiple regulatory and catalytic functions of ChlH of oxygenic photosynthetic organisms and for a chaperoning function that sequesters the enzyme-bound magnesium protoporphyrin product prior to its delivery to the next enzyme in the chlorophyll biosynthetic pathway, magnesium protoporphyrin methyltransferase.
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http://dx.doi.org/10.1074/jbc.M111.308239DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3281664PMC
February 2012

Evolution in a family of chelatases facilitated by the introduction of active site asymmetry and protein oligomerization.

Proc Natl Acad Sci U S A 2011 Jan 20;108(1):97-102. Epub 2010 Dec 20.

Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa, Avenida da República (EAN), 2780-157 Oeiras, Portugal.

The class II chelatases associated with heme, siroheme, and cobalamin biosynthesis are structurally related enzymes that insert a specific metal ion (Fe(2+) or Co(2+)) into the center of a modified tetrapyrrole (protoporphyrin or sirohydrochlorin). The structures of two related class II enzymes, CbiX(S) from Archaeoglobus fulgidus and CbiK from Salmonella enterica, that are responsible for the insertion of cobalt along the cobalamin biosynthesis pathway are presented in complex with their metallated product. A further structure of a CbiK from Desulfovibrio vulgaris Hildenborough reveals how cobalt is bound at the active site. The crystal structures show that the binding of sirohydrochlorin is distinctly different to porphyrin binding in the protoporphyrin ferrochelatases and provide a molecular overview of the mechanism of chelation. The structures also give insights into the evolution of chelatase form and function. Finally, the structure of a periplasmic form of Desulfovibrio vulgaris Hildenborough CbiK reveals a novel tetrameric arrangement of its subunits that are stabilized by the presence of a heme b cofactor. Whereas retaining colbaltochelatase activity, this protein has acquired a central cavity with the potential to chaperone or transport metals across the periplasmic space, thereby evolving a new use for an ancient protein subunit.
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http://dx.doi.org/10.1073/pnas.1014298108DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3017170PMC
January 2011

NirJ, a radical SAM family member of the d1 heme biogenesis cluster.

FEBS Lett 2010 Jun 24;584(11):2461-6. Epub 2010 Apr 24.

Department of Biosciences, University of Kent, Canterbury, Kent, UK.

NirJ is involved in the transformation of precorrin-2 into heme d(1), although its precise role in the process has not been established. The purified protein was found to contain a 4Fe-4S centre, in line with the prediction that it belongs to the radical SAM class of enzymes. This was further confirmed by binding of S-adenosyl-L-methionine (SAM) to dithionite-reduced NirJ, which resulted in a decrease in the signal intensity and in a shift to higher field of the [4Fe-4S](1+) EPR signal. Significantly, though, this approach also led to the appearance of a small but reproducible organic radical signal that was associated with about 2% of the NirJ molecules and was affected by the incorporation of SAM deuterated at the 5' adenosyl group.
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http://dx.doi.org/10.1016/j.febslet.2010.04.053DOI Listing
June 2010

d(1) haem biogenesis - assessing the roles of three nir gene products.

FEBS J 2009 Nov 1;276(21):6399-411. Epub 2009 Oct 1.

Department of Biochemistry, University of Oxford, UK.

The synthesis of the modified tetrapyrrole known as d(1) haem requires several dedicated proteins which are coded for by a set of genes that are often found adjacent to the structural gene, nirS, for cytochrome cd(1) nitrite reductase. NirE, the product of the first gene in the nir biogenesis operon, was anticipated to catalyse the conversion of uroporphyrinogen III into precorrin-2; this was confirmed, but it was shown that this enzyme is less sensitive to product inhibition than similar enzymes that function in other biosynthetic pathways. Sequence analysis suggesting that one of these proteins, NirN, is a c-type cytochrome, and has similarity to the part of cytochrome cd(1) that binds d(1), was validated by recombinant production and characterization of NirN. A NirN-d(1) haem complex was demonstrated to release the cofactor to a semi-apo form of cytochrome cd(1) from which d(1) was extracted, suggesting a role for NirN in the assembly of cytochrome cd(1) (NirS). However, inactivation of nirN surprisingly led to only a marginal attenuation of growth of Paracoccus pantotrophus under anaerobic denitrifying conditions. As predicted, NirC is a c-type cytochrome; it was shown in vitro to be an electron donor to the NirN-d(1) complex.
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http://dx.doi.org/10.1111/j.1742-4658.2009.07354.xDOI Listing
November 2009

Structure and function of SirC from Bacillus megaterium: a metal-binding precorrin-2 dehydrogenase.

Biochem J 2008 Oct;415(2):257-63

Department of Biochemistry, University of Utah, Salt Lake City, UT 84112, USA.

In Bacillus megaterium, the synthesis of vitamin B(12) (cobalamin) and sirohaem diverges at sirohydrochlorin along the branched modified tetrapyrrole biosynthetic pathway. This key intermediate is made by the action of SirC, a precorrin-2 dehydrogenase that requires NAD(+) as a cofactor. The structure of SirC has now been solved by X-ray crystallography to 2.8 A (1 A = 0.1 nm) resolution. The protein is shown to consist of three domains and has a similar topology to the multifunctional sirohaem synthases Met8p and the N-terminal region of CysG, both of which catalyse not only the dehydrogenation of precorrin-2 but also the ferrochelation of sirohydrochlorin to give sirohaem. Guided by the structure, in the present study a number of active-site residues within SirC were investigated by site-directed mutagenesis. No active-site general base was identified, although surprisingly some of the resulting protein variants were found to have significantly enhanced catalytic activity. Unexpectedly, SirC was found to bind metal ions such as cobalt and copper, and to bind them in an identical fashion with that observed in Met8p. It is suggested that SirC may have evolved from a Met8p-like protein by loss of its chelatase activity. It is proposed that the ability of SirC to act as a single monofunctional enzyme, in conjunction with an independent chelatase, may provide greater control over the intermediate at this branchpoint in the synthesis of sirohaem and cobalamin.
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http://dx.doi.org/10.1042/BJ20080785DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2857972PMC
October 2008

Two distinct roles for two functional cobaltochelatases (CbiK) in Desulfovibrio vulgaris hildenborough.

Biochemistry 2008 May 6;47(21):5851-7. Epub 2008 May 6.

Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa, Avenida da Republica (EAN), 2780-157 Oeiras, Portugal.

The sulfate-reducing bacterium Desulfovibrio vulgaris Hildenborough possesses a large number of porphyrin-containing proteins whose biosynthesis is poorly characterized. In this work, we have studied two putative CbiK cobaltochelatases present in the genome of D. vulgaris. The assays revealed that both enzymes insert cobalt and iron into sirohydrochlorin, with specific activities with iron lower than that measured with cobalt. Nevertheless, the two D. vulgaris chelatases complement an E. coli cysG mutant strain showing that, in vivo, they are able to load iron into sirohydrochlorin. The results showed that the functional cobaltochelatases have distinct roles with one, CbiK(C), likely to be the enzyme associated with cytoplasmic cobalamin biosynthesis, while the other, CbiK(P), is periplasmic located and possibly associated with an iron transport system. Finally, the ability of D. vulgaris to produce vitamin B 12 was also demonstrated in this work.
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http://dx.doi.org/10.1021/bi800342cDOI Listing
May 2008

Enzyme sequence and its relationship to hyperbaric stability of artificial and natural fish lactate dehydrogenases.

PLoS One 2008 Apr 30;3(4):e2042. Epub 2008 Apr 30.

Department of Biosciences, University of Kent, Canterbury, Kent, United Kingdom.

The cDNAs of lactate dehydrogenase b (LDH-b) from both deep-sea and shallow living fish species, Corphaenoides armatus and Gadus morhua respectively, have been isolated, sequenced and their encoded products overproduced as recombinant enzymes in E. coli. The proteins were characterised in terms of their kinetic and physical properties and their ability to withstand high pressures. Although the two proteins are very similar in terms of their primary structure, only 21 differences at the amino acid level exist between them, the enzyme from the deep-sea species has a significantly increased tolerance to pressure and a higher thermostability. It was possible to investigate whether the changes in the N-terminal or C-terminal regions played a greater role in barophilic adaptation by the construction of two chimeric enzymes by use of a common restriction site within the cDNAs. One of these hybrids was found to have even greater pressure stability than the recombinant enzyme from the deep-living fish species. It was possible to conclude that the major adaptive changes to pressure tolerance must be located in the N-terminal region of the protein. The types of changes that are found and their spatial location within the protein structure are discussed. An analysis of the kinetic parameters of the enzymes suggests that there is clearly a trade off between K(m) and k(cat) values, which likely reflects the necessity of the deep-sea enzyme to operate at low temperatures.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0002042PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2323112PMC
April 2008

Biochemical and structural insights into bacterial organelle form and biogenesis.

J Biol Chem 2008 May 10;283(21):14366-75. Epub 2008 Mar 10.

Protein Science Group, Department of Biochemistry, University of Kent, Canterbury, Kent, UK.

Many heterotrophic bacteria have the ability to make polyhedral structures containing metabolic enzymes that are bounded by a unilamellar protein shell (metabolosomes or enterosomes). These bacterial organelles contain enzymes associated with a specific metabolic process (e.g. 1,2-propanediol or ethanolamine utilization). We show that the 21 gene regulon specifying the pdu organelle and propanediol utilization enzymes from Citrobacter freundii is fully functional when cloned in Escherichia coli, both producing metabolosomes and allowing propanediol utilization. Genetic manipulation of the level of specific shell proteins resulted in the formation of aberrantly shaped metabolosomes, providing evidence for their involvement as delimiting entities in the organelle. This is the first demonstration of complete recombinant metabolosome activity transferred in a single step and supports phylogenetic evidence that the pdu genes are readily horizontally transmissible. One of the predicted shell proteins (PduT) was found to have a novel Fe-S center formed between four protein subunits. The recombinant model will facilitate future experiments establishing the structure and assembly of these multiprotein assemblages and their fate when the specific metabolic function is no longer required.
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http://dx.doi.org/10.1074/jbc.M709214200DOI Listing
May 2008

Elucidation of substrate specificity in the cobalamin (vitamin B12) biosynthetic methyltransferases. Structure and function of the C20 methyltransferase (CbiL) from Methanothermobacter thermautotrophicus.

J Biol Chem 2007 Aug 13;282(33):23957-69. Epub 2007 Jun 13.

Protein Science Group, Department of Biosciences, University of Kent, Canterbury, Kent, CT2 7NJ, United Kingdom.

Ring contraction during cobalamin (vitamin B12) biosynthesis requires a seemingly futile methylation of the C20 position of the tetrapyrrole framework. Along the anaerobic route, this reaction is catalyzed by CbiL, which transfers a methyl group from S-adenosyl-L-methionine to cobalt factor II to generate cobalt factor III. CbiL belongs to the class III methyltransferases and displays similarity to other cobalamin biosynthetic methyltransferases that are responsible for the regiospecific methylation of a number of positions on the tetrapyrrole molecular canvas. In an attempt to understand how CbiL selectively methylates the C20 position, a detailed structure function analysis of the enzyme has been undertaken. In this paper, we demonstrate that the enzyme methylates the C20 position, that its preferred substrate is cobalt factor II, and that the metal ion does not undergo any oxidation change during the course of the reaction. The enzyme was crystallized, and its structure was determined by x-ray crystallography, revealing that the 26-kDa protein has a similar overall topology to other class III enzymes. This helped in the identification of some key amino acid residues (Asp(104), Lys(176), and Tyr(220)). Analysis of mutant variants of these groups has allowed us to suggest potential roles that these side chains may play in substrate binding and catalysis. EPR analysis of binary and ternary complexes indicate that the protein donates a fifth ligand to the cobalt ion via a gated mechanism to prevent transfer of the methyl group to water. The chemical logic underpinning the methylation is discussed.
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http://dx.doi.org/10.1074/jbc.M703827200DOI Listing
August 2007

Structure/function studies on a S-adenosyl-L-methionine-dependent uroporphyrinogen III C methyltransferase (SUMT), a key regulatory enzyme of tetrapyrrole biosynthesis.

J Mol Biol 2004 Nov;344(2):419-33

Structural Biology Laboratory, Department of Chemistry, University of York, Heslington, York YO10 5YW, UK.

The crystallographic structure of the Pseudomonas denitrificans S-adenosyl-L-methionine-dependent uroporphyrinogen III methyltransferase (SUMT), which is encoded by the cobA gene, has been solved by molecular replacement to 2.7A resolution. SUMT is a branchpoint enzyme that plays a key role in the biosynthesis of modified tetrapyrroles by controlling flux to compounds such as vitamin B(12) and sirohaem, and catalysing the transformation of uroporphyrinogen III into precorrin-2. The overall topology of the enzyme is similar to that of the SUMT module of sirohaem synthase (CysG) and the cobalt-precorrin-4 methyltransferase CbiF and, as with the latter structures, SUMT has the product S-adenosyl-L-homocysteine bound in the crystal. The roles of a number of residues within the SUMT structure are discussed with respect to their conservation either across the broader family of cobalamin biosynthetic methyltransferases or within the sub-group of SUMT members. The D47N, L49A, F106A, T130A, Y183A and M184A variants of SUMT were generated by mutagenesis of the cobA gene, and tested for SAM binding and enzymatic activity. Of these variants, only D47N and L49A bound the co-substrate S-adenosyl-L-methionine. Consequently, all the mutants were severely restricted in their capacity to synthesise precorrin-2, although both the D47N and L49A variants produced significant quantities of precorrin-1, the monomethylated derivative of uroporphyrinogen III. The activity of these variants is interpreted with respect to the structure of the enzyme.
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http://dx.doi.org/10.1016/j.jmb.2004.09.020DOI Listing
November 2004

Crystallization and preliminary X-ray diffraction studies of a fungal hydrolase from Ophiostoma novo-ulmi.

Acta Crystallogr D Biol Crystallogr 2004 Oct 23;60(Pt 10):1879-82. Epub 2004 Sep 23.

Henry Wellcome Building for Biocatalysis, School of Biological and Chemical Sciences, University of Exeter, Exeter EX4 4QD, England.

Dutch elm disease fungus Ophiostoma novo-ulmi contains a hydrolase activity which catalyses the resolution of racemic ethyl naproxen to the corresponding acid. The recombinant enzyme has been crystallized by the vapour-diffusion method in two crystal forms. The crystals of the first form belong to space group P2(1)2(1)2, with unit-cell parameters a = 115.9, b = 174.4, c = 62.1 A. The enzyme also crystallizes in space group P2(1)2(1)2, with unit-cell parameters a = 72.9, b = 212.7, c = 61.7 A. Synchrotron data have been collected for both crystal forms to 2.6 and 2.3 A, respectively. A molecular-replacement solution has been found using a remote starting model of a bacterial esterase (23% sequence identity) for both crystal forms. Multicrystal averaging has resulted in interpretable electron-density maps.
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http://dx.doi.org/10.1107/S0907444904018153DOI Listing
October 2004

X-ray structure of a putative reaction intermediate of 5-aminolaevulinic acid dehydratase.

Biochem J 2003 Aug;373(Pt 3):733-8

Division of Biochemistry and Molecular Biology, School of Biological Sciences, University of Southampton, Bassett Crescent East, Southampton, SO16 7PX, UK.

The X-ray structure of yeast 5-aminolaevulinic acid dehydratase, in which the catalytic site of the enzyme is complexed with a putative cyclic intermediate composed of both substrate moieties, has been solved at 0.16 nm (1.6 A) resolution. The cyclic intermediate is bound covalently to Lys(263) with the amino group of the aminomethyl side chain ligated to the active-site zinc ion in a position normally occupied by a catalytic hydroxide ion. The cyclic intermediate is catalytically competent, as shown by its turnover in the presence of added substrate to form porphobilinogen. The findings, combined with those of previous studies, are consistent with a catalytic mechanism in which the C-C bond linking both substrates in the intermediate is formed before the C-N bond.
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http://dx.doi.org/10.1042/BJ20030513DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1223560PMC
August 2003

A story of chelatase evolution: identification and characterization of a small 13-15-kDa "ancestral" cobaltochelatase (CbiXS) in the archaea.

J Biol Chem 2003 Jun 9;278(25):22388-95. Epub 2003 Apr 9.

School of Biological Sciences, Queen Mary, University of London, Mile End Road, London E1 4NS, United Kingdom.

The cobaltochelatase required for the synthesis of vitamin B12 (cobalamin) in the archaeal kingdom has been identified as CbiX through similarity searching with the CbiX from Bacillus megaterium. However, the CbiX proteins in the archaea are much shorter than the CbiX proteins found in eubacteria, typically containing less than half the number of amino acids in their primary structure. For this reason the shorter CbiX proteins have been termed CbiXS and the longer versions CbiXL. The CbiXS proteins from Methanosarcina barkeri and Methanobacter thermoautotrophicum were overproduced in Escherichia coli as recombinant proteins and characterized. Through complementation studies of a defined chelatase-deficient strain of E. coli and by direct in vitro assays the function of CbiXS as a sirohydrochlorin cobaltochelatase has been demonstrated. On the basis of sequence alignments and conserved active site residues we suggest that CbiXS may represent a primordial chelatase, giving rise to larger chelatases such as CbiXL, SirB, CbiK, and HemH through gene duplication and subsequent variation and selection. A classification scheme for chelatases is proposed.
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http://dx.doi.org/10.1074/jbc.M302468200DOI Listing
June 2003