Molybdoenzymes are involved in a large number of enzymatic reactions in the nitrogen, carbon and sulfur cycles. They occur in all the kingdoms of life. With the exception of nitrogenase, all molybdoenzymes carry the molybdenum cofactor (Moco), where the molybdenum atom is coordinated to the unique dithiolene moiety of a conserved tricyclic pyranopterin cofactor called molybdopterin (MPT). Depending on the remaining ligands of the molybdenum center, molybdoenzymes are classified into three families: (a) the xanthine oxidase (XO) family, characterized by a cyanolyzable equatorial sulfur ligand coordinated to the molybdenum atom; (b) the sulfite oxidase family, with two oxo ligands at the molybdenum center; and Three DNA regions carrying genes encoding putative homologs of xanthine dehydrogenases were identified in Escherichia coli, named xdhABC, xdhD, and yagTSRQ. Here, we describe the purification and characterization of gene products of the yagTSRQ operon, a molybdenum-containing ironsulfur flavoprotein from E. coli, which is located in the periplasm. The 135 kDa enzyme comprised a noncovalent (abc) heterotrimer with a large (78.1 kDa) molybdenum cofactor (Moco)-containing YagR subunit, a medium (33.9 kDa) FAD-containing YagS subunit, and a small (21.0 kDa) 2 · [2Fe2S]-containing YagT subunit. YagQ is not a subunit of the mature enzyme, and the protein is expected to be involved in Moco modification and insertion into YagTSR. Analysis of the form of Moco present in YagTSR revealed the presence of the molybdopterin cytosine dinucleotide cofactor. Two different [2Fe2S] clusters, typical for this class of enzyme, were identified by EPR. YagTSR represents the first example of a molybdopterin cytosine dinucleotide-containing protein in E. coli. Kinetic characterization of the enzyme revealed that YagTSR converts a broad spectrum of aldehydes, with a preference for aromatic aldehydes. Ferredoxin instead of NAD + or molecular oxygen was used as terminal electron acceptor. Complete growth inhibition of E. coli cells devoid of genes from the yagTSRQ operon was observed by the addition of cinnamaldehyde to a low-pH medium. This finding shows that YagTSR might have a role in the detoxification of aromatic aldehydes for E. coli under certain growth conditions. Abbreviations ICP-OES, inductively coupled plasma optical emission spectroscopy; MCD, molybdopterin cytosine dinucleotide; MGD, molybdopterin guanine dinucleotide; Moco, molybdenum cofactor; MPT, molybdopterin; Tat, twin arginine protein transport; XDH, xanthine dehydrogenase; XO, xanthine oxidase.

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Metal-containing formate dehydrogenases (FDH) catalyse the reversible oxidation of formate to carbon dioxide at their molybdenum or tungsten active site. They display a diverse subunit and cofactor composition, but structural information on these enzymes is limited. Here we report the cryo-electron microscopic structures of the soluble Rhodobacter capsulatus FDH (RcFDH) as isolated and in the presence of reduced nicotinamide adenine dinucleotide (NADH). RcFDH assembles into a 360 kDa dimer of heterotetramers revealing a putative interconnection of electron pathway chains. In the presence of NADH, the RcFDH structure shows charging of cofactors, indicative of an increased electron load.

Adenosine triphosphate phosphoribosyltransferase (ATP-PRT) catalyzes the first committed step of the histidine biosynthesis in plants and microorganisms. Here, we present the functional and structural characterization of the ATP-PRT from the pathogenic e-proteobacteria Campylobacter jejuni (CjeATP-PRT). This enzyme is a member of the long form (HisG L ) ATP-PRT and is allosterically inhibited by histidine, which binds to a remote regulatory domain, and competitively inhibited by AMP. In the crystalline form, CjeATP-PRT was found to adopt two distinctly different hexameric conformations, with an open homohexameric structure observed in the presence of substrate ATP, and a more compact closed form present when inhibitor histidine is bound. CjeATP-PRT was observed to adopt only a hexameric quaternary structure in solution, contradicting previous hypotheses favoring an allosteric mechanism driven by an oligomer equilibrium.Abbreviations: ATP-PRT, adenosine triphosphate phosphoribosyltransferase; BTP, 1,3-bis[tris(hydroxymethyl)methylamino]propane; CjeATP-PRT, ATP-PRT from Campylobacter jejuni; EcoATP-PRT, Escherichia coli ATP-PRT; His, l-histidine; ITC, isothermal titration calorimetry; MtuATP-PRT, Mycobacterium tuberculosis ATP-PRT; PDB, Protein Data Bank; PR-ATP, phosphoribosyl ATP; PRPP, phosphoribosyl pyrophosphate; PRT, phosphoribosyltransferase; RMSD, root-mean-square difference; SenATP-PRT, Salmonella enterica subsp. enterica Typhimurium ATP-PRT Additional Supporting Information may be found in the online version of this article.Significance Statement: ATP-phosphoribosyltransferase catalyzes the first dedicated step in the biosynthesis of the essential amino acid histidine in microorganisms. We report the functional characterization of this enzyme from human pathogen Campylobacter jejuni. The enzyme is inhibited by histidine, allowing for tuned production of histidine in response to cellular demands. Our results reveal how the enzyme structure becomes compressed when histidine binds and exposes the molecular details of how this enzyme performs its function. Instead, this study supports the conclusion that the ATP-PRT long form hexamer is the active species; the tightening of this structure in response to remote histidine binding results in an inhibited enzyme.

We have purified and characterized a specific CTP: molybdopterin cytidylyltransferase for the biosynthesis of the molybdopterin (MPT) cytosine dinucleotide (MCD) cofactor in Escherichia coli. The protein, named MocA, shows 22% amino acid sequence identity to E. coli MobA, the specific GTP: molybdopterin guanylyltransferase for molybdopterin guanine dinucleotide biosynthesis. MocA is essential for the activity of the MCD-containing enzymes aldehyde oxidoreductase Yag-TSR and the xanthine dehydrogenases XdhABC and XdhD. Using a fully defined in vitro assay, we showed that MocA, Mo-MPT, CTP, and MgCl 2 are required and sufficient for MCD biosynthesis in vitro. The activity of MocA is specific for CTP; other nucleotides such as ATP and GTP were not utilized. In the defined in vitro system a turnover number of 0.37 ؎ 0.01 min ؊1 was obtained. A 1:1 binding ratio of MocA to Mo-MPT and CTP was determined to monomeric MocA with dissociation constants of 0.23 ؎ 0.02 M for CTP and 1.17 ؎ 0.18 M for Mo-MPT. We showed that MocA was also able to convert MPT to MCD in the absence of molybdate, however, with only one catalytic turnover. The addition of molybdate after one turnover gave rise to a higher MCD production, revealing that MCD remains bound to MocA in the absence of molybdate. This work presents the first characterization of a specific enzyme involved in MCD biosynthesis in bacteria.

Edited by Michael IbbaATP-phosphoribosyltransferase (ATP-PRT) catalyses the first step of histidine biosynthesis. Two different forms of ATP-PRT have been described; the homo-hexameric long form, and the hetero-octameric short form. Lactococcus lactis possesses the short form ATP-PRT comprising four subunits of HisG S , the catalytic subunit, and four subunits of HisZ, a histidyl-tRNA synthetase paralogue. Previous studies have suggested that HisG S requires HisZ for catalysis. Here, we reveal that the dimeric HisG S does display ATP-PRT activity in the absence of HisZ. This result reflects the evolutionary relationship between the long and short form ATP-PRT, which acquired allosteric inhibition and enhanced catalysis via two divergent strategies.

Adenosine triphosphate (ATP) phosphoribosyltransferase (ATP-PRT) catalyses the first committed step of histidine biosynthesis in plants and microorganisms. Two forms of ATP-PRT have been reported, which differ in their molecular architecture and mechanism of allosteric regulation. The short-form ATP-PRT is a hetero-octamer, with four HisG chains that comprise only the catalytic domains and four separate chains of HisZ required for allosteric regulation by histidine. The long-form ATP-PRT is homo-hexameric, with each chain comprising two catalytic domains and a covalently linked regulatory domain that binds histidine as an allosteric inhibitor. Here, we describe a truncated long-form ATP-PRT from devoid of its regulatory domain (ATP-PRT). Results showed that ATP-PRT is dimeric, exhibits attenuated catalytic activity, and is insensitive to histidine, indicating that the covalently linked regulatory domain plays a role in both catalysis and regulation. Crystal structures were obtained for ATP-PRT in complex with both substrates, and for the first time, the complete product of the reaction. These structures reveal the key features of the active site and provide insights into how substrates move into position during catalysis.

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