Functionally redundant formate dehydrogenases enable formate-dependent growth in Methanococcus maripaludis.

Methanogens are essential for the complete remineralization of organic matter in anoxic environments. Most cultured methanogens are hydrogenotrophic, using H2 as an electron donor to reduce CO2 to CH4, but in the absence of H2 many can also use formate. Formate dehydrogenase (Fdh) is essential for formate oxidation, where it transfers electrons for the reduction of coenzyme F420 or to a flavin-based electron bifurcating reaction catalyzed by heterodisulfide reductase (Hdr), the terminal reaction of methanogenesis. Furthermore, methanogens that use formate encode at least two isoforms of Fdh in their genomes, but how these different isoforms participate in methanogenesis is unknown. Using Methanococcus maripaludis, we undertook a biochemical characterization of both Fdh isoforms involved in methanogenesis. Both Fdh1 and Fdh2 interacted with Hdr to catalyze the flavin-based electron bifurcating reaction, and both reduced F420 at similar rates. F420 reduction preceded flavin-based electron bifurcation activity for both enzymes. In a Δfdh1 mutant background, a suppressor mutation was required for Fdh2 activity. Genome sequencing revealed that this mutation resulted in the loss of a specific molybdopterin transferase (moeA), allowing for Fdh2-dependent growth, and the metal content of the proteins suggested that isoforms are dependent on either molybdenum or tungsten for activity. These data suggest that both isoforms of Fdh are functionally redundant, but their activities in vivo may be limited by gene regulation or metal availability under different growth conditions. Together these results expand our understanding of formate oxidation and the role of Fdh in methanogenesis.

A heterodimeric hyaluronate lyase secreted by the activated sludge bacterium Haliscomenobacter hydrossis.

Haliscomenobacter hydrossis is a filamentous bacterium common in activated sludge. The bacterium was found to utilize hyaluronic acid, and hyaluronate lyase activity was detected in its culture. However, no hyaluronate lyase gene was found in the genome, suggesting the bacterium secretes a novel hyaluronate lyase. The purified enzyme exhibited two bands on SDS-PAGE and a single peak on gel filtration chromatography, suggesting a heterodimeric composition. N-terminal amino acid sequence and mass spectrometric analyses suggested that the subunits are molybdopterin-binding and [2Fe-2S]-binding subunits of a xanthine oxidase family protein. The presence of the cofactors was confirmed using spectrometric analysis. Oxidase activity was not detected, revealing that the enzyme is not an oxidase but a hyaluronate lyase. Nuclear magnetic resonance analysis of the enzymatic digest revealed that the enzyme breaks hyaluronic acid to 3-(4-deoxy-ß-d-gluc-4-enuronosyl)-N-acetyl-d-glucosamine. As hyaluronate lyases (EC 4.2.2.1) are monomeric or trimeric, the enzyme is the first heterodimeric hyaluronate lyase.

Making Moco: A Personal History.

This contribution describes the path of my nearly forty-year quest to understand the special ligand coordinated to molybdenum and tungsten ions in their respective enzymes. Through this quest, I aimed to discover why nature did not simply use a methyl group on the dithiolene that chelates Mo and W but instead chose a complicated pyranopterin. My journey sought answers through the synthesis of model Mo compounds that allowed systematic investigations of the interactions between molybdenum and pterin and molybdenum and pterin-dithiolene and revealed special features of the pyranopterin dithiolene chelate bound to molybdenum.

Advancing Our Understanding of Pyranopterin-Dithiolene Contributions to Moco Enzyme Catalysis.

The pyranopterin dithiolene ligand is remarkable in terms of its geometric and electronic structure and is uniquely found in mononuclear molybdenum and tungsten enzymes. The pyranopterin dithiolene is found coordinated to the metal ion, deeply buried within the protein, and non-covalently attached to the protein via an extensive hydrogen bonding network that is enzyme-specific. However, the function of pyranopterin dithiolene in enzymatic catalysis has been difficult to determine. This focused account aims to provide an overview of what has been learned from the study of pyranopterin dithiolene model complexes of molybdenum and how these results relate to the enzyme systems. This work begins with a summary of what is known about the pyranopterin dithiolene ligand in the enzymes. We then introduce the development of inorganic small molecule complexes that model aspects of a coordinated pyranopterin dithiolene and discuss the results of detailed physical studies of the models by electronic absorption, resonance Raman, X-ray absorption and NMR spectroscopies, cyclic voltammetry, X-ray crystallography, and chemical reactivity.

XDH and XO Research and Drug Discovery-Personal History.

The author will outline the research history of the main issues addressed in this paper. The author has worked on this research himself. XDH, which is responsible for purine degradation, is present in various organisms. However, conversion to XO only occurs in mammals. The molecular mechanism of this conversion was elucidated in this study. The physiological and pathological significance of this conversion is presented. Finally, enzyme inhibitors were successfully developed, two of which are used as therapeutic agents for gout. Their wide application potential is also discussed.

Bioelectrocatalytic CO2 Reduction by Mo-Dependent Formylmethanofuran Dehydrogenase.

Massive efforts are invested in developing innovative CO2 -sequestration strategies to counter climate change and transform CO2 into higher-value products. CO2 -capture by reduction is a chemical challenge, and attention is turned toward biological systems that selectively and efficiently catalyse this reaction under mild conditions and in aqueous solvents. While a few reports have evaluated the effectiveness of isolated bacterial formate dehydrogenases as catalysts for the reversible electrochemical reduction of CO2 , it is imperative to explore other enzymes among the natural reservoir of potential models that might exhibit higher turnover rates or preferential directionality for the reductive reaction. Here, we present electroenzymatic catalysis of formylmethanofuran dehydrogenase, a CO2 -reducing-and-fixing biomachinery isolated from a thermophilic methanogen, which was deposited on a graphite rod electrode to enable direct electron transfer for electroenzymatic CO2 reduction. The gas is reduced with a high Faradaic efficiency (109±1 %), where a low affinity for formate prevents its electrochemical reoxidation and favours formate accumulation. These properties make the enzyme an excellent tool for electroenzymatic CO2 -fixation and inspiration for protein engineering that would be beneficial for biotechnological purposes to convert the greenhouse gas into stable formate that can subsequently be safely stored, transported, and used for power generation without energy loss.

Tracking W-Formate Dehydrogenase Structural Changes During Catalysis and Enzyme Reoxidation.

Metal-dependent formate dehydrogenases (Fdh) catalyze the reversible conversion of CO2 to formate, with unrivalled efficiency and selectivity. However, the key catalytic aspects of these enzymes remain unknown, preventing us from fully benefiting from their capabilities in terms of biotechnological applications. Here, we report a time-resolved characterization by X-ray crystallography of the Desulfovibrio vulgaris Hildenborough SeCys/W-Fdh during formate oxidation. The results allowed us to model five different intermediate structures and to chronologically map the changes occurring during enzyme reduction. Formate molecules were assigned for the first time to populate the catalytic pocket of a Fdh. Finally, the redox reversibility of DvFdhAB in crystals was confirmed by reduction and reoxidation structural studies.

The History of the Molybdenum Cofactor-A Personal View.

The transition element molybdenum (Mo) is an essential micronutrient for plants, animals, and microorganisms, where it forms part of the active center of Mo enzymes. To gain biological activity in the cell, Mo has to be complexed by a pterin scaffold to form the molybdenum cofactor (Moco). Mo enzymes and Moco are found in all kingdoms of life, where they perform vital transformations in the metabolism of nitrogen, sulfur, and carbon compounds. In this review, I recall the history of Moco in a personal view, starting with the genetics of Moco in the 1960s and 1970s, followed by Moco biochemistry and the description of its chemical structure in the 1980s. When I review the elucidation of Moco biosynthesis in the 1990s and the early 2000s, I do it mainly for eukaryotes, as I worked with plants, human cells, and filamentous fungi. Finally, I briefly touch upon human Moco deficiency and whether there is life without Moco.

Synthesis, Redox and Spectroscopic Properties of Pterin of Molybdenum Cofactors.

Pterins are bicyclic heterocycles that are found widely across Nature and are involved in a variety of biological functions. Notably, pterins are found at the core of molybdenum cofactor (Moco) containing enzymes in the molybdopterin (MPT) ligand that coordinates molybdenum and facilitates cofactor activity. Pterins are diverse and can be widely functionalized to tune their properties. Herein, the general methods of synthesis, redox and spectroscopic properties of pterin are discussed to provide more insight into pterin chemistry and their importance to biological systems.

Molybdenum Cofactor Deficiency in Humans.

Molybdenum cofactor (Moco) deficiency (MoCD) is characterized by neonatal-onset myoclonic epileptic encephalopathy and dystonia with cerebral MRI changes similar to hypoxic-ischemic lesions. The molecular cause of the disease is the loss of sulfite oxidase (SOX) activity, one of four Moco-dependent enzymes in men. Accumulating toxic sulfite causes a secondary increase of metabolites such as S-sulfocysteine and thiosulfate as well as a decrease in cysteine and its oxidized form, cystine. Moco is synthesized by a three-step biosynthetic pathway that involves the gene products of MOCS1, MOCS2, MOCS3, and GPHN. Depending on which synthetic step is impaired, MoCD is classified as type A, B, or C. This distinction is relevant for patient management because the metabolic block in MoCD type A can be circumvented by administering cyclic pyranopterin monophosphate (cPMP). Substitution therapy with cPMP is highly effective in reducing sulfite toxicity and restoring biochemical homeostasis, while the clinical outcome critically depends on the degree of brain injury prior to the start of treatment. In the absence of a specific treatment for MoCD type B/C and SOX deficiency, we summarize recent progress in our understanding of the underlying metabolic changes in cysteine homeostasis and propose novel therapeutic interventions to circumvent those pathological changes.

Microbe-Mineral Interaction and Novel Proteins for Iron Oxide Mineral Reduction in the Hyperthermophilic Crenarchaeon Pyrodictium delaneyi.

Dissimilatory iron reduction by hyperthermophilic archaea occurs in many geothermal environments and generally relies on microbe-mineral interactions that transform various iron oxide minerals. In this study, the physiology of dissimilatory iron and nitrate reduction was examined in the hyperthermophilic crenarchaeon type strain Pyrodictium delaneyi Su06. Iron barrier experiments showed that P. delaneyi required direct contact with the Fe(III) oxide mineral ferrihydrite for reduction. The separate addition of an exogenous electron shuttle (anthraquinone-2,6-disulfonate), a metal chelator (nitrilotriacetic acid), and 75% spent cell-free supernatant did not stimulate growth with or without the barrier. Protein electrophoresis showed that the c-type cytochrome and general protein compositions of P. delaneyi changed when grown on ferrihydrite relative to nitrate. Differential proteomic analyses using tandem mass tagged protein fragments and mass spectrometry detected 660 proteins and differential production of 127 proteins. Among these, two putative membrane-bound molybdopterin-dependent oxidoreductase complexes increased in relative abundance 60- to 3,000-fold and 50- to 100-fold in cells grown on iron oxide. A putative 8-heme c-type cytochrome was 60-fold more abundant in iron-grown cells and was unique to the Pyrodictiaceae There was also a >14,700-fold increase in a membrane transport protein in iron-grown cells. For flagellin proteins and a putative nitrate reductase, there were no changes in abundance, but a membrane nitric oxide reductase was more abundant on nitrate. These data help to elucidate the mechanisms by which hyperthermophilic crenarchaea generate energy and transfer electrons across the membrane to iron oxide minerals.IMPORTANCE Understanding iron reduction in the hyperthermophilic crenarchaeon Pyrodictium delaneyi provides insight into the diversity of mechanisms used for this process and its potential impact in geothermal environments. The ability of P. delaneyi to reduce Fe(III) oxide minerals through direct contact potentially using a novel cytochrome respiratory complex and a membrane-bound molybdopterin respiratory complex sets iron reduction in this organism apart from previously described iron reduction processes. A model is presented where obligatory H2 oxidation on the membrane coupled with electron transport and either Fe(III) oxide or nitrate reduction leads to the generation of a proton motive force and energy generation by oxidative phosphorylation. However, P. delaneyi cannot fix CO2 and relies on organic compounds from its environment for biosynthesis.

Major aldehyde dehydrogenase AldFGH of Gluconacetobacter diazotrophicus is independent of pyrroloquinoline quinone but dependent on molybdopterin for acetic acid fermentation.

Acetic acid fermentation involves the oxidation of ethanol to acetic acid via acetaldehyde as the intermediate and is catalyzed by the membrane-bound alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH) of acetic acid bacteria. Although ADH depends on pyrroloquinoline quinone (PQQ), the prosthetic group associated with ALDH remains a matter of debate. This study aimed to address the dependency of ALDH of Gluconacetobacter diazotrophicus strain PAL5 on PQQ and the physiological role of ALDH in acetic acid fermentation. We constructed deletion mutant strains for both the ALDH gene clusters of PAL5, aldFGH and aldSLC. In addition, the adhAB operon for ADH was eliminated, since it shows ALDH activity. The triple-deletion derivative ΔaldFGH ΔaldSLC ΔadhAB failed to show ALDH activity, which suggested that ALDH activity in PAL5 is derived from these three enzyme complexes. Since the single-gene cluster deletion derivative ΔaldFGH lost most ALDH activity, and accumulated much higher acetaldehyde than wild type under acetic acid fermentation conditions, we concluded that AldFGH functions as the major ALDH in PAL5. Furthermore, deletion of the PQQ biosynthesis gene cluster (pqqABCDE) abolished ADH activity completely, but did not affect ALDH activity. Instead, the molybdopterin biosynthesis gene deletion derivatives lost ALDH activity. Thus, we concluded that the AldFGH and AldSLC complexes of Ga. diazotrophicus PAL5 require a form of molybdopterin but not PQQ for ALDH activity. KEY POINTS: • AldFGH is the major aldehyde dehydrogenase in Gluconacetobacter diazotrophicus PAL5. • Acetaldehyde accumulated from ethanol in the absence of AldFGH. • Molybdopterin, rather than pyrroloquinoline quinone, is required for AldFGH.

Model Substrate/Inactivation Reactions for MoaA and Ribonucleotide Reductases: Loss of Bromo, Chloro, or Tosylate Groups from C2 of 1,5-Dideoxyhomoribofuranoses upon Generation of an α-Oxy Radical at C3.

We report studies on radical-initiated fragmentations of model 1,5-dideoxyhomoribofuranose derivatives with bromo, chloro, and tosyloxy substituents on C2. The effects of stereochemical inversion at C2 were probed with the corresponding arabino epimers. In all cases, the elimination of bromide, chloride, and tosylate anions occurred when the 3-hydroxyl group was unprotected. The isolation of deuterium-labeled furanone products established heterolytic cleavage followed by the transfer of deuterium from labeled tributylstannane. In contrast, 3-O-methyl derivatives underwent the elimination of bromine or chlorine radicals to give the 2,3-alkene with no incorporation of label in the methyl vinyl ether. More drastic fragmentation occurred with both of the 3-O-methyl-2-tosyloxy epimers to give an aromatized furan derivative with no deuterium label. Contrasting results observed with the present anhydroalditol models relative to our prior studies with analogously substituted nucleoside models have demonstrated that insights from biomimetic chemical reactions can provide illumination of mechanistic pathways employed by ribonucleotide reductases (RNRs) and the MoaA enzyme involved in the biosynthesis of molybdopterin.

Structural analysis of molybdopterin synthases from two mycobacterial pathogens.

The molybdenum cofactor, composed of molybdopterin and molybdenum, is a necessary compound for the catalytic activity of molybdenum enzymes. Molybdenum cofactor biosynthesis is a conserved multi-step process involving several enzymes. Molybdopterin synthase, a hetero-tetrameric enzyme composed of a pair of MoaE-MoaD subunits, catalyzes the generation of the cis-dithiolene group of molybdopterin in the second step of the process. The cis-dithiolene group can covalently bind molybdenum. Most mycobacterial species possess several genes encoding the full pathway of molybdenum cofactor biosynthesis. In M. smegmatis, the moaD2 and moaE2 genes encode the functional molybdopterin synthase. However, M. tuberculosis has genes encoding several molybdopterin synthase subunit homologs, including moaD1, moaD2, moaE1, moaE2, and moaX, which encodes a MoaD-MoaE fusion protein. Previous studies have shown that moaD2 and moaE2 encode functional molybdopterin synthase. Here, we report the crystal structures of two substrate-free molybdopterin synthases from two different mycobacterial pathogens, M. tuberculosis and M. smegmatis, at 2.1 Šand 2.6 Šresolutions, respectively. The overall structure of both molybdopterin synthases was hetero-tetrameric, consisting of a MoaE2 dimer flanked on either side by single MoaD2 subunits. The carboxyl-terminal domain of MoaD2 inserted into MoaE2, forming the active pocket. A comparison with previously reported molybdopterin synthase structures showed that substrate-binding and catalytic residues were conserved, despite low sequence similarity among these enzymes. The low sequence identity at the MoaE-MoaD heterodimer interface may provide the structural basis to explore mycobacterial inhibitors.

Prokaryotic ubiquitin-like protein modification.

Prokaryotes form ubiquitin (Ub)-like isopeptide bonds on the lysine residues of proteins by at least two distinct pathways that are reversible and regulated. In mycobacteria, the C-terminal Gln of Pup (prokaryotic ubiquitin-like protein) is deamidated and isopeptide linked to proteins by a mechanism distinct from ubiquitylation in enzymology yet analogous to ubiquitylation in targeting proteins for destruction by proteasomes. Ub-fold proteins of archaea (SAMPs, small archaeal modifier proteins) and Thermus (TtuB, tRNA-two-thiouridine B) that differ from Ub in amino acid sequence, yet share a common ß-grasp fold, also form isopeptide bonds by a mechanism that appears streamlined compared with ubiquitylation. SAMPs and TtuB are found to be members of a small group of Ub-fold proteins that function not only in protein modification but also in sulfur-transfer pathways associated with tRNA thiolation and molybdopterin biosynthesis. These multifunctional Ub-fold proteins are thought to be some of the most ancient of Ub-like protein modifiers.

Deconvolution of reduction potentials of formate dehydrogenase from Cupriavidus necator.

The formate dehydrogenase enzyme from Cupriavidus necator (FdsABG) carries out the two-electron oxidation of formate to CO2, but is also capable of reducing CO2 back to formate, a potential biofuel. FdsABG is a heterotrimeric enzyme that performs this transformation using nine redox-active cofactors: a bis(molybdopterin guanine dinucleotide) (bis-MGD) at the active site coupled to seven iron-sulfur clusters, and one equivalent of flavin mononucleotide (FMN). To better understand the pathway of electron flow in FdsABG, the reduction potentials of the various cofactors were examined through direct electrochemistry. Given the redundancy of cofactors, a truncated form of the FdsA subunit was developed that possesses only the bis-MGD active site and a singular [4Fe-4S] cluster. Electrochemical characterization of FdsABG compared to truncated FdsA shows that the measured reduction potentials are remarkably similar despite the truncation with two observable features at - 265 mV and - 455 mV vs SHE, indicating that the voltammetry of the truncated enzyme is representative of the reduction potentials of the intact heterotrimer. By producing truncated FdsA without the necessary maturation factors required for bis-MGD insertion, a form of the truncated FdsA that possesses only the [4Fe-4S] was produced, which gives a single voltammetric feature at - 525 mV, allowing the contributions of the molybdenum cofactor to be associated with the observed feature at - 265 mV. This method allowed for the deconvolution of reduction potentials for an enzyme with highly complex cofactor content to know more about the thermodynamic landscape of catalysis.

Cleavage of molybdopterin synthase MoaD-MoaE linear fusion by JAMM/MPN+ domain containing metalloprotease DR0402 from Deinococcus radiodurans.

Molybdenum cofactor (Moco), molybdopterin (MPT) complexed with molybdenum, is an essential cofactor required for the catalytic center of diverse enzymes in all domains of life. Since Moco cannot be taken up as a nutrient unlike many other cofactors, Moco requires de novo biosynthesis. During the synthesis of MPT, the sulfur atom on the C-terminus of MoaD is transferred to cyclic pyranopterin monophosphate (cPMP) which is bound in the substrate pocket of MoaE. MoaD is a ubiquitin-like (Ubl) protein and has a C-terminal di-Gly motif which is a common feature of Ubl proteins. Despite the importance of free C terminal di-Gly motif of MoaD as a sulfur carrier, some bacteria encode a fused MPT synthase in which MoaD- and MoaE-like domains are located on a single peptide. Although it has recently been reported that the fused MPT synthase MoaX from Mycobacterium tuberculosis is posttranslationally cleaved into functional MoaD and MoaE in M. smegmatis, the protease responsible for the cleavage of MoaD-MoaE fusion protein has remained unknown to date. Here we report that the JAMM/MPN+ domain containing metalloprotease DR0402 (JAMMDR) from Deinococcus radiodurans can cleave the MoaD-MoaE fusion protein DR2607, the sole MPT synthase in D. radiodurans, generating the MoaD having a C-terminal di-Gly motif. Furthermore, JAMMDR can also cleave off the MoaD from MoaD-eGFP fusion protein suggesting that JAMMDR recognizes the MoaD region rather than MoaE region in the cleaving process of MoaD-MoaE fusion protein.

Role of a membrane-bound aldehyde dehydrogenase complex AldFGH in acetic acid fermentation with Acetobacter pasteurianus SKU1108.

Acetic acid fermentation is widely considered a consequence of ethanol oxidation by two membrane-bound enzymes-alcohol dehydrogenase and aldehyde dehydrogenase (ALDH)-of acetic acid bacteria. Here, we used a markerless gene disruption method to construct a mutant of the Acetobacter pasteurianus strain SKU1108 with a deletion in the aldH gene, which encodes the large catalytic subunit of a heterotrimeric ALDH complex (AldFGH), to examine the role of AldFGH in acetic acid fermentation. The ΔaldH strain grew less on ethanol-containing medium, i.e., acetic acid fermentation conditions, than the wild-type strain and significantly accumulated acetaldehyde in the culture medium. Unexpectedly, acetaldehyde oxidase activity levels of the intact ΔaldH cells and the ΔaldH cell membranes were similar to those of the wild-type strain, which might be attributed to an additional ALDH isozyme (AldSLC). The apparent KM values of the wild-type and ΔaldH membranes for acetaldehyde were similar to each other, when the cells were cultured in nonfermentation conditions, where ΔaldH cells grow as well as the wild-type cells. However, the membranes of the wild-type cells grown under fermentation conditions showed a 10-fold lower apparent KM value than those of the cells grown under nonfermentation conditions. Under fermentation conditions, transcriptional levels of a gene for AldSLC were 10-fold lower than those under nonfermentation conditions, whereas aldH transcript levels were not dramatically changed under the two conditions. We suggest that A. pasteurianus SKU1108 has two ALDHs, and the AldFGH complex is indispensable for acetic acid fermentation and is the major enzyme under fermentation conditions.

Radical S-adenosylmethionine (SAM) enzymes in cofactor biosynthesis: a treasure trove of complex organic radical rearrangement reactions.

In this minireview, we describe the radical S-adenosylmethionine enzymes involved in the biosynthesis of thiamin, menaquinone, molybdopterin, coenzyme F420, and heme. Our focus is on the remarkably complex organic rearrangements involved, many of which have no precedent in organic or biological chemistry.

Human FAD synthase is a bi-functional enzyme with a FAD hydrolase activity in the molybdopterin binding domain.

FAD synthase (FMN:ATP adenylyl transferase, FMNAT or FADS, EC 2.7.7.2) is involved in the biochemical pathway for converting riboflavin into FAD. Human FADS exists in different isoforms. Two of these have been characterized and are localized in different subcellular compartments. hFADS2 containing 490 amino acids shows a two domain organization: the 3'-phosphoadenosine-5'-phosphosulfate (PAPS) reductase domain, that is the FAD-forming catalytic domain, and a resembling molybdopterin-binding (MPTb) domain. By a multialignment of hFADS2 with other MPTb containing proteins of various organisms from bacteria to plants, the critical residues for hydrolytic function were identified. A homology model of the MPTb domain of hFADS2 was built, using as template the solved structure of a T. acidophilum enzyme. The capacity of hFADS2 to catalyse FAD hydrolysis was revealed. The recombinant hFADS2 was able to hydrolyse added FAD in a Co(2+) and mersalyl dependent reaction. The recombinant PAPS reductase domain is not able to perform the same function. The mutant C440A catalyses the same hydrolytic function of WT with no essential requirement for mersalyl, thus indicating the involvement of C440 in the control of hydrolysis switch. The enzyme C440A is also able to catalyse hydrolysis of FAD bound to the PAPS reductase domain, which is quantitatively converted into FMN.