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The antibiotic roseoflavin is a riboflavin (vitamin B 2 ) analog. One step of the roseoflavin biosynthetic pathway is catalyzed by the phosphatase RosC, which dephosphorylates 8-demethyl-8-amino-riboflavin-5'-phosphate (AFP) to 8-demethyl-8-amino-riboflavin (AF). RosC also catalyzes the potentially cell-damaging dephosphorylation of the AFP analog riboflavin-5'-phosphate also called "flavin mononucleotide" (FMN), however, with a lower efficiency. We performed X-ray structural analyses and mutagenesis studies on RosC from Streptomyces davaonensis to understand binding of the flavin substrates, the distinction between AFP and FMN and the catalytic mechanism of this enzyme. This work is the first structural analysis of an AFP phosphatase. Each monomer of the RosC dimer consists of an α/β-fold core, which is extended by three specific elongated strand-to-helix sections and a specific N-terminal helix. Altogether these segments envelope the flavin thereby forming a novel flavin-binding site. We propose that distinction between AFP and FMN is provided by substrate-induced rigidification of the four RosC specific supplementary segments mentioned above and by an interaction between the amino group at C8 of AFP and the β-carboxylate of D166. This key amino acid is involved in binding the ring system of AFP and positioning its ribitol phosphate part. Accordingly, site-specific exchanges at D166 disturbed the active site geometry of the enzyme and drastically reduced the catalytic activity. Based on the structure of the catalytic core we constructed a whole series of RosC variants but a disturbing, FMN dephosphorylating "killer enzyme", was not generated., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Methylene-tetrahydropterin reductases catalyze the reduction of a methylene to a methyl group bound to a reduced pterin as C 1 carrier in various one-carbon (C 1 ) metabolisms. F 420 -dependent methylene-tetrahydromethanopterin (methylene-H 4 MPT) reductase (Mer) and the flavin-independent methylene-tetrahydrofolate (methylene-H 4 F) reductase (Mfr) use a ternary complex mechanism for the direct transfer of a hydride from F 420 H 2 and NAD(P)H to the respective methylene group, whereas FAD-dependent methylene-H 4 F reductase (MTHFR) uses FAD as prosthetic group and a ping-pong mechanism to catalyze the reduction of methylene-H 4 F. A ternary complex structure and a thereof derived catalytic mechanism of MTHFR is available, while no ternary complex structures of Mfr or Mer are reported. Here, Mer from Methanocaldococcus jannaschii (jMer) was heterologously produced and the crystal structures of the enzyme with and without F 420 were determined. A ternary complex of jMer was modeled on the basis of the jMer-F 420 structure and the ternary complex structure of MTHFR by superimposing the polypeptide after fixing hydride-transferring atoms of the flavins on each other, and by the subsequent transfer of the methyl-tetrahydropterin from MTHFR to jMer. Mutational analysis of four functional amino acids, which are similarly positioned in the three reductase structures, indicated despite the insignificant sequence identity, a common catalytic mechanism with a 5-iminium cation of methylene-tetrahydropterin as intermediate protonated by a shared glutamate. According to structural, mutational and phylogenetic analysis, the evolution of the three reductases most likely proceeds via a convergent development although a divergent scenario requiring drastic structural changes of the common ancestor cannot be completely ruled out., (© 2024 The Authors. Protein Science published by Wiley Periodicals LLC on behalf of The Protein Society.)

Methanogenic archaea inhabiting anaerobic environments play a crucial role in the global biogeochemical material cycle. The most universal electrogenic reaction of their methane-producing energy metabolism is catalyzed by N     5 -methyl-tetrahydromethanopterin: coenzyme M methyltransferase (MtrABCDEFGH), which couples the vectorial Na + transport with a methyl transfer between the one-carbon carriers tetrahydromethanopterin and coenzyme M via a vitamin B 12 derivative (cobamide) as prosthetic group. We present the 2.08 Å cryo-EM structure of Mtr(ABCDEFG) 3 composed of the central Mtr(ABFG) 3 stalk symmetrically flanked by three membrane-spanning MtrCDE globes. Tetraether glycolipids visible in the map fill gaps inside the multisubunit complex. Putative coenzyme M and Na + were identified inside or in a side-pocket of a cytoplasmic cavity formed within MtrCDE. Its bottom marks the gate of the transmembrane pore occluded in the cryo-EM map. By integrating Alphafold2 information, functionally competent MtrA-MtrH and MtrA-MtrCDE subcomplexes could be modeled and thus the methyl-tetrahydromethanopterin demethylation and coenzyme M methylation half-reactions structurally described. Methyl-transfer-driven Na + transport is proposed to be based on a strong and weak complex between MtrCDE and MtrA carrying vitamin B 12 , the latter being placed at the entrance of the cytoplasmic MtrCDE cavity. Hypothetically, strongly attached methyl-cob(III)amide (His-on) carrying MtrA induces an inward-facing conformation, Na + flux into the membrane protein center and finally coenzyme M methylation while the generated loosely attached (or detached) MtrA carrying cob(I)amide (His-off) induces an outward-facing conformation and an extracellular Na + outflux. Methyl-cob(III)amide (His-on) is regenerated in the distant active site of the methyl-tetrahydromethanopterin binding MtrH implicating a large-scale shuttling movement of the vitamin B 12 -carrying domain., Competing Interests: Competing interests statement:The authors declare no competing interest.

FAD-independent methylene-tetrahydrofolate (methylene-H 4 F) reductase (Mfr), recently identified in mycobacteria, catalyzes the reduction of methylene-H 4 F to methyl-H 4 F with NADH as hydride donor by a ternary complex mechanism. This biochemical reaction corresponds to that of the ubiquitous FAD-dependent methylene-H 4 F reductase (MTHFR), although the latter uses a ping-pong mechanism with the prosthetic group as intermediate hydride carrier. Comparative genomics and genetic analyses indicated that Mfr is indispensable for the growth of Mycobacterium tuberculosis, which lacks the MTHFR encoding gene. Therefore, Mfr appears to be an excellent target for the design of antimycobacterial drugs. Here, we report the heterologous production, enzymological characterization, and the crystal structure of Mfr from the thermophilic mycobacterium Mycobacterium hassiacum (hMfr), which shows 78% sequence identity to Mfr from M. tuberculosis. Although hMfr and MTHFR have minor sequence identity and different catalytic mechanisms, their structures are highly similar, thus suggesting a divergent evolution of Mfr and MTHFR from a common ancestor. Most of the important active site residues of MTHFR are conserved and equivalently positioned in the tertiary structure of hMfr. The Glu9Gln variant of hMfr exhibits a drastic reduction of the catalytic activity, which supports the predicted function of the glutamate residue as proton donor in both hMfr and MTHFR. Thus, highly similar binding modes for the C 1 -carriers and the reducing agents in hMfr and MTHFR are assumed., (© 2023 The Authors. Proteins: Structure, Function, and Bioinformatics published by Wiley Periodicals LLC.)

Various microbial metabolisms use H + /Na + -translocating ferredoxin:NAD + reductase (Rnf) either to exergonically oxidize reduced ferredoxin by NAD + for generating a transmembrane electrochemical potential or reversely to exploit the latter for producing reduced ferredoxin. For cryo-EM structural analysis, we elaborated a quick four-step purification protocol for the Rnf complex from Clostridium tetanomorphum and integrated the homogeneous and active enzyme into a nanodisc. The obtained 4.27 Å density map largely allows chain tracing and redox cofactor identification complemented by biochemical data from entire Rnf and single subunits RnfB, RnfC and RnfG. On this basis, we postulated an electron transfer route between ferredoxin and NAD via eight [4Fe-4S] clusters, one Fe ion and four flavins crossing the cell membrane twice related to the pathway of NADH:ubiquinone reductase. Redox-coupled Na + translocation is provided by orchestrating Na + uptake/release, electrostatic effects of the assumed membrane-integrated FMN semiquinone anion and accompanied polypeptide rearrangements mediated by different redox steps., (© 2022. The Author(s).)

Bacteria utilize small extracellular molecules to communicate in order to collectively coordinate their behaviors in response to the population density. Autoinducer-2 (AI-2), a universal molecule for both intra- and inter-species communication, is involved in the regulation of biofilm formation, virulence, motility, chemotaxis, and antibiotic resistance. While many studies have been devoted to understanding the biosynthesis and sensing of AI-2, very little information is available on its export. The protein TqsA from Escherichia coli, which belongs to the AI-2 exporter superfamily, has been shown to export AI-2. Here, we report the cryogenic electron microscopic structures of two AI-2 exporters (TqsA and YdiK) from E. coli at 3.35 Å and 2.80 Å resolutions, respectively. Our structures suggest that the AI-2 exporter exists as a homo-pentameric complex. In silico molecular docking and native mass spectrometry experiments were employed to demonstrate the interaction between AI-2 and TqsA, and the results highlight the functional importance of two helical hairpins in substrate binding. We propose that each monomer works as an independent functional unit utilizing an elevator-type transport mechanism., (© 2022 The Authors. Published under the terms of the CC BY NC ND 4.0 license.)

Anaerobic toluene degradation involves β-oxidation of the first intermediate (R)-2-benzylsuccinate to succinyl-CoA and benzoyl-CoA. Here, we characterize the last enzyme of this pathway, (S)-2-benzoylsuccinyl-CoA thiolase (BbsAB). Although benzoylsuccinyl-CoA is not available for enzyme assays, the recombinantly produced enzymes from two different species showed the reverse activity, benzoylsuccinyl-CoA formation from benzoyl-CoA and succinyl-CoA. Activity depended on the presence of both subunits, the thiolase family member BbsB and the Zn-finger protein BbsA, which is affiliated to the DUF35 family of unknown function. We determined the structure of BbsAB from Geobacter metallireducens with and without bound CoA at 1.7 and 2.0 Å resolution, respectively. CoA binding into the well-known thiolase cavity triggers an induced-fit movement of the highly disordered covering loop, resulting in its rigidification by forming multiple interactions to the outstretched CoA moiety. This event is coupled with an 8 Å movement of an adjacent hairpin loop of BbsB and the C-terminal domain of BbsA. Thereby, CoA is placed into a catalytically productive conformation, and a putative second CoA binding site involving BbsA and the partner BbsB' subunit is simultaneously formed that also reaches the active center. Therefore, while maintaining the standard thioester-dependent Claisen-type mechanism, BbsAB represents a new type of thiolase., (© 2022 The Authors. The FEBS Journal published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

Some N 2 -fixing bacteria store Mo to maintain the formation of the vital FeMo-cofactor dependent nitrogenase under Mo depleting conditions. The Mo storage protein (MoSto), developed for this purpose, has the unique capability to compactly deposit molybdate as polyoxometalate (POM) clusters in a (αβ) 3 hexameric cage; the same occurs with the physicochemically related tungstate. To explore the structural diversity of W-based POM clusters, MoSto loaded under different conditions with tungstate and two site-specifically modified MoSto variants were structurally characterized by X-ray crystallography or single-particle cryo-EM. The MoSto cage contains five major locations for POM clusters occupied among others by heptanuclear, Keggin ion and even Dawson-like species also found in bulk solvent under defined conditions. We found both lacunary derivatives of these archetypical POM clusters with missing WO x units at positions exposed to bulk solvent and expanded derivatives with additional WO x units next to protecting polypeptide segments or other POM clusters. The cryo-EM map, unexpectedly, reveals a POM cluster in the cage center anchored to the wall by a WO x linker. Interestingly, distinct POM cluster structures can originate from identical, highly occupied core fragments of three to seven WO x units that partly correspond to those found in MoSto loaded with molybdate. These core fragments are firmly bound to the complementary protein template in contrast to the more variable, less occupied residual parts of the visible POM clusters. Due to their higher stability, W-based POM clusters are, on average, larger and more diverse than their Mo-based counterparts., (Copyright © 2022. Published by Elsevier Inc.)

Lactate oxidation with NAD + as electron acceptor is a highly endergonic reaction. Some anaerobic bacteria overcome the energetic hurdle by flavin-based electron bifurcation/confurcation (FBEB/FBEC) using a lactate dehydrogenase (Ldh) in concert with the electron-transferring proteins EtfA and EtfB. The electron cryo-microscopically characterized (Ldh-EtfAB) 2 complex of Acetobacterium woodii at 2.43 Å resolution consists of a mobile EtfAB shuttle domain located between the rigid central Ldh and the peripheral EtfAB base units. The FADs of Ldh and the EtfAB shuttle domain contact each other thereby forming the D (dehydrogenation-connected) state. The intermediary Glu37 and Glu139 may harmonize the redox potentials between the FADs and the pyruvate/lactate pair crucial for FBEC. By integrating Alphafold2 calculations a plausible novel B (bifurcation-connected) state was obtained allowing electron transfer between the EtfAB base and shuttle FADs. Kinetic analysis of enzyme variants suggests a correlation between NAD + binding site and D-to-B-state transition implicating a 75° rotation of the EtfAB shuttle domain. The FBEC inactivity when truncating the ferredoxin domain of EtfA substantiates its role as redox relay. Lactate oxidation in Ldh is assisted by the catalytic base His423 and a metal center. On this basis, a comprehensive catalytic mechanism of the FBEC process was proposed., Competing Interests: KK, AK, CH, SW, JS, UE, VM No competing interests declared, (© 2022, Kayastha, Katsyv et al.)

Despite the plethora of information on ( S )-selective amine transaminases, the ( R )-selective ones are still not well-studied; only a few structures are known to date, and their substrate scope is limited, apart from a few stellar works in the field. Herein, the structure of Luminiphilus syltensis ( R )-selective amine transaminase is elucidated to facilitate engineering towards variants active on bulkier substrates. The V37A variant exhibited increased activity towards 1-phenylpropylamine and to activity against 1-butylamine. In contrast, the S248 and T249 positions, located on the β-turn in the P-pocket, seem crucial for maintaining the activity of the enzyme.

Multidrug and toxic compound extrusion (MATE) transporters are widespread in all domains of life. Bacterial MATE transporters confer multidrug resistance by utilizing an electrochemical gradient of H + or Na + to export xenobiotics across the membrane. Despite the availability of X-ray structures of several MATE transporters, a detailed understanding of the transport mechanism has remained elusive. Here we report the crystal structure of a MATE transporter from Aquifex aeolicus at 2.0-Å resolution. In light of its phylogenetic placement outside of the diversity of hitherto-described MATE transporters and the lack of conserved acidic residues, this protein may represent a subfamily of prokaryotic MATE transporters, which was proven by phylogenetic analysis. Furthermore, the crystal structure and substrate docking results indicate that the substrate binding site is located in the N bundle. The importance of residues surrounding this binding site was demonstrated by structure-based site-directed mutagenesis. We suggest that Aq_128 is functionally similar but structurally diverse from DinF subfamily transporters. Our results provide structural insights into the MATE transporter, which further advances our global understanding of this important transporter family., Competing Interests: The authors declare no competing interest.

The biologically important, FAD-containing acyl-coenzyme A (CoA) dehydrogenases (ACAD) usually catalyze the anti-1,2-elimination of a proton and a hydride of aliphatic CoA thioesters. Here, we report on the structure and function of an ACAD from anaerobic bacteria catalyzing the unprecedented 1,4-elimination at C3 and C6 of cyclohex-1-ene-1-carboxyl-CoA (Ch1CoA) to cyclohex-1,5-diene-1-carboxyl-CoA (Ch1,5CoA) and at C3 and C4 of the latter to benzoyl-CoA. Based on high-resolution Ch1CoA dehydrogenase crystal structures, the unorthodox reactivity is explained by the presence of a catalytic aspartate base (D91) at C3, and by eliminating the catalytic glutamate base at C1. Moreover, C6 of Ch1CoA and C4 of Ch1,5CoA are positioned towards FAD-N5 to favor the biologically relevant C3,C6- over the C3,C4-dehydrogenation activity. The C1,C2-dehydrogenation activity was regained by structure-inspired amino acid exchanges. The results provide the structural rationale for the extended catalytic repertoire of ACADs and offer previously unknown biocatalytic options for the synthesis of cyclic 1,3-diene building blocks., (© 2021 The Authors. ChemBioChem published by Wiley-VCH GmbH.)

The discovery of a new energy-coupling mechanism termed flavin-based electron bifurcation (FBEB) in 2008 revealed a novel field of application for flavins in biology. The key component is the bifurcating flavin endowed with strongly inverted one-electron reduction potentials (FAD/FAD• - ≪ FAD• - /FADH - ) that cooperatively transfers in its reduced state one low and one high-energy electron into different directions and thereby drives an endergonic with an exergonic reduction reaction. As energy splitting at the bifurcating flavin apparently implicates one-electron chemistry, the FBEB machinery has to incorporate prior to and behind the central bifurcating flavin 2e-to-1e and 1e-to-2e switches, frequently also flavins, for oxidizing variable medium-potential two-electron donating substrates and for reducing high-potential two-electron accepting substrates. The one-electron carriers ferredoxin or flavodoxin serve as low-potential (high-energy) electron acceptors, which power endergonic processes almost exclusively in obligate anaerobic microorganisms to increase the efficiency of their energy metabolism. In this review, we outline the global organization of FBEB enzymes, the functions of the flavins therein and the surrounding of the isoalloxazine rings by which their reduction potentials are specifically adjusted in a finely tuned energy landscape., (Copyright © 2021 Elsevier Inc. All rights reserved.)

In methanogenic archaea, the archetypical complex of heterodisulfide reductase (HdrABC) and hydrogenase (MvhAGD) couples the endergonic reduction of CO 2 by H 2 to the exergonic reduction of the CoB-S-S-CoM heterodisulfide by H 2 via flavin-based electron bifurcation. Presently known enzymes containing HdrA(BC)-like components play key roles in methanogenesis, acetogenesis, respiratory sulfate reduction, lithotrophic reduced sulfur compound oxidation, aromatic compound degradation, fermentations, and probably many further processes. This functional diversity is achieved by a modular architecture of HdrA(BC) enzymes, where a big variety of electron input/output modules may be connected either directly or via adaptor modules to the HdrA(BC) components. Many, but not all HdrA(BC) complexes are proposed to catalyse a flavin-based electron bifurcation/confurcation. Despite the availability of HdrA(BC) crystal structures, fundamental questions of electron transfer and energy coupling processes remain. Here, we address the common properties and functional diversity of HdrA(BC) core modules integrated into electron-transfer machineries of outstanding complexity., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Many bacteria and archaea employ a novel pathway of sulfur oxidation involving an enzyme complex that is related to the heterodisulfide reductase (Hdr or HdrABC) of methanogens. As a first step in the biochemical characterization of Hdr-like proteins from sulfur oxidizers (sHdr), we structurally analyzed the recombinant sHdrA protein from the Alphaproteobacterium Hyphomicrobium denitrificans at 1.4 Å resolution. The sHdrA core structure is similar to that of methanogenic HdrA (mHdrA) which binds the electron-bifurcating flavin adenine dinucleotide (FAD), the heart of the HdrABC-[NiFe]-hydrogenase catalyzed reaction. Each sHdrA homodimer carries two FADs and two [4Fe-4S] clusters being linked by electron conductivity. Redox titrations monitored by electron paramagnetic resonance and visible spectroscopy revealed a redox potential between -203 and -188 mV for the [4Fe-4S] center. The potentials for the FADH•/FADH - and FAD/FADH• pairs reside between -174 and -156 mV and between -81 and -19 mV, respectively. The resulting stable semiquinone FADH• species already detectable in the visible and electron paramagnetic resonance spectra of the as-isolated state of sHdrA is incompatible with basic principles of flavin-based electron bifurcation such that the sHdr complex does not apply this new mode of energy coupling. The inverted one-electron FAD redox potentials of sHdr and mHdr are clearly reflected in the different FAD-polypeptide interactions. According to this finding and the assumption that the sHdr complex forms an asymmetric HdrAA'B1C1B2C2 hexamer, we tentatively propose a mechanism that links protein-bound sulfane oxidation to sulfite on HdrB1 with NAD + reduction via lipoamide disulfide reduction on HdrB2. The FAD of HdrA thereby serves as an electron storage unit. DATABASE: Structural data are available in PDB database under the accession number 6TJR., (© 2020 The Authors. The FEBS Journal published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

Most methanogenic archaea use the rudimentary hydrogenotrophic pathway-from CO 2 and H 2 to methane-as the terminal step of microbial biomass degradation in anoxic habitats. The barely exergonic process that just conserves sufficient energy for a modest lifestyle involves chemically challenging reactions catalyzed by complex enzyme machineries with unique metal-containing cofactors. The basic strategy of the methanogenic energy metabolism is to covalently bind C 1 species to the C 1 carriers methanofuran, tetrahydromethanopterin, and coenzyme M at different oxidation states. The four reduction reactions from CO 2 to methane involve one molybdopterin-based two-electron reduction, two coenzyme F 420 -based hydride transfers, and one coenzyme F 430 -based radical process. For energy conservation, one ion-gradient-forming methyl transfer reaction is sufficient, albeit supported by a sophisticated energy-coupling process termed flavin-based electron bifurcation for driving the endergonic CO 2 reduction and fixation. Here, we review the knowledge about the structure-based catalytic mechanism of each enzyme of hydrogenotrophic methanogenesis.

Certain anaerobic microorganisms evolved a mechanism to use H 2 as a reductant in their energy metabolisms. For these purposes, the microorganisms developed H 2 -activating enzymes, which are aspirational catalysts in a sustainable hydrogen economy. In the case of the hydrogenotrophic pathway performed by methanogenic archaea, 8e - are extracted from 4H 2 and used as reducing equivalents to convert CO 2 into CH 4 . Under standard cultivation conditions, these archaea express [NiFe]-hydrogenases, which are Ni-dependent and Fe-dependent enzymes and heterolytically cleave H 2 into 2H + and 2e - , the latter being supplied into the central metabolism. Under Ni-limiting conditions, F 420 -reducing [NiFe]-hydrogenases are downregulated and their functions are predominantly taken over by an upregulated [Fe]-hydrogenase. Unique in biology, this Fe-dependent hydrogenase cleaves H 2 and directly transfers H - to an imidazolium-containing substrate. [Fe]-hydrogenase activates H 2 at an Fe cofactor ligated by two CO molecules, an acyl group, a pyridinol N atom and a cysteine thiolate as the central constituent. This Fe centre has inspired chemists to not only design synthetic mimics to catalytically cleave H 2 in solution but also for incorporation into apo-[Fe]-hydrogenase to give semi-synthetic proteins. This Perspective describes the enzymes involved in hydrogenotrophic methanogenesis, with a focus on those performing the reduction steps. Of these, we describe [Fe]-hydrogenases in detail and cover recent progress in their synthetic modelling., (© 2020. Springer Nature Limited.)

Some anaerobic bacteria use biotin-dependent Na + -translocating decarboxylases (Bdc) of β-keto acids or their thioester analogs as key enzymes in their energy metabolism. Glutaconyl-CoA decarboxylase (Gcd), a member of this protein family, drives the endergonic translocation of Na + across the membrane with the exergonic decarboxylation of glutaconyl-CoA (Δ G 0 ' ≈-30 kJ/mol) to crotonyl-CoA. Here, we report on the molecular characterization of Gcd from Clostridium symbiosum based on native PAGE, size exclusion chromatography (SEC) and laser-induced liquid bead ion desorption mass spectrometry (LILBID-MS). The obtained molecular mass of ca. 400 kDa fits to the DNA sequence-derived mass of 379 kDa with a subunit composition of 4 GcdA (65 kDa), 2 GcdB (35 kDa), GcdC1 (15 kDa), GcdC2 (14 kDa), and 2 GcdD (10 kDa). Low-resolution structural information was achieved from preliminary electron microscopic (EM) measurements, which resulted in a 3D reconstruction model based on negative-stained particles. The Gcd structure is built up of a membrane-spanning base primarily composed of the GcdB dimer and a solvent-exposed head with the GcdA tetramer as major component. Both globular parts are bridged by a linker presumably built up of segments of GcdC1, GcdC2 and the 2 GcdDs. The structure of the highly mobile Gcd complex represents a template for the global architecture of the Bdc family., (Copyright © 2020 Vitt, Prinz, Hellwig, Morgner, Ermler and Buckel.)

NADP-dependent methylene-tetrahydromethanopterin (methylene-H 4 MPT) dehydrogenase (MtdA) catalyzes the reversible dehydrogenation of methylene-H 4 MPT to form methenyl-H 4 MPT + by using NADP + as a hydride acceptor. This hydride transfer reaction is involved in the oxidative metabolism from formaldehyde to CO 2 in methylotrophic and methanotrophic bacteria. Here, we report on the crystal structures of the ternary MtdA-substrate complexes from Methylorubrum extorquens AM1 obtained in open and closed forms. Their conversion is accomplished by opening/closing the active site cleft via a 15° rotation of the NADP, relative to the pterin domain. The 1.08 Å structure of the closed and active enzyme-NADP-methylene-H 4 MPT complex allows a detailed geometric analysis of the bulky substrates and a precise prediction of the hydride trajectory. Upon domain closure, the bulky substrate rings become compressed resulting in a tilt of the imidazolidine group of methylene-H 4 MPT that optimizes the geometry for hydride transfer. An additional 1.5 Å structure of MtdA in complex with the nonreactive NADP + and methenyl-H 4 MPT + revealed an extremely short distance between nicotinamide-C4 and imidazoline-C14a of 2.5 Å, which demonstrates the strong pressure imposed. The pterin-imidazolidine-phenyl butterfly angle of methylene-H 4 MPT bound to MtdA is smaller than that in the enzyme-free state but is similar to that in H 2 - and F 420 -dependent methylene-H 4 MPT dehydrogenases. The concept of compression-driven hydride transfer including quantum mechanical hydrogen tunneling effects, which are established for flavin- and NADP-dependent enzymes, can be expanded to hydride-transferring H 4 MPT-dependent enzymes., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

The molybdenum storage protein (MoSto) deposits large amounts of molybdenum as polyoxomolybdate clusters in a heterohexameric (αβ) 3 cage-like protein complex under ATP consumption. Here, we suggest a unique mechanism for the ATP-powered molybdate pumping process based on X-ray crystallography, cryoelectron microscopy, hydrogen-deuterium exchange mass spectrometry, and mutational studies of MoSto from Azotobacter vinelandii . First, we show that molybdate, ATP, and Mg 2+ consecutively bind into the open ATP-binding groove of the β-subunit, which thereafter becomes tightly locked by fixing the previously disordered N-terminal arm of the α-subunit over the β-ATP. Next, we propose a nucleophilic attack of molybdate onto the γ-phosphate of β-ATP, analogous to the similar reaction of the structurally related UMP kinase. The formed instable phosphoric-molybdic anhydride becomes immediately hydrolyzed and, according to the current data, the released and accelerated molybdate is pressed through the cage wall, presumably by turning aside the Metβ149 side chain. A structural comparison between MoSto and UMP kinase provides valuable insight into how an enzyme is converted into a molecular machine during evolution. The postulated direct conversion of chemical energy into kinetic energy via an activating molybdate kinase and an exothermic pyrophosphatase reaction to overcome a proteinous barrier represents a novelty in ATP-fueled biochemistry, because normally, ATP hydrolysis initiates large-scale conformational changes to drive a distant process.

Hydride transfers play a crucial role in a multitude of biological redox reactions and are mediated by flavin, deazaflavin or nicotinamide adenine dinucleotide cofactors at standard redox potentials ranging from 0 to -340 mV. 2-Naphthoyl-CoA reductase, a key enzyme of oxygen-independent bacterial naphthalene degradation, uses a low-potential one-electron donor for the two-electron dearomatization of its substrate below the redox limit of known biological hydride transfer processes at E°' = -493 mV. Here we demonstrate by X-ray structural analyses, QM/MM computational studies, and multiple spectroscopy/activity based titrations that highly cooperative electron transfer (n = 3) from a low-potential one-electron (FAD) to a two-electron (FMN) transferring flavin cofactor is the key to overcome the resonance stabilized aromatic system by hydride transfer in a highly hydrophobic pocket. The results evidence how the protein environment inversely functionalizes two flavins to switch from low-potential one-electron to hydride transfer at the thermodynamic limit of flavin redox chemistry.

[Fe]-hydrogenase (Hmd) catalyzes the reversible hydrogenation of methenyl-tetrahydromethanopterin (methenyl-H 4 MPT + ) with H 2 . H 4 MPT is a C1-carrier of methanogenic archaea. One bacterial genus, Desulfurobacterium, contains putative genes for the Hmd paralog, termed HmdII, and the HcgA-G proteins. The latter are required for the biosynthesis of the prosthetic group of Hmd, the iron-guanylylpyridinol (FeGP) cofactor. This finding is intriguing because Hmd and HmdII strictly use H 4 MPT derivatives that are absent in most bacteria. We identified the presence of the FeGP cofactor in D. thermolithotrophum. The bacterial HmdII reconstituted with the FeGP cofactor catalyzed the hydrogenation of derivatives of tetrahydrofolate, the bacterial C1-carrier, albeit with low enzymatic activities. The crystal structures show how Hmd recognizes tetrahydrofolate derivatives. These findings have an impact on future biotechnology by identifying a bacterial Hmd paralog., (© 2019 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

A continuous FeMo cofactor supply for nitrogenase maturation is ensured in Azotobacter vinelandii by developing a cage-like molybdenum storage protein (MoSto) capable to store ca. 120 molybdate molecules ( MoO 4 2 - ) as discrete polyoxometalate (POM) clusters. To gain mechanistic insight into this process, MoSto was characterized by Mo and ATP/ADP content, structural, and kinetic analysis. We defined three functionally relevant states specified by the presence of both ATP/ADP and POM clusters (MoSto funct ), of only ATP/ADP (MoSto basal ) and of neither ATP/ADP nor POM clusters (MoSto zero ), respectively. POM clusters are only produced when ATP is hydrolyzed to ADP and phosphate. V max was ca. 13 μmol phosphate ·min -1 ·mg -1 and K m for molybdate and ATP/Mg 2+ in the low micromolar range. ATP hydrolysis presumably proceeds at subunit α, inferred from a highly occupied α-ATP/Mg 2+ and a weaker occupied β-ATP/no Mg 2+ -binding site found in the MoSto funct structure. Several findings indicate that POM cluster storage is separated into a rapid ATP hydrolysis-dependent molybdate transport across the protein cage wall and a slow molybdate assembly induced by combined auto-catalytic and protein-driven processes. The cage interior, the location of the POM cluster depot, is locked in all three states and thus not rapidly accessible for molybdate from the outside. Based on V max , the entire Mo storage process should be completed in less than 10 s but requires, according to the molybdate content analysis, ca. 15 min. Long-time incubation of MoSto basal with nonphysiological high molybdate amounts implicates an equilibrium in and outside the cage and POM cluster self-formation without ATP hydrolysis. DATABASES: The crystal structures MoSto in the MoSto-F6, MoSto-F7, MoSto basal , MoSto zero , and MoSto-F1 vitro states were deposited to PDB under the accession numbers PDB 6GU5, 6GUJ, 6GWB, 6GWV, and 6GX4., (© 2018 Federation of European Biochemical Societies.)

[Fe]-hydrogenase (Hmd) catalyzes the reversible hydrogenation of methenyltetrahydromethanopterin (methenyl-H 4 MPT + ) with H 2 . Hmd contains the iron-guanylylpyridinol (FeGP) cofactor, which is sensitive to light and oxidative stress. A natural protection mechanism is reported for Hmd based on structural and biophysical data. Hmd from Methanothermobacter marburgensis (mHmd) was found in a hexameric state, where an expanded oligomerization loop is detached from the dimer core and intrudes into the active site of a neighboring dimer. An aspartic acid residue from the loop ligates to Fe II of the FeGP cofactor and thus blocks the postulated H 2 -binding site. In solution, this enzyme is in a hexamer-to-dimer equilibrium. Lower enzyme concentrations, and the presence of methenyl-H 4 MPT + , shift the equilibrium toward the active dimer side. At higher enzyme concentrations-as present in the cell-the enzyme is predominantly in the inactive hexameric state and is thereby protected against light and oxidative stress., (© 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Mono-iron hydrogenase ([Fe]-hydrogenase) reversibly catalyzes the transfer of a hydride ion from H 2 to methenyltetrahydromethanopterin (methenyl-H 4 MPT + ) to form methylene-H 4 MPT. Its iron guanylylpyridinol (FeGP) cofactor plays a key role in H 2 activation. Evidence is presented for O 2 sensitivity of [Fe]-hydrogenase under turnover conditions in the presence of reducing substrates, methylene-H 4 MPT or methenyl-H 4 MPT + /H 2 . Only then, H 2 O 2 is generated, which decomposes the FeGP cofactor; as demonstrated by spectroscopic analyses and the crystal structure of the deactivated enzyme. O 2 reduction to H 2 O 2 requires a reductant, which can be a catalytic intermediate transiently formed during the [Fe]-hydrogenase reaction. The most probable candidate is an iron hydride species; its presence has already been predicted by theoretical studies of the catalytic reaction. The findings support predictions because the same type of reduction reaction is described for ruthenium hydride complexes that hydrogenate polar compounds., (© 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The electron transferring flavoprotein/butyryl-CoA dehydrogenase (EtfAB/Bcd) catalyzes the reduction of one crotonyl-CoA and two ferredoxins by two NADH within a flavin-based electron-bifurcating process. Here we report on the X-ray structure of the Clostridium difficile (EtfAB/Bcd) 4 complex in the dehydrogenase-conducting D-state, α-FAD (bound to domain II of EtfA) and δ-FAD (bound to Bcd) being 8 Å apart. Superimposing Acidaminococcus fermentans EtfAB onto C. difficile EtfAB/Bcd reveals a rotation of domain II of nearly 80°. Further rotation by 10° brings EtfAB into the bifurcating B-state, α-FAD and β-FAD (bound to EtfB) being 14 Å apart. This dual binding mode of domain II, substantiated by mutational studies, resembles findings in non-bifurcating EtfAB/acyl-CoA dehydrogenase complexes. In our proposed mechanism, NADH reduces β-FAD, which bifurcates. One electron goes to ferredoxin and one to α-FAD, which swings over to reduce δ-FAD to the semiquinone. Repetition affords a second reduced ferredoxin and δ-FADH - , which reduces crotonyl-CoA.

Common structural elements in proteins such as α-helices or β-sheets are characterized by uniformly repeating, energetically favorable main chain conformations which additionally exhibit a completely saturated hydrogen-bonding network of the main chain NH and CO groups. Although polyproline or polyglycine type II helices (PP II or PG II ) are frequently found in proteins, they are not considered as equivalent secondary structure elements because they do not form a similar self-contained hydrogen-bonding network of the main chain atoms. In this context our finding of an unusual motif of glycine-rich PG II -like helices in the structure of the acetophenone carboxylase core complex is of relevance. These PG II -like helices form hexagonal bundles which appear to fulfill the criterion of a (largely) saturated hydrogen-bonding network of the main-chain groups and therefore may be regarded in this sense as a new secondary structure element. It consists of a central PG II -like helix surrounded by six nearly parallel PG II -like helices in a hexagonal array, plus an additional PG II -like helix extending the array outwards. Very related structural elements have previously been found in synthetic polyglycine fibers. In both cases, all main chain NH and CO groups of the central PG II -helix are saturated by either intra- or intermolecular hydrogen-bonds, resulting in a self-contained hydrogen-bonding network. Similar, but incomplete PG II -helix patterns were also previously identified in a GTP-binding protein and an antifreeze protein., (© 2017 Wiley Periodicals, Inc.)

Historically, room-temperature structure determination was succeeded by cryo-crystallography to mitigate radiation damage. Here, we demonstrate that serial millisecond crystallography at a synchrotron beamline equipped with high-viscosity injector and high frame-rate detector allows typical crystallographic experiments to be performed at room-temperature. Using a crystal scanning approach, we determine the high-resolution structure of the radiation sensitive molybdenum storage protein, demonstrate soaking of the drug colchicine into tubulin and native sulfur phasing of the human G protein-coupled adenosine receptor. Serial crystallographic data for molecular replacement already converges in 1,000-10,000 diffraction patterns, which we collected in 3 to maximally 82 minutes. Compared with serial data we collected at a free-electron laser, the synchrotron data are of slightly lower resolution, however fewer diffraction patterns are needed for de novo phasing. Overall, the data we collected by room-temperature serial crystallography are of comparable quality to cryo-crystallographic data and can be routinely collected at synchrotrons.Serial crystallography was developed for protein crystal data collection with X-ray free-electron lasers. Here the authors present several examples which show that serial crystallography using high-viscosity injectors can also be routinely employed for room-temperature data collection at synchrotrons.

[Fe]-hydrogenase hosts an iron-guanylylpyridinol (FeGP) cofactor. The FeGP cofactor contains a pyridinol ring substituted with GMP, two methyl groups, and an acylmethyl group. HcgC, an enzyme involved in FeGP biosynthesis, catalyzes methyl transfer from S-adenosylmethionine (SAM) to C3 of 6-carboxymethyl-5-methyl-4-hydroxy-2-pyridinol (2). We report on the ternary structure of HcgC/S-adenosylhomocysteine (SAH, the demethylated product of SAM) and 2 at 1.7 Å resolution. The proximity of C3 of substrate 2 and the S atom of SAH indicates a catalytically productive geometry. The hydroxy and carboxy groups of substrate 2 are hydrogen-bonded with I115 and T179, as well as through a series of water molecules linked with polar and a few protonatable groups. These interactions stabilize the deprotonated state of the hydroxy groups and a keto form of substrate 2, through which the nucleophilicity of C3 is increased by resonance effects. Complemented by mutational analysis, a structure-based catalytic mechanism was proposed., (© 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

In methanogenic archaea, the carbon dioxide (CO 2 ) fixation and methane-forming steps are linked through the heterodisulfide reductase (HdrABC)-[NiFe]-hydrogenase (MvhAGD) complex that uses flavin-based electron bifurcation to reduce ferredoxin and the heterodisulfide of coenzymes M and B. Here, we present the structure of the native heterododecameric HdrABC-MvhAGD complex at 2.15-angstrom resolution. HdrB contains two noncubane [4Fe-4S] clusters composed of fused [3Fe-4S]-[2Fe-2S] units sharing 1 iron (Fe) and 1 sulfur (S), which were coordinated at the CCG motifs. Soaking experiments showed that the heterodisulfide is clamped between the two noncubane [4Fe-4S] clusters and homolytically cleaved, forming coenzyme M and B bound to each iron. Coenzymes are consecutively released upon one-by-one electron transfer. The HdrABC-MvhAGD atomic model serves as a structural template for numerous HdrABC homologs involved in diverse microbial metabolic pathways., (Copyright © 2017 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

The phylogenetically diverse family of methanogenic archaea universally use methyl coenzyme M reductase (MCR) for catalyzing the final methane-forming reaction step of the methanogenic energy metabolism. Some methanogens of the orders Methanobacteriales and Methanococcales contain two isoenzymes. Comprehensive phylogenetic analyses on the basis of all three subunits grouped MCRs from Methanobacteriales and Methanococcales into three distinct types: (i) MCRs from Methanobacteriales , (ii) MCRs from Methanobacteriales and Methanococcales , and (iii) MCRs from Methanococcales The first and second types contain MCR isoenzymes I and II from Methanothermobacter marburgensis , respectively; therefore, they were designated MCR type I and type II and accordingly; the third one was designated MCR type III. For comparison with the known MCR type I and type II structures, we determined the structure of MCR type III from Methanotorris formicicus and Methanothermococcus thermolithotrophicus As predicted, the three MCR types revealed highly similar overall structures and virtually identical active site architectures reflecting the chemically challenging mechanism of methane formation. Pronounced differences were found at the protein surface with respect to loop geometries and electrostatic properties, which also involve the entrance of the active-site funnel. In addition, the C-terminal end of the γ-subunit is prolonged by an extra helix after helix γ8 in MCR type II and type III, which is, however, differently arranged in the two MCR types. MCR types I, II, and III share most of the posttranslational modifications which appear to fine-tune the enzymatic catalysis. Interestingly, MCR type III lacks the methyl-cysteine but possesses in subunit α of M. formicicus a 6-hydroxy-tryptophan, which thus far has been found only in the α-amanitin toxin peptide but not in proteins. IMPORTANCE Methyl coenzyme M reductase (MCR) represents a prime target for the mitigation of methane releases. Phylogenetic analyses of MCRs suggested several distinct sequence clusters; those from Methanobacteriales and Methanococcales were subdivided into three types: MCR type I from Methanobacteriales , MCR type II from Methanobacteriales and Methanococcales , and the newly designated MCR type III exclusively from Methanococcales We determined the first X-ray structures for an MCR type III. Detailed analyses revealed substantial differences between the three types only in the peripheral region. The subtle modifications identified and electrostatic profiles suggested enhanced substrate binding for MCR type III. In addition, MCR type III from Methanotorris formicicus contains 6-hydroxy-tryptophan, a new posttranslational modification that thus far has been found only in the α-amanitin toxin., (Copyright © 2017 American Society for Microbiology.)

The greenhouse gas and energy carrier methane is produced on Earth mainly by methanogenic archaea. In the hydrogenotrophic methanogenic pathway the reduction of one CO 2 to one methane molecule requires four molecules of H 2 containing eight electrons. Four of the electrons from two H 2 are supplied for reduction of an electron carrier F 420 , which is catalyzed by F 420 -reducing [NiFe]-hydrogenase under nickel-sufficient conditions. The same reaction is catalysed under nickel-limiting conditions by [Fe]-hydrogenase coupled with a reaction catalyzed by F 420 -dependent methylene tetrahydromethanopterin dehydrogenase. [Fe]-hydrogenase contains an iron-guanylylpyridinol (FeGP) cofactor for H 2 activation at the active site. Fe II of FeGP is coordinated to a pyridinol-nitrogen, an acyl-carbon, two CO and a cysteine-thiolate. We report here on comparative genomic analyses of biosynthetic genes of the FeGP cofactor, which are primarily located in a hmd-co-occurring (hcg) gene cluster. One of the gene products is HcgB which transfers the guanosine monophosphate (GMP) moiety from guanosine triphosphate (GTP) to a pyridinol precursor. Crystal structure analysis of HcgB from Methanococcus maripaludis and its complex with 6-carboxymethyl-3,5-dimethyl-4-hydroxy-2-pyridinol confirmed the physiological guanylyltransferase reaction. Furthermore, we tested the properties of semi-synthetic [Fe]-hydrogenases using the [Fe]-hydrogenase apoenzyme from several methanogenic archaea and a mimic of the FeGP cofactor. On the basis of the enzymatic reactions involved in the methanogenic pathway, we came up with an idea how the methanogenic pathway could be simplified to develop an artificial methanogenesis system.

8-demethyl-8-aminoriboflavin-5'-phosphate (AFP) synthase (RosB) catalyzes the key reaction of roseoflavin biosynthesis by forming AFP from riboflavin-5'-phosphate (RP) and glutamate via the intermediates 8-demethyl-8-formylriboflavin-5'-phosphate (OHC-RP) and 8-demethyl-8-carboxylriboflavin-5'-phosphate (HO 2 C-RP). To understand this reaction in which a methyl substituent of an aromatic ring is replaced by an amine we structurally characterized RosB in complex with OHC-RP (2.0 Å) and AFP (1.7 Å). RosB is composed of four flavodoxin-like subunits which have been upgraded with specific extensions and a unique C-terminal arm. It appears that RosB has evolved from an electron- or hydride-transferring flavoprotein to a sophisticated multi-step enzyme which uses RP as a substrate (and not as a cofactor). Structure-based active site analysis was complemented by mutational and isotope-based mass-spectrometric data to propose an enzymatic mechanism on an atomic basis., (© 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Degradation of the aromatic ketone acetophenone is initiated by its carboxylation to benzoylacetate catalyzed by acetophenone carboxylase (Apc) in a reaction dependent on the hydrolysis of two ATP to ADP and P i . Apc is a large protein complex which dissociates during purification into a heterooctameric Apc(αα'βγ) 2 core complex of 482 kDa and Apcε of 34 kDa. In this report, we present the X-ray structure of the Apc(αα'βγ) 2 core complex from Aromatoleum aromaticum at ca. 3 Å resolution which reveals a unique modular architecture and serves as model of a new enzyme family. Apcβ contains a novel domain fold composed of two β-sheets in a barrel-like arrangement running into a bundle of eight short polyproline (type II)-like helical segments. Apcα and Apcα' possess ATP binding modules of the ASKHA superfamily integrated into their multidomain structures and presumably operate as ATP-dependent kinases for acetophenone and bicarbonate, respectively. Mechanistic aspects of the novel carboxylation reaction requiring massive structural rearrangements are discussed and criteria for specifically annotating the family members Apc, acetone carboxylase and hydantoinase are defined.

Biological methane formation starts with a challenging adenosine triphosphate (ATP)-independent carbon dioxide (CO 2 ) fixation process. We explored this enzymatic process by solving the x-ray crystal structure of formyl-methanofuran dehydrogenase, determined here as Fwd(ABCDFG) 2 and Fwd(ABCDFG) 4 complexes, from Methanothermobacter wolfeii The latter 800-kilodalton apparatus consists of four peripheral catalytic sections and an electron-supplying core with 46 electronically coupled [4Fe-4S] clusters. Catalysis is separately performed by subunits FwdBD (FwdB and FwdD), which are related to tungsten-containing formate dehydrogenase, and subunit FwdA, a binuclear metal center carrying amidohydrolase. CO 2 is first reduced to formate in FwdBD, which then diffuses through a 43-angstrom-long tunnel to FwdA, where it condenses with methanofuran to formyl-methanofuran. The arrangement of [4Fe-4S] clusters functions as an electron relay but potentially also couples the four tungstopterin active sites over 206 angstroms., (Copyright © 2016, American Association for the Advancement of Science.)

Methanogenic archaea share one ion gradient forming reaction in their energy metabolism catalyzed by the membrane-spanning multisubunit complex N(5)-methyl-tetrahydromethanopterin: coenzyme M methyltransferase (MtrABCDEFGH or simply Mtr). In this reaction the methyl group transfer from methyl-tetrahydromethanopterin to coenzyme M mediated by cobalamin is coupled with the vectorial translocation of Na(+) across the cytoplasmic membrane. No detailed structural and mechanistic data are reported about this process. In the present work we describe a procedure to provide a highly pure and homogenous Mtr complex on the basis of a selective removal of the only soluble subunit MtrH with the membrane perturbing agent dimethyl maleic anhydride and a subsequent two-step chromatographic purification. A molecular mass determination of the Mtr complex by laser induced liquid bead ion desorption mass spectrometry (LILBID-MS) and size exclusion chromatography coupled with multi-angle light scattering (SEC-MALS) resulted in a (MtrABCDEFG)3 heterotrimeric complex of ca. 430kDa with both techniques. Taking into account that the membrane protein complex contains various firmly bound small molecules, predominantly detergent molecules, the stoichiometry of the subunits is most likely 1:1. A schematic model for the subunit arrangement within the MtrABCDEFG protomer was deduced from the mass of Mtr subcomplexes obtained by harsh IR-laser LILBID-MS., (Copyright © 2016. Published by Elsevier B.V.)

All methanogenic and methanotrophic archaea known to date contain methyl-coenzyme M reductase (MCR) that catalyzes the reversible reduction of methyl-coenzyme M to methane. This enzyme contains the nickel porphinoid F430 as a prosthetic group and, highly conserved, a thioglycine and four methylated amino acid residues near the active site. We describe herein the presence of a novel post-translationally modified amino acid, didehydroaspartate, adjacent to the thioglycine as revealed by mass spectrometry and high-resolution X-ray crystallography. Upon chemical reduction, the didehydroaspartate residue was converted into aspartate. Didehydroaspartate was found in MCR I and II from Methanothermobacter marburgensis and in MCR of phylogenetically distantly related Methanosarcina barkeri but not in MCR I and II of Methanothermobacter wolfeii, which indicates that didehydroaspartate is dispensable but might have a role in fine-tuning the active site to increase the catalytic efficiency., (© 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Previous retrosynthetic and isotope-labeling studies have indicated that biosynthesis of the iron guanylylpyridinol (FeGP) cofactor of [Fe]-hydrogenase requires a methyltransferase. This hypothetical enzyme covalently attaches the methyl group at the 3-position of the pyridinol ring. We describe the identification of HcgC, a gene product of the hcgA-G cluster responsible for FeGP cofactor biosynthesis. It acts as an S-adenosylmethionine (SAM)-dependent methyltransferase, based on the crystal structures of HcgC and the HcgC/SAM and HcgC/S-adenosylhomocysteine (SAH) complexes. The pyridinol substrate, 6-carboxymethyl-5-methyl-4-hydroxy-2-pyridinol, was predicted based on properties of the conserved binding pocket and substrate docking simulations. For verification, the assumed substrate was synthesized and used in a kinetic assay. Mass spectrometry and NMR analysis revealed 6-carboxymethyl-3,5-dimethyl-4-hydroxy-2-pyridinol as the reaction product, which confirmed the function of HcgC., (© 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

In the three domains of life, vitamin B12 (cobalamin) is primarily used in methyltransferase and isomerase reactions. The methyltransferase complex MtrA-H of methanogenic archaea has a key function in energy conservation by catalysing the methyl transfer from methyl-tetrahydromethanopterin to coenzyme M and its coupling with sodium-ion translocation. The cobalamin-binding subunit MtrA is not homologous to any known B12-binding proteins and is proposed as the motor of the sodium-ion pump. Here, we present crystal structures of the soluble domain of the membrane-associated MtrA from Methanocaldococcus jannaschii and the cytoplasmic MtrA homologue/cobalamin complex from Methanothermus fervidus. The MtrA fold corresponds to the Rossmann-type α/β fold, which is also found in many cobalamin-containing proteins. Surprisingly, the cobalamin-binding site of MtrA differed greatly from all the other cobalamin-binding sites. Nevertheless, the hydrogen-bond linkage at the lower axial-ligand site of cobalt was equivalently constructed to that found in other methyltransferases and mutases. A distinct polypeptide segment fixed through the hydrogen-bond linkage in the relaxed Co(III) state might be involved in propagating the energy released upon corrinoid demethylation to the sodium-translocation site by a conformational change.

Hydrocarbons are abundant in anoxic environments and pose biochemical challenges to their anaerobic degradation by microorganisms. Within the framework of the Priority Program 1319, investigations funded by the Deutsche Forschungsgemeinschaft on the anaerobic microbial degradation of hydrocarbons ranged from isolation and enrichment of hitherto unknown hydrocarbon-degrading anaerobic microorganisms, discovery of novel reactions, detailed studies of enzyme mechanisms and structures to process-oriented in situ studies. Selected highlights from this program are collected in this synopsis, with more detailed information provided by theme-focused reviews of the special topic issue on 'Anaerobic biodegradation of hydrocarbons' [this issue, pp. 1-244]. The interdisciplinary character of the program, involving microbiologists, biochemists, organic chemists and environmental scientists, is best exemplified by the studies on alkyl-/arylalkylsuccinate synthases. Here, research topics ranged from in-depth mechanistic studies of archetypical toluene-activating benzylsuccinate synthase, substrate-specific phylogenetic clustering of alkyl-/arylalkylsuccinate synthases (toluene plus xylenes, p-cymene, p-cresol, 2-methylnaphthalene, n-alkanes), stereochemical and co-metabolic insights into n-alkane-activating (methylalkyl)succinate synthases to the discovery of bacterial groups previously unknown to possess alkyl-/arylalkylsuccinate synthases by means of functional gene markers and in situ field studies enabled by state-of-the-art stable isotope probing and fractionation approaches. Other topics are Mo-cofactor-dependent dehydrogenases performing O2-independent hydroxylation of hydrocarbons and alkyl side chains (ethylbenzene, p-cymene, cholesterol, n-hexadecane), degradation of p-alkylated benzoates and toluenes, glycyl radical-bearing 4-hydroxyphenylacetate decarboxylase, novel types of carboxylation reactions (for acetophenone, acetone, and potentially also benzene and naphthalene), W-cofactor-containing enzymes for reductive dearomatization of benzoyl-CoA (class II benzoyl-CoA reductase) in obligate anaerobes and addition of water to acetylene, fermentative formation of cyclohexanecarboxylate from benzoate, and methanogenic degradation of hydrocarbons., (© 2016 S. Karger AG, Basel.)

In biology, tungsten (W) is exclusively found in microbial enzymes bound to a bis-pyranopterin cofactor (bis-WPT). Previously known W enzymes catalyze redox oxo/hydroxyl transfer reactions by directly coordinating their substrates or products to the metal. They comprise the W-containing formate/formylmethanofuran dehydrogenases belonging to the dimethyl sulfoxide reductase (DMSOR) family and the aldehyde:ferredoxin oxidoreductase (AOR) families, which form a separate enzyme family within the Mo/W enzymes. In the last decade, initial insights into the structure and function of two unprecedented W enzymes were obtained: the acetaldehyde forming acetylene hydratase (ACH) belongs to the DMSOR and the class II benzoyl-coenzyme A (CoA) reductase (BCR) to the AOR family. The latter catalyzes the reductive dearomatization of benzoyl-CoA to a cyclic diene. Both are key enzymes in the degradation of acetylene (ACH) or aromatic compounds (BCR) in strictly anaerobic bacteria. They are unusual in either catalyzing a nonredox reaction (ACH) or a redox reaction without coordinating the substrate or product to the metal (BCR). In organic chemical synthesis, analogous reactions require totally nonphysiological conditions depending on Hg2+ (acetylene hydration) or alkali metals (benzene ring reduction). The structural insights obtained pave the way for biological or biomimetic approaches to basic reactions in organic chemistry., (© 2016 S. Karger AG, Basel.)

Short-chain fatty acids such as acetic, propionic, butyric or lactic acids are typical primary fermentation products in the anaerobic feeding chain. Fifteen years ago, a novel fermentation type was discovered in the obligately anaerobic Deltaproteobacterium Syntrophus aciditrophicus. During fermentative growth with crotonate and/or benzoate, acetate is formed in the oxidative branch and cyclohexane carboxylate in the reductive branch. In both cases cyclohexa-1,5-diene-1-carboxyl-CoA (Ch1,5CoA) is a central intermediate that is either formed by a class II benzoyl-CoA reductase (fermentation of benzoate) or by reverse reactions of the benzoyl-CoA degradation pathway (fermentation of crotonate). Here, we summarize the current knowledge of the enzymology involved in fermentations yielding cyclohexane carboxylate as an excreted product. The characteristic enzymes involved are two acyl-CoA dehydrogenases specifically acting on Ch1,5CoA and cyclohex-1-ene-1-carboxyl-CoA. Both enzymes are also employed during the syntrophic growth of S. aciditrophicus with cyclohexane carboxylate as the carbon source in coculture with a methanogen. An investigation of anabolic pathways in S. aciditrophicus revealed a rather unusual pathway for glutamate synthesis involving a Re-citrate synthase. Future work has to address the unresolved question concerning which components are involved in reoxidation of the NADH formed in the oxidative branch of the unique cyclohexane carboxylate fermentation pathway in S. aciditrophicus., (© 2016 S. Karger AG, Basel.)

NADH-dependent reduced ferredoxin:NADP oxidoreductase (NfnAB) is found in the cytoplasm of various anaerobic bacteria and archaea. The enzyme reversibly catalyzes the endergonic reduction of ferredoxin with NADPH driven by the exergonic transhydrogenation from NADPH onto NAD(+). Coupling is most probably accomplished via the mechanism of flavin-based electron bifurcation. To understand this process on a structural basis, we heterologously produced the NfnAB complex of Thermotoga maritima in Escherichia coli, provided kinetic evidence for its bifurcating behavior, and determined its x-ray structure in the absence and presence of NADH. The structure of NfnAB reveals an electron transfer route including the FAD (a-FAD), the [2Fe-2S] cluster of NfnA and the FAD (b-FAD), and the two [4Fe-4S] clusters of NfnB. Ferredoxin is presumably docked onto NfnB close to the [4Fe-4S] cluster distal to b-FAD. NAD(H) binds to a-FAD and NADP(H) consequently to b-FAD, which is positioned in the center of the NfnAB complex and the site of electron bifurcation. Arg(187) is hydrogen-bonded to N5 and O4 of the bifurcating b-FAD and might play a key role in adjusting a low redox potential of the FADH(•)/FAD pair required for ferredoxin reduction. A mechanism of FAD-coupled electron bifurcation by NfnAB is proposed., (© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.)

Unlabelled: [Fe]-hydrogenase (Hmd), an enzyme of the methanogenic energy metabolism, harbors an iron-guanylylpyridinol (FeGP) cofactor used for H2 cleavage. The generated hydride is transferred to methenyl-tetrahydromethanopterin (methenyl-H4MPT(+)). Most hydrogenotrophic methanogens contain the hmd-related genes hmdII and hmdIII. Their function is still elusive. We were able to reconstitute the HmdII holoenzyme of Methanocaldococcus jannaschii with recombinantly produced apoenzyme and the FeGP cofactor, which is a prerequisite for in vitro functional analysis. Infrared spectroscopic and X-ray structural data clearly indicated binding of the FeGP cofactor. Methylene-H4MPT binding was detectable in the significantly altered infrared spectra of the HmdII holoenzyme and in the HmdII apoenzyme-methylene-H4 MPT complex structure. The related binding mode of the FeGP cofactor and methenyl-H4MPT(+) compared with Hmd and their multiple contacts to the polypeptide highly suggest a biological role in HmdII. However, holo-HmdII did not catalyze the Hmd reaction, not even in a single turnover process, as demonstrated by kinetic measurements. The found inactivity can be rationalized by an increased contact area between the C- and N-terminal folding units in HmdII compared with in Hmd, which impairs the catalytically necessary open-to-close transition, and by an exchange of a crucial histidine to a tyrosine. Mainly based on the presented data, a function of HmdII as Hmd isoenzyme, H2 sensor, FeGP-cofactor storage protein and scaffold protein for FeGP-cofactor biosynthesis could be excluded. Inspired by the recently found binding of HmdII to aminoacyl-tRNA synthetases and tRNA, we tentatively consider HmdII as a regulatory protein for protein synthesis that senses the intracellular methylene-H4 MPT concentration., Database: Structural data are available in the Protein Data Bank under the accession numbers 4YT8; 4YT2; 4YT4 and 4YT5., (© 2015 FEBS.)