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Methylene‐tetrahydropterin reductases catalyze the reduction of a methylene to a methyl group bound to a reduced pterin as C1 carrier in various one‐carbon (C1) metabolisms. F420‐dependent methylene‐tetrahydromethanopterin (methylene‐H4MPT) reductase (Mer) and the flavin‐independent methylene‐tetrahydrofolate (methylene‐H4F) reductase (Mfr) use a ternary complex mechanism for the direct transfer of a hydride from F420H2 and NAD(P)H to the respective methylene group, whereas FAD‐dependent methylene‐H4F reductase (MTHFR) uses FAD as prosthetic group and a ping–pong mechanism to catalyze the reduction of methylene‐H4F. 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 F420 were determined. A ternary complex of jMer was modeled on the basis of the jMer‐F420 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. [ABSTRACT FROM AUTHOR]

Crystallization is the limiting step in X-ray structure determination of biological macromolecules. As crystallization experiments can be largely automatized, the diversity of precipitant solutions is often the determinant factor to obtain crystals of high quality. Here, we introduce a 96-well screening kit of crystallization conditions, centered on three ethoxylate-based organic polymers as precipitants and various additional compounds to promote crystal formation. This crystallization screen was tested on various non-standard proteins from bacteria and archaea. Structure determination succeeded for seven out of thirteen targets based on crystals that frequently diffracted to a higher resolution than those obtained with commercially available screening kits. Crystallization hits were rarely similar among the three ethoxylate-based organic polymers and, in comparison, with already available crystallization screens. Hence, the presented crystallization screen is an efficient tool to complement other screens and increase the likelihood of growing crystals suitable for X-ray structure determination. [ABSTRACT FROM AUTHOR]

The anaerobic oxidation of methane (AOM) with sulphate, an area currently generating great interest in microbiology, is accomplished by consortia of methanotrophic archaea (ANME) and sulphate-reducing bacteria (1,2). The enzyme activating methane in methanotrophic archaea has tentatively been identified as a homologue of methyl-coenzyme M reductase (MCR) that catalyses the methane-forming step in methanogenic archaea (3,4).Here we report an X-ray structure of the 280 kDa heterohexameric ANME-1MCR complex. It was crystallized uniquely from a protein ensemble purified from consortia of microorganisms collected with a submersible from a Black Sea mat catalysing AOM with sulphate (4). Crystals grown from the heterogeneous sample diffract to 2.1 A resolution and consist of a single ANME-1 MCR population, demonstrating the strong selective power of crystallization. The structure revealed ANME-1 MCR in complex with coenzyme M and coenzyme B, indicating the same substrates for MCR from methanotrophic and methanogenic archaea. Differences between the highly similar structures of ANME-1 MCR and methanogenic MCR include a [F.sub.430] modification, a cysteine-rich patch and an altered post-translational amino acid modification pattern, which may tune the enzymes for their functions in different biological contexts., Large amounts (up to 300 Tg per year) of the potent greenhouse gas, methane, are oxidized to C[O.sub.2] (reaction 1) in marine sediments by consortia of methanotrophic archaea (ANME-1, ANME-2 [...]

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. [ABSTRACT FROM AUTHOR]

The crystal structure of the obligately anaerobic bacteria Desulfococcus multivorans ([GDH.sub.Des]) with and without glutaconyl-coenzyme A is determined in order to understand the distinct catalytic behavior of two glutaryl-coenzyme A dehydrogenase (GDH) types on an atomic basis. The structural changes have prevented decarboxylation by strengthening the C4-C5 bond of glutaconyl-coenzyme A, by reducing the leaving group potential of C[O.sub.2] and by increasing the distance between the C4 atom and the adjacent glutamic acid.

The structural characterization of methylenetetrahydromethanopterin Mtd-methylene-[H.sub.4]MPT, Mtd-methenyl-[[H.sub.4]MPT.sup.+], and the Mtd-methenyl-[[H.sub.4]MPT.sup.+]-[F.sub.420][H.sub.2] complexes of Methanopyrus kandleri at 2.1, 2.0, and 1.8 [Angstrom] resolution, respectively is reported. The polypeptide scaffold did not reveal any significant conformational change upon binding of the bulky substrates but in turn changed the conformations of the substrate rings either to avoid clashes between certain ring atoms or to adjust the rings involved in hydride transfer for providing an optimal catalytic efficiency.

The X-ray crystallographic structure of the enzyme (E)-4-hydroxy-3-methyl-but-2-enyl diphosphate (HMBPP) reductase, LytB, from Aquifex aeolicus and a structure-based model for catalysis are described. The results have shown that E126 is involved, with the ca. 5:1 isopentenyl diphosphate (IPP)/dimethylallyl diphosphate (DMAPP) product ratio being related to the E126 side-chain conformations, as there are no other candidates for protonating C4.

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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 (HO2C-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. [ABSTRACT FROM AUTHOR]

ATP sulfurylase (ATPS) catalyzes a key reaction in the global sulfur cycle by reversibly converting inorganic sulfate (SO42−) with ATP to adenosine 5′-phosphosulfate (APS) and pyrophosphate (PPi). In this work we report on the sat encoded dissimilatory ATP sulfurylase from the sulfur-oxidizing purple sulfur bacterium Allochromatium vinosum. In this organism, the sat gene is located in one operon and co-transcribed with the aprMBA genes for membrane-bound APS reductase. Like APS reductase, Sat is dispensible for growth on reduced sulfur compounds due to the presence of an alternate, so far unidentified sulfite-oxidizing pathway in A. vinosum. Sulfate assimilation also proceeds independently of Sat by a separate pathway involving a cysDN-encoded assimilatory ATP sulfurylase. We produced the purple bacterial sat-encoded ATP sulfurylase as a recombinant protein in E. coli, determined crucial kinetic parameters and obtained a crystal structure in an open state with a ligand-free active site. By comparison with several known structures of the ATPS-APS complex in the closed state a scenario about substrate-induced conformational changes was worked out. Despite different kinetic properties ATPS involved in sulfur-oxidizing and sulfate-reducing processes are not distinguishable on a structural level presumably due to the interference between functional and evolutionary processes. [ABSTRACT FROM AUTHOR]

ATP sulfurylase (ATPS) catalyzes a key reaction in the global sulfur cycle by reversibly converting inorganic sulfate (SO42−) with ATP to adenosine 5′-phosphosulfate (APS) and pyrophosphate (PPi). In this work we report on the sat encoded dissimilatory ATP sulfurylase from the sulfur-oxidizing purple sulfur bacterium Allochromatium vinosum. In this organism, the sat gene is located in one operon and co-transcribed with the aprMBA genes for membrane-bound APS reductase. Like APS reductase, Sat is dispensible for growth on reduced sulfur compounds due to the presence of an alternate, so far unidentified sulfite-oxidizing pathway in A. vinosum. Sulfate assimilation also proceeds independently of Sat by a separate pathway involving a cysDN-encoded assimilatory ATP sulfurylase. We produced the purple bacterial sat-encoded ATP sulfurylase as a recombinant protein in E. coli, determined crucial kinetic parameters and obtained a crystal structure in an open state with a ligand-free active site. By comparison with several known structures of the ATPS-APS complex in the closed state a scenario about substrate-induced conformational changes was worked out. Despite different kinetic properties ATPS involved in sulfur-oxidizing and sulfate-reducing processes are not distinguishable on a structural level presumably due to the interference between functional and evolutionary processes. [ABSTRACT FROM AUTHOR]

Autotrophic members of the Sulfolobales (crenarchaeota) use the 3-hydroxypropionate/4-hydroxybutyrate cycle to assimilate CO2 into cell material. The product of the initial acetyl-CoA carboxylation with CO2, malonyl-CoA, is further reduced to malonic semialdehyde by an NADPH-dependent malonyl-CoA reductase (MCR); the enzyme also catalyzes the reduction of succinyl-CoA to succinic semialdehyde onwards in the cycle. Here, we present the crystal structure of Sulfolobus tokodaii malonyl-CoA reductase in the substrate-free state and in complex with NADP+ and CoA. Structural analysis revealed an unexpected reaction cycle in which NADP+ and CoA successively occupy identical binding sites. Both coenzymes are pressed into an S-shaped, nearly superimposable structure imposed by a fixed and preformed binding site. The template- governed cofactor shaping implicates the same binding site for the 3'- and 2'-ribose phosphate group of CoA and NADP+, respectively, but a different one for the common ADP part: the β-phosphate of CoA aligns with theβ-phosphate of NADP+. Evolution from an NADP+ to a bispecific NADP+ and CoA binding site involves many amino acid exchanges within a complex process by which constraints of the CoA structure also influence NADP+ binding. Based on the paralogous aspartate-β-semialdehyde dehydrogenase structurally characterized with a covalent Cys-aspartyl adduct, a malonyl/succinyl group can be reliably modeled into MCR and discussed regarding its binding mode, the malonyl/succinyl specificity, and the catalyzed reaction. The modified polypeptide surrounding around the absent ammonium group in malonate/ succinate compared with aspartate provides the structural basis for engineering a methylmalonyl-CoA reductase applied for biotechnical polyester building block synthesis. [ABSTRACT FROM AUTHOR]