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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]

FAD‐independent methylene‐tetrahydrofolate (methylene‐H4F) reductase (Mfr), recently identified in mycobacteria, catalyzes the reduction of methylene‐H4F to methyl‐H4F with NADH as hydride donor by a ternary complex mechanism. This biochemical reaction corresponds to that of the ubiquitous FAD‐dependent methylene‐H4F 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 C1‐carriers and the reducing agents in hMfr and MTHFR are assumed. [ABSTRACT FROM AUTHOR]

Flavin‐based electron bifurcation (FBEB) is a recently discovered mode of energy coupling in anaerobic microorganisms. The electron‐bifurcating caffeyl‐CoA reductase (CarCDE) catalyzes the reduction of caffeyl‐CoA and ferredoxin by oxidizing NADH. The 3.5 Å structure of the heterododecameric Car(CDE)4 complex of Acetobacterium woodii, presented here, reveals compared to other electron‐transferring flavoprotein/acyl dehydrogenase family members an additional ferredoxin‐like domain with two [4Fe–4S] clusters N‐terminally fused to CarE. It might serve, in vivo, as specific adaptor for the physiological electron acceptor. Kinetic analysis of a CarCDE(∆Fd) complex indicates the bypassing of the ferredoxin‐like domain by artificial electron acceptors. Site‐directed mutagenesis studies substantiated the crucial role of the C‐terminal arm of CarD and of ArgE203, hydrogen‐bonded to the bifurcating FAD, for FBEB. [ABSTRACT FROM AUTHOR]

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]

Flavohemoglobins (flavoHbs) serve various microorganisms as the major protective enzymes against NO˙-mediated toxicity. FlavoHbs dominantly function as an NO˙ dioxygenase ( [ABSTRACT FROM AUTHOR]

The molybdenum storage protein (MoSto) can store more than 100 Mo or W atoms as discrete polyoxometalate (POM) clusters. Here, we describe the three POM cluster sites along the threefold axis of the protein complex based on four X-ray structures with slightly different polyoxomolybdate compositions between 1.35 and 2 Å resolution. In contrast to the Moα-out binding site occupied by an Mo3 cluster, the Moα-in and Moβ binding sites contain rather weak and non-uniform electron density for the Mo atoms (but clearly identifiable by anomalous data), suggesting the presence of POM cluster ensembles and/or degradation products of larger aggregates. The "Moα-in cluster ensemble" was interpreted as an antiprism-like Mo6 species superimposed with an Mo7 pyramide and the "Moβ cluster ensemble" as an Mo13 cluster (present mostly in a degraded form) composed of a pyramidal Mo7 and a Mo3 building block linked by three spatially separated MoOx units. Inside the ball-shaped Mo13 cluster sits an occluded central atom, perhaps a metal ion. POM cluster formation at the Moα-in and Moβ sites appears to be driven by filtering out and binding/protecting self-assembled transient species complementary to the protein template. [ABSTRACT FROM AUTHOR]

NADP-dependent methylene-H4MPT dehydrogenase, MtdA, from Methylobacterium extorquens AM1 catalyzes the dehydrogenation of methylene-tetrahydromethanopterin and methylene-tetrahydrofolate with NADP+ as cosubstrate. The X-ray structure of MtdA with and without NADP bound was established at 1.9 A˚ resolution. The enzyme is present as a homotrimer. The α,β fold of the monomer is related to that of methylene-H4F dehydrogenases, suggesting a common evolutionary origin. The position of the active site is located within a large crevice built up by the two domains of one subunit and one domain of a second subunit. Methylene-H4MPT could be modeled into the cleft, and crucial active site residues such as Phe18, Lys256, His260, and Thr102 were identified. The molecular basis of the different substrate specificities and different catalytic demands of MtdA compared to methylene-H4F dehydrogenases are discussed. [Copyright &y& Elsevier]