Search

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Highlights: [•] LytB of the malaria pathogen Plasmodium falciparum was structurally characterized. [•] The substrate and electron donor binding sites serve as targets for a search for antimalarian drugs. [•] A LytB-[2Fe–2S] ferredoxin complex was modelled by docking calculations. [ABSTRACT FROM AUTHOR]

The article presents a study which examines the implication of the crystal structures of iron (Fe)-hydrogenase for the activation site of H2. The study involves several methods including the purification of Fe-hydrogenase from Methanothermobacter marburgensis, crystallization of protein-isocyanide complexes, and X-ray diffraction. The study reveals that the mode of isocyanide-binding to Fe-dehydrogenase shows that H2 is bound to the iron site trans to the acyl ligand.

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

Abstract: [Fe]-hydrogenase is one of three types of enzymes known to activate H2. Crystal structure analysis recently revealed that its active site iron is ligated square-pyramidally by Cys176-sulfur, two CO, an “unknown” ligand and the sp2-hybridized nitrogen of a unique iron–guanylylpyridinol-cofactor. We report here on the structure of the C176A mutated enzyme crystallized in the presence of dithiothreitol (DTT). It suggests an iron center octahedrally coordinated by one DTT-sulfur and one DTT-oxygen, two CO, the 2-pyridinol’s nitrogen and the 2-pyridinol’s 6-formylmethyl group in an acyl-iron ligation. This result led to a re-interpretation of the iron ligation in the wild-type. [Copyright &y& Elsevier]

[Display omitted] • RosC is the first roseoflavin specific phosphatase whose structure has been solved. • The reaction mechanism of AFP dephosphorylation is now clear. • RosC has evolved to contain specially designed strand-to-helix sections and a unique N-terminal helical extension to carry out its functions. • Distinction between AFP and FMN is supported by the aspartate residue D166. • The crystal structure of RosC gives an idea how cell-damaging dephosphorylation of FMN may be avoided. 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. [ABSTRACT FROM AUTHOR]

Highlights • The mode of Mo storage protein preparation affects the polyoxomolybdate (POMo) composition. • The POMo cluster sets were identified by several high-resolution X-ray structures. • Neighbored POMo clusters mutually influence their type and positions. • A site-specifically modified cage surface can create new POMo cluster binding sites. • POMo cluster building blocks can fuse to larger aggregates up to 14 and 18 MoO x units. [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]

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]

Methenyltetrahydromethanopterin (methenyl- H4MPT+) cydohydrolase (Mch) catalyzes the interconversion of methenyl-H4MPT+ and formyl-H4MPT in the one-carbon energy metabolism of methanogenic, methanotrophic, and sulfate-reducing archaea and of methylotrophic bacteria. To understand the catalytic mechanism of this reaction, we kinetically characterized site-specific variants of Mch from Arrhaeoglobus fulgidus (aMch) and determined the X-ray structures of the substrate-free aMch(E186Q), the aMch,H4MPT complex, and the aMch(E186Q):formyl-H4MPT complex. (Formyl-)H4MPT is embedded inside a largely preformed, interdomain pocket of the homotrimeric enzyme with the pterin and benzyl rings being oriented nearly perpendicular to each other. The active site is primarily built up by the segment 93:95, Arg183 and Glu 186 that either interact with the catalytic water attacking methenyl-H4MPT+ or with the formyl oxygen of forinyl-t 4MPT. The catalytic function of the strictly conserved Arg183 and G1ul86 was substantiated by the low enzymatic activities of the E186A, E 186D, E 186N, E186Q R183A, R183Q R1S3E, R183K, and R183E-E186Q variants. G1u186 most likely acts as a general base. Axg183 decisively influences the pKa value of Glu186 and the proposed catalytic water mainly by its positive charge In addition, G1u186 appears to be also responsible for product specificity by donating a proton to the directly neighbored N'° tertiary amine of H4MPT. Thus, N10 becomes a better leaving group than N5 which implies the generation of N5-formyl-H4MPT. For comparison, methenyltetrahydrofolate (H4F) cyclohydrolase produces N5-formyl-H4F in an analogous reaction. An enzymatic mechanism of Mch is postulated and compared with that of other cyclohydrolases. [ABSTRACT FROM AUTHOR]