RESUMO
Thiamine pyrophosphate (TPP)-dependent oxalate oxidoreductase (OOR) metabolizes oxalate, generating two molecules of CO2 and two low-potential electrons, thus providing both the carbon and reducing equivalents for operation of the Wood-Ljungdahl pathway of acetogenesis. Here we present structures of OOR in which two different reaction intermediate bound states have been trapped: the covalent adducts between TPP and oxalate and between TPP and CO2. These structures, along with the previously determined structure of substrate-free OOR, allow us to visualize how active site rearrangements can drive catalysis. Our results suggest that OOR operates via a bait-and-switch mechanism, attracting substrate into the active site through the presence of positively charged and polar residues, and then altering the electrostatic environment through loop and side chain movements to drive catalysis. This simple but elegant mechanism explains how oxalate, a molecule that humans and most animals cannot break down, can be used for growth by acetogenic bacteria.
Assuntos
Carbono/metabolismo , Moorella/enzimologia , Oxirredutases/metabolismo , Domínio Catalítico , Cristalografia por Raios X , Modelos Moleculares , Oxalatos/metabolismo , Oxirredução , Oxirredutases/química , Estrutura Terciária de Proteína , SolventesRESUMO
Microbes use siderophores to access essential iron resources in the environment. Over 500 siderophores are known, but they utilize a small set of common moieties to bind iron. Azotobacter chroococcum expresses iron-rich nitrogenases, with which it reduces N2 . Though an important agricultural inoculant, the structures of its iron-binding molecules remain unknown. Here, the "chelome" of A. chroococcum is examined using small molecule discovery and bioinformatics. The bacterium produces vibrioferrin and amphibactins as well as a novel family of siderophores, the crochelins. Detailed characterization shows that the most abundant member, crochelinâ A, binds iron in a hexadentate fashion using a new iron-chelating γ-amino acid. Insights into the biosynthesis of crochelins and the mechanism by which iron may be removed upon import of the holo-siderophore are presented. This work expands the repertoire of iron-chelating moieties in microbial siderophores.
Assuntos
Azotobacter/metabolismo , Quelantes de Ferro/química , Fixação de Nitrogênio , Sideróforos/química , Cromatografia Líquida de Alta Pressão , Espectrometria de Massas/métodos , Estrutura MolecularRESUMO
Thiamine pyrophosphate (TPP), a derivative of vitamin B1, is a versatile and ubiquitous cofactor. When coupled with [4Fe-4S] clusters in microbial 2-oxoacid:ferredoxin oxidoreductases (OFORs), TPP is involved in catalyzing low-potential redox reactions that are important for the synthesis of key metabolites and the reduction of N2, H(+), and CO2. We have determined the high-resolution (2.27 Å) crystal structure of the TPP-dependent oxalate oxidoreductase (OOR), an enzyme that allows microbes to grow on oxalate, a widely occurring dicarboxylic acid that is found in soil and freshwater and is responsible for kidney stone disease in humans. OOR catalyzes the anaerobic oxidation of oxalate, harvesting the low-potential electrons for use in anaerobic reduction and fixation of CO2. We compare the OOR structure to that of the only other structurally characterized OFOR family member, pyruvate:ferredoxin oxidoreductase. This side-by-side structural analysis highlights the key similarities and differences that are relevant for the chemistry of this entire class of TPP-utilizing enzymes.
Assuntos
Moorella/enzimologia , Moorella/metabolismo , Oxalatos/metabolismo , Oxirredutases/química , Oxirredutases/metabolismo , Modelos Moleculares , Moorella/química , Conformação Proteica , Tiamina Pirofosfato/metabolismoRESUMO
The title complex, [Pt(2)(CH(3))(4)(C(19)H(15)N(2)Si)(2)I][B(C(6)F(5))(4)]·0.66CH(2)Cl(2), resulted from an attempt to synthesize a stable five-coordinate platinum species via ligand abstraction of a six-coordinate platinum precursor. However, dimerization occurred after ligand abstraction, thereby yielding the compound described in this study. The cation is a dinuclear Pt(IV) organometallic complex, in which the metal centers are bridged by an I(-) anion. Both metal centers display a coordination geometry close to octa-hedral, including cis-arranged quinoline ligands connected by Si atoms, which form Pt-Si bonds, two cis-methyl groups, and the bridging I(-) anion. In the crystal structure, voids between cations and anions are partially filled with an average of 0.66â mol-ecules of dichloro-methane solvent.
RESUMO
Glycopeptide antibiotics (GPAs) have served as potent clinical drugs and as an inspiration to chemists in various disciplines. Among known GPAs, complestatin, chloropeptin, and kistamicin are unique in that they contain an unusual indole-phenol crosslink. The mechanism of formation of this linkage is unknown, and to date, the biosynthetic gene cluster of only one GPA with an indole-phenol crosslink, that of complestatin, has been identified. Here, we report the genome sequence of the kistamicin producer Nonomuraea sp. ATCC 55076. We find that this strain harbours the largest actinobacterial chromosome to date, consisting of a single linear chromosome of â¼13.1 Mbp. AntiSMASH analysis shows that â¼32 biosynthetic gene clusters and â¼10% of the genome are devoted to production of secondary metabolites, which include 1,6-dihydroxyphenazine and nomuricin, a new anthraquinone-type pentacyclic compound that we report herein. The kistamicin gene cluster (kis) was identified bioinformatically. A unique feature of kis is that it contains two cytochrome P450 enzymes, which likely catalyze three crosslinking reactions. These findings set the stage for examining the biosynthesis of kistamicin and its unusual indole-phenol crosslink in the future.
RESUMO
Studies on the biosynthesis of glycopeptide antibiotics have provided many insights into the strategies that Nature employs to build architecturally strained molecules. A key structural feature of vancomycin, the founding member of this class, is a set of three aromatic cross-links that are introduced via yet unknown mechanisms. Previous reports have identified three cytochrome P450 enzymes involved in this process and demonstrated enzymatic activity for OxyB, which installs the first aromatic cross-link. However, the activities of the remaining two P450 enzymes have not been recapitulated. Herein, we show that OxyA generates the second bis-aryl ether bond in vancomycin and that it exhibits strict substrate specificity toward the chlorinated, OxyB-cross-linked product. No OxyA product is detected with the unchlorinated substrate. Together with previous results, these data suggest that chlorination occurs after OxyB- but before OxyA-catalyzed cross-link formation. Our results have important implications for the chemo-enzymatic synthesis of vancomycin and its analogs.
Assuntos
Actinomycetales/enzimologia , Antibacterianos/metabolismo , Vias Biossintéticas , Sistema Enzimático do Citocromo P-450/metabolismo , Vancomicina/metabolismo , Actinomycetales/química , Actinomycetales/metabolismo , Antibacterianos/química , Halogenação , Especificidade por Substrato , Vancomicina/químicaRESUMO
2-Oxoacid:ferredoxin oxidoreductases (OFORs) are essential enzymes in microbial one-carbon metabolism. They use thiamine pyrophosphate to reversibly cleave carbon-carbon bonds, generating low potential (â¼-500mV) electrons. Crystallographic analysis of a recently discovered OFOR, an oxalate oxidoreductase (OOR), has provided a second view of OFOR architecture and active site composition. Using these recent structural data along with the previously determined structures of pyruvate:ferredoxin oxidoreductase, structure-function relationships in this superfamily have been expanded and re-evaluated. Additionally, structural motifs have been defined that better serve to distinguish one OFOR subfamily from another and potentially uncover novel OFORs.