RESUMO
Gentamicins are heavily methylated, clinically valuable pseudotrisaccharide antibiotics produced by Micromonospora echinospora. GenN has been characterized as an S-adenosyl-l-methionine-dependent methyltransferase with low sequence similarity to other enzymes. It is responsible for the 3â³-N-methylation of 3â³-dehydro-3â³-amino-gentamicin A2, an essential modification of ring III in the biosynthetic pathway to the gentamicin C complex. Purified recombinant GenN also efficiently catalyzes 3â³-N-methylation of related aminoglycosides kanamycin B and tobramycin, which both contain an additional hydroxymethyl group at the C5â³ position in ring III. We have obtained eight cocrystal structures of GenN, at a resolution of 2.2 Å or better, including the binary complex of GenN and S-adenosyl-l-homocysteine (SAH) and the ternary complexes of GenN, SAH, and several aminoglycosides. The GenN structure reveals several features not observed in any other N-methyltransferase that fit it for its role in gentamicin biosynthesis. These include a novel N-terminal domain that might be involved in protein:protein interaction with upstream enzymes of the gentamicin X2 biosynthesis and two long loops that are involved in aminoglycoside substrate recognition. In addition, the analysis of structures of GenN in complex with different ligands, supported by the results of active site mutagenesis, has allowed us to propose a catalytic mechanism and has revealed the structural basis for the surprising ability of native GenN to act on these alternative substrates.
Assuntos
Aminoglicosídeos/metabolismo , Antibacterianos/metabolismo , Proteínas de Bactérias/metabolismo , Gentamicinas/metabolismo , Metiltransferases/metabolismo , Micromonospora/enzimologia , Proteínas de Bactérias/química , Cristalografia por Raios X , Canamicina/análogos & derivados , Canamicina/metabolismo , Metiltransferases/química , Micromonospora/química , Micromonospora/metabolismo , Modelos Moleculares , Conformação Proteica , Especificidade por Substrato , Tobramicina/metabolismoRESUMO
Two putative C3-ketoreductases, MegBIIa and MegBIIb (formerly MegBII and MegDVII, respectively), homologues to members of the family 12 of aldo-keto reductase (AKR12) superfamily of enzymes, were identified in the megalomicin gene cluster from Micromonospora megalomicea. Proteins from this family are involved in the metabolism of TDP-sugars by actinomycetes. MegBIIa was originally proposed to be involved in the l-mycarose biosynthetic pathway, while MegBIIb in the l-megosamine biosynthetic pathway. In this work we have investigated the role of these proteins in the biosynthesis of dTDP-l-mycarose. In vivo analysis of the dTDP-sugar intermediates indicated that neither MegBIIa nor its homologue, MegBIIb, was a fully active enzyme by itself. Surprisingly, C3-ketoreductase activity was observed only in the presence of both MegBIIa and MegBIIb, suggesting the formation of an active complex. Copurification and size exclusion chromatography experiments confirmed that MegBIIa and MegBIIb interact forming a 1:1 heterodimeric complex. Finally, a mycarose operon containing megBIIa and megBIIb together with the other biosynthetic genes of the l-mycarose pathway was constructed and tested by bioconversion experiments in Escherichia coli. High levels of mycarosyl-erythronolide B were produced under the condition tested, confirming the role of these two proteins in this metabolic pathway.
Assuntos
Oxirredutases do Álcool/metabolismo , Hexoses/biossíntese , Micromonospora/metabolismo , Oxirredutases do Álcool/química , Oxirredutases do Álcool/isolamento & purificação , Aldeído Redutase , Aldo-Ceto Redutases , Sequência de Aminoácidos , Sequência de Bases , Cromatografia em Gel , Primers do DNA , Dimerização , Dados de Sequência Molecular , Homologia de Sequência de Aminoácidos , Espectrometria de Massas em TandemRESUMO
It was possible to isolate several strains of Micromonospora sp. from waters of Rio Reconquista. They all were filamentous bacteria with lateral spores, highly resistant to Cr(III) and another heavy metal cations. All these strains were able to grow on naphthalene-2-sulphonate as sole carbon source in a mineral medium. The biodegradation of the xenobiotic proceeds via the formation of salicylate and gentisate. These compounds have been isolated and mass spectrometry identified.