Characterization of the heterodimeric MegBIIa:MegBIIb aldo-keto reductase involved in the biosynthesis of L-mycarose from Micromonospora megalomicea.
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.
Peirú
Salvador
S
Microbiology Division, Instituto de Biología Molecular y Celular de Rosario, Consejo Nacional de Investigaciones Científicas y Técnicas, Facultad de Ciencias, Bioquímicas y Farmacéuticas, Universidad Nacional de Rosario, Argentina.
Rodríguez
Eduardo
E
Tran
Chau Q
CQ
Carney
John R
JR
Gramajo
Hugo
H
D000429:Alcohol Oxidoreductases / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D000449:Aldehyde Reductase; D000074408:Aldo-Keto Reductases; D000595:Amino Acid Sequence; D001483:Base Sequence; D002850:Chromatography, Gel; D017931:DNA Primers; D019281:Dimerization; D006601:Hexoses / Q000096:biosynthesis*; D008847:Micromonospora / Q000378:metabolism*; D008969:Molecular Sequence Data; D017386:Sequence Homology, Amino Acid; D053719:Tandem Mass Spectrometry
D017931:DNA Primers; D006601:Hexoses; C045835:mycarose; D000429:Alcohol Oxidoreductases; D000074408:Aldo-Keto Reductases; D000449:Aldehyde Reductase