Characterization of the heterodimeric MegBIIa:MegBIIb aldo-keto reductase involved in the biosynthesis of L-mycarose from Micromonospora megalomicea

S. PEIRÚ, E. RODRÍGUEZ, C.Q. TRAN, J.E. CARNNEY AND H. GRAMAJO

BIOCHEMISTRY

Año: 2007 vol. 46 p. 4100 - 4109

0006-2960

Two putative C3-ketoreductases, MegBIIa and MegBIIb (formerly MegBII and MegDVII), homologues to members of the family 12 of aldo-keto reductases (AKR12) superfamily of enzymes, were identified in the megalomicin gene cluster from Micromonospora megalomicea. Proteins from this family are involved in the TDP-sugar metabolism from 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, were fully active enzymes by themselves.  Surprisingly, C3-ketoreductase activity was observed only in the presence of both MegBIIa and MegBIIb, suggesting the formation of an active complex. Co-purification 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.