MioC Is an FMN-binding Protein That Is Essential forEscherichia coli Biotin Synthase Activity in Vitro

Olwen M Birch,Kirsty S Hewitson,Martin Fuhrmann,Knut Burgdorf,Jack E Baldwin,Peter L Roach,Nicholas M Shaw

doi:10.1074/jbc.m004497200

Abstract

Biotin synthase is required for the conversion of dethiobiotin to biotin and requires a number of accessory proteins and small molecule cofactors for activity in vitro. We have previously identified two of these proteins as flavodoxin and ferredoxin (flavodoxin) NADP(+) reductase. We now report the identification of MioC as a third essential protein, together with its cloning, purification, and characterization. Purified MioC has a UV-visible spectrum characteristic of a flavoprotein and contains flavin mononucleotide. The presence of flavin mononucleotide and the primary sequence similarity to flavodoxin suggest that MioC may function as an electron transport protein. The role of MioC in the biotin synthase reaction is discussed, and the structure and function of MioC is compared with that of flavodoxin.

Highlights

Biotin synthase is required for the conversion of dethiobiotin to biotin and requires a number of accessory proteins and small molecule cofactors for activity in vitro
Evidence That the MioC Protein Is Required for Biotin Synthase Activity in Vitro—We have shown previously that a number of low molecular weight compounds plus at least flavodoxin, ferredoxin NADPϩ reductase, and a third protein are essential for biotin synthase activity in vitro [6, 7]
When partially purified biotin synthase, flavodoxin, and ferredoxin NADPϩ reductase fractions prepared by ion exchange chromatography on a Q Sepharose Fast Flow column plus the essential low molecular weight compounds were incubated with dethiobiotin, no biotin was formed

Summary

Introduction

Biotin synthase is required for the conversion of dethiobiotin to biotin and requires a number of accessory proteins and small molecule cofactors for activity in vitro. Two have been identified as flavodoxin and ferredoxin (flavodoxin) NADPϩ reductase [6, 7, 9] These proteins are thought to provide an electron transfer pathway from NADPH to the Fe-S cluster of biotin synthase. A third protein required for biotin synthase activity in vitro has previously been reported [6, 7] but not identified. This protein eluted from a Q-Sepharose column at a NaCl concentration of ϳ0.5 M and required TPP to stabilize its activity during purification. In this paper we report the cloning, overexpression, and characterization of MioC

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Results

Conclusion