Identification, Characterization, and Structure/Function Analysis of a Corrin Reductase Involved in Adenosylcobalamin Biosynthesis

Andrew D Lawrence,Evelyne Deery,Kirsty J Mclean,Andrew W Munro,Richard W Pickersgill,Stephen E.J Rigby,Martin J Warren

doi:10.1074/jbc.m710431200

Andrew D Lawrence, Evelyne Deery + Show 5 more

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https://doi.org/10.1074/jbc.m710431200

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Abstract

Vitamin B(12), the antipernicious anemia factor, is the cyano derivative of adenosylcobalamin, which is one of nature's most complex coenzymes. Adenosylcobalamin is made along one of two similar yet distinct metabolic pathways, which are referred to as the aerobic and anaerobic routes. The aerobic pathway for cobalamin biosynthesis proceeds via cobalt insertion into a ring-contracted macrocycle, which is closely followed by adenosylation of the cobalt ion. An important prerequisite for adenosylation is the reduction of the centrally chelated metal from Co(II) to a highly nucleophilic Co(I) form. We have cloned a gene, cobR, encoding a biosynthetic enzyme with this co(II)rrin reductase activity from Brucella melitensis. The protein has been overproduced, and the resulting flavoprotein has been purified, characterized, and crystallized and its structure determined to 1.6A resolution. Kinetic and EPR analysis reveals that the enzyme proceeds via a semiquinone form. It is proposed that CobR may interact with the adenosyltransferase to overcome the large thermodynamic barrier required for co(II)rrin reduction.

Highlights

Cobalamin is a coenzyme that is associated with a range of isomerization, methylation, and dehalogenation reactions, utilizing the unique chemistry of the carbon-cobalt bond that is characteristic of this molecule [1]
Central to the role played in catalysis by cobalamin-containing enzymes is the cobalt ion, which forms a unique cobaltcarbon bond with either an upper adenosyl or methyl group
This led to the identification of SMc00514 in S. meliloti, which was annotated as being a putative monooxygenase. This sequence was used in further BLAST searches that produced a list of analogous proteins from a wide range of organisms. From this list a gene encoding an orthologue from B. melitensis (BMEI0709) was identified within a cobalamin biosynthetic operon

Summary

Introduction

Cobalamin (vitamin B12) is a coenzyme that is associated with a range of isomerization, methylation, and dehalogenation reactions, utilizing the unique chemistry of the carbon-cobalt bond that is characteristic of this molecule [1]. This gene, which has been termed cobR, encodes a protein consisting of 173 amino acids with a predicted molecular mass of 18.7 kDa and has primary sequence similarity to a number of flavoproteins, including the smaller reductive component of the 4-hydroxyphenylacetate 3-monooxygenase (either HpaC from E. coli or Phe(A2) from Geobacillus thermoglucosidasius A7) (supplemental Fig. S1) [18, 19].

Results

Conclusion