Ethylmalonyl-CoA Mutase from Rhodobacter sphaeroides Defines a New Subclade of Coenzyme B12-dependent Acyl-CoA Mutases

Janos Rétey,Tobias J. Erb,Birgit E. Alber,Georg Fuchs

doi:10.1074/jbc.m805527200

Janos Rétey, Tobias J. Erb + Show 2 more

Open Access

https://doi.org/10.1074/jbc.m805527200

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Abstract

Coenzyme B(12)-dependent mutases are radical enzymes that catalyze reversible carbon skeleton rearrangement reactions. Here we describe Rhodobacter sphaeroides ethylmalonyl-CoA mutase (Ecm), a novel member of the family of coenzyme B(12)-dependent acyl-CoA mutases, that operates in the recently discovered ethylmalonyl-CoA pathway for acetate assimilation. Ecm is involved in the central reaction sequence of this novel pathway and catalyzes the transformation of ethylmalonyl-CoA to methylsuccinyl-CoA in combination with a second enzyme that was further identified as promiscuous ethylmalonyl-CoA/methylmalonyl-CoA epimerase. In contrast to the epimerase, Ecm is highly specific for its substrate, ethylmalonyl-CoA, and accepts methylmalonyl-CoA only at 0.2% relative activity. Sequence analysis revealed that Ecm is distinct from (2R)-methylmalonyl-CoA mutase as well as isobutyryl-CoA mutase and defines a new subfamily of coenzyme B(12)-dependent acyl-CoA mutases. In combination with molecular modeling, two signature sequences were identified that presumably contribute to the substrate specificity of these enzymes.

Highlights

We proposed the so-called ethylmalonyl-CoA pathway for acetyl-CoA assimilation by Rhodobacter sphaeroides, which lacks isocitrate lyase, the key enzyme of the glyoxylate cycle [3, 4]
We conclude that a coenzyme B12-dependent ethylmalonyl-CoA mutase catalyzed this reaction in cell extracts of R. sphaeroides
Rearrangement of Ethylmalonyl-CoA to MethylsuccinylCoA—The assimilation of C1 and C2 compounds via the recently described ethylmalonyl-CoA pathway depends on the conversion of ethylmalonyl-CoA into methylsuccinylCoA, a central reaction in the unique reaction sequence from crotonyl-CoA to ␤-methylmalyl-CoA (Fig. 1)

Summary

Introduction

We proposed the so-called ethylmalonyl-CoA pathway for acetyl-CoA assimilation by Rhodobacter sphaeroides, which lacks isocitrate lyase, the key enzyme of the glyoxylate cycle [3, 4]. Crotonyl-CoA is further transformed in a unique reaction sequence to ␤-methylmalylCoA that is cleaved in the latter part of the pathway to glyoxylate and propionyl-CoA Glyoxylate condenses with another molecule of acetyl-CoA to form L-malyl-CoA that is in turn hydrolyzed by a so far unknown thioesterase to malate. Propionyl-CoA is assimilated by carboxylation followed by a carbon skeleton rearrangement step catalyzed by coenzyme B12-dependent (2R)-methylmalonyl-CoA mutase yielding succinylCoA. This novel assimilation pathway is limited to R. sphaeroides but seems to operate in a number of other bacteria that have been reported to lack a functional glyoxylate cycle, like Methylobacterium extorquens and Streptomyces coelicolor [3, 4, 6, 7]. Ethylmalonyl-CoA Mutase from R. sphaeroides cinnamonensis revealed interesting structural and functional aspects with implications for the substrate specificities of these coenzyme B12dependent acyl-CoA mutases

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