Glyoxylate regeneration pathway in the methylotroph Methylobacterium extorquens AM1

Abstract

Most serine cycle methylotrophic bacteria lack isocitrate lyase and convert acetyl coenzyme A (acetyl-CoA) to glyoxylate via a novel pathway thought to involve butyryl-CoA and propionyl-CoA as intermediates. In this study we have used a genome analysis approach followed by mutation to test a number of genes for involvement in this novel pathway. We show that methylmalonyl-CoA mutase, an R-specific crotonase, isobutyryl-CoA dehydrogenase, and a GTPase are involved in glyoxylate regeneration. We also monitored the fate of (14)C-labeled carbon originating from acetate, butyrate, or bicarbonate in mutants defective in glyoxylate regeneration and identified new potential intermediates in the pathway: ethylmalonyl-CoA, methylsuccinyl-CoA, isobutyryl-CoA, methacrylyl-CoA, and beta-hydroxyisobutyryl-CoA. A new scheme for the pathway is proposed based on these data.

FIG. 1.

Pathway of valine degradation in P. aeruginosa (34, 37). Enzymes: 1, IBD; 2, (S)-specific enoyl-CoA hydratase; 3, β-hydroxyisobutyryl-CoA hydrolase; 4, HID; 5, methylmalonic semialdehyde dehydrogenase.

FIG. 2.

Autoradiograph of labeled intermediates separated by TLC, which have accumulated after exposure of whole cells of an meaA mutant to [¹⁴C]butyrate and methanol during 10, 30, 60, 90, 240, and 420 s. Intermediates: 1, methylsuccinate; 2, unknown compound; 3, β-hydroxyisobutyrate; 4, β-hydroxybutyrate.

FIG. 3.

Pathways of glyoxylate regeneration in M. extorquens AM1. Enzymes: 1, β-ketothiolase; 2, NADPH-linked acetoacetyl-CoA reductase; 3, (R)-specific enoyl-CoA hydratase; 4, crotonyl-CoA reductase; 5, butyryl-CoA carboxylase; 6, IBD; 7, (S)-specific enoyl-CoA hydratase; 8, PCC; 9, methylmalonyl-CoA epimerase; 10, MCM. Dotted lines reflect enzymes not yet known or detected.

FIG. 4.

Production of ¹⁴CO₂ from 1-¹⁴C-labeled butyrate in wild-type and mutant M. extorquens AM1, as a percentage of total incorporation.

FIG. 5.

Schematic diagram showing intermediates of the proposed route for conversion of acetyl-CoA to glyoxylate in M. extorquens AM1. Colors trace carbons from the acetyl moiety of acetyl-CoA. The CO₂ fixed early in the pathway (red carbon) is released, while the second CO₂ released is derived from the C-1 carbon of butyryl-CoA (green carbon). Dotted lines reflect enzymes not yet known or detected.