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

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.The citric acid cycle has a dual role in central carbon metabolism. On the one hand, it is the terminal respiratory pathway and is used for the complete oxidation of acetyl-CoA to CO 2 . On the other hand, the citric acid cycle is the source of carbon precursors for the synthesis of cell constituents. However, net synthesis of carbon precursors via the citric acid cycle is not possible. Intermediates that are drained from the cycle must be replenished using anaplerotic reaction sequences (1). Therefore, growth on substrates that enter the central carbon metabolism on the level of acetyl-CoA, such as fatty acids, alcohols, and esters but also waxes, alkenes, or C 1 compounds, requires an additional pathway for the assimilation of acetyl-CoA. The glyoxylate cycle that is used for the net conversion of two acetylCoA molecules to malate provides a solution for this problem and has been the only known route for 50 years (2).Recently 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). The new pathway converts three molecules of acetyl-CoA, one molecule of CO 2 , and one molecule of bicarbonate to the citric acid cycle intermediates malate and succinyl-CoA (see Fig. 1). Initially two molecules of acetyl-CoA are converted into crotonyl-CoA involving steps that are common to polyhydroxybutyrate synthesis. 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 B 12 -dependent (2R)-methylmalonyl-CoA mutase yielding suc...

Carboxylation mechanism and stereochemistry of crotonyl-CoA carboxylase/reductase, a carboxylating enoyl-thioester reductase

“…Recombinant Ccr was produced in 200-L scale and purified from cell extracts (15 mL, 1.6 g of protein) as described (8). Epi and Mcm were prepared as described previously (50). A histidine-tagged version of Ccr(Ccr his) was produced as described in detail in SI Methods.…”

“…Butyryl-CoA dehydrogenase was prepared from pig liver as described (51,52) with minor modifications (see SI Methods). Crotonyl-CoA and acryloyl-CoA were synthesized as reported before (50 Pollock and Barber (53) with some modifications (see SI Methods for details). The purified products were characterized by NMR (Fig.…”

Carboxylases in Natural and Synthetic Microbial Pathways

“…This reaction is catalyzed by crotonyl-CoA carboxylase/reductase (CCR), which represents a novel class of biotin-independent reductive acyl-CoA carboxylases that use NADPH as a reductant (42). In the ensuing process, ethylmalonyl-CoA is cleaved into the C 3 unit propionyl-CoA and a C 2 intermediate (glyoxylate), which are subsequently transformed into two C 4 building blocks (succinyl-CoA and malate, respectively) (43)(44)(45)136). Transformation of propionyl-CoA into succinyl-CoA involves biotin-dependent propionyl-CoA carboxylase, which catalyzes the second essential carboxylation reaction of the ethylmalonylCoA pathway.…”

Study of the distribution of autotrophic CO2 fixation cycles in Crenarchaeota

“…14 C-labelled products of the propionyl-CoA carboxylase reaction were analysed by TLC with CH 3 Cl : acetic acid (5 : 1, v/v) and detected by phosphorimaging (Erb et al, 2008).…”