Carboxymethylproline synthase from Pectobacterium carotorova: a multifaceted member of the crotonase superfamily.
The simplest carbapenem antibiotic, (5R)-carbapen-2-em-3-carboxylic acid, is biosynthesized from primary metabolites in Pectobacterium carotorova by the action of three enzymes, carboxymethylproline synthase (hereafter named CarB), carbapenam synthetase, and carbapenem synthase. CarB, a member of the crotonase superfamily, catalyzes the formation of (2S,5S)-5-carboxymethylproline from malonyl-CoA and l-pyrroline-5-carboxylate. In this study we show that, in addition, CarB catalyzes the independent decarboxylation of malonyl-CoA and methylmalonyl-CoA and the hydrolysis of CoA esters such as acetyl-CoA and propionyl-CoA. The steady-state rate constants for these reactions are reported. We have identified the intermediates in the CarB reactions with l-pyrroline-5-carboxylate and malonyl-CoA or methylmalonyl-CoA as the CoA esters of (2S,5S)-5-carboxymethylproline and (2S,5S)-6-methyl-5-carboxymethylproline, respectively. The data provided indicate that these intermediates partition between completing turnover and dissociating from the enzyme. On the basis of the steady-state rate constants measured for the CarB-catalyzed hydrolysis of synthetic (2S,5S)-5-carboxymethylprolyl-CoA and for the CarB reaction with malonyl-CoA and l-pyrroline-5-carboxylate, we have calculated the rate constants for each step of these reactions. The results identify CarB as a particularly interesting member of the crotonase superfamily that combines in one net reaction three activities of this superfamily, decarboxylation, C-C bond formation, and CoA ester hydrolysis.
Gerratana
Barbara
B
Department of Chemistry, The Johns Hopkins University, 3400 North Charles Street, Baltimore, Maryland 21218, USA.
Arnett
Samantha O
SO
Stapon
Anthony
A
Townsend
Craig A
CA
D000214:Acyl Coenzyme A / Q000378:metabolism; D019755:Carbon-Carbon Lyases / Q000145:classification / Q000378:metabolism*; D002384:Catalysis; D003653:Decarboxylation; D004746:Enoyl-CoA Hydratase / Q000145:classification / Q000378:metabolism*; D006868:Hydrolysis; D007700:Kinetics; D011758:Pyrroles / Q000378:metabolism; D013379:Substrate Specificity
D000214:Acyl Coenzyme A; D011758:Pyrroles; C015485:delta-1-pyrroline-5-carboxylate; D019755:Carbon-Carbon Lyases; C484323:carboxymethylproline synthase; D004746:Enoyl-CoA Hydratase