Carboxymethylproline synthase (CarB), an unusual carbon-carbon bond-forming enzyme of the crotonase superfamily involved in carbapenem biosynthesis.
Carboxymethylproline synthase (CarB) catalyzes the committed step in the biosynthesis of (R)-1-carbapen-2-em-3-carboxylate, the simplest member of the carbapenem family of beta-lactam antibiotics, some of which are used clinically. CarB displays sequence homology with members of the crotonase family including enoyl-CoA hydratase (crotonase) and methylmalonyl-CoA decarboxylase. The CarB reaction has been proposed to comprise condensation of acetyl coenzyme A (AcCoA) and glutamate semi-aldehyde to give (2S,5S)-carboxymethylproline ((2S,5S)-CMP). (2S,5S)-CMP is then cyclized in an ATP-driven reaction catalyzed by CarA to give a carbapenam, which is subsequently epimerized and desaturated to give a carbapenem in a CarC-mediated reaction. Here we report the purification of recombinant CarB and that it exists predominantly in a trimeric form as do other members of the crotonase family. AcCoA was not found to be a substrate for CarB. Instead malonyl-CoA was found to be a substrate, efficiently producing (2S,5S)-CMP in the presence of glutamate semi-aldehyde. In the absence of glutamate semi-aldehyde, mass spectrometric analysis indicated that CarB catalyzed the decarboxylation of malonyl-CoA to AcCoA. The reactions of CarB, CarA, and CarC were coupled in vitro demonstrating the viability of malonyl-CoA as a carbapenem precursor. CarB was also shown to accept methylmalonyl CoA as a substrate to form 6-methyl-(2S,5S)CMP, which in turn is a substrate for CarA. The implications of the results for the biosynthesis of both carbapenem-3-carboxylate and C-2/C-6-substituted carbapenems, such as thienamycin, are discussed.
Sleeman
Mark C
MC
Department of Chemistry, University of Oxford, Chemistry Research Laboratory, Mansfield Road, Oxford OX1 3TA, UK.
Schofield
Christopher J
CJ
D000105:Acetyl Coenzyme A / Q000737:chemistry; D000595:Amino Acid Sequence; D015780:Carbapenems / Q000737:chemistry*; D002244:Carbon / Q000737:chemistry*; D019755:Carbon-Carbon Lyases / Q000737:chemistry* / Q000378:metabolism; D002850:Chromatography, Gel; D002942:Circular Dichroism; D003001:Cloning, Molecular; D004228:Dithionitrobenzoic Acid / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel; D004746:Enoyl-CoA Hydratase / Q000737:chemistry*; D005971:Glutamates / Q000737:chemistry; D018698:Glutamic Acid / Q000737:chemistry; D007700:Kinetics; D007769:Lactams / Q000737:chemistry; D009682:Magnetic Resonance Spectroscopy; D008316:Malonyl Coenzyme A / Q000737:chemistry; D013058:Mass Spectrometry; D008956:Models, Chemical; D008969:Molecular Sequence Data; D044043:Pectobacterium / Q000201:enzymology; D011485:Protein Binding; D011994:Recombinant Proteins / Q000737:chemistry; D017386:Sequence Homology, Amino Acid; D013237:Stereoisomerism; D013845:Thienamycins / Q000737:chemistry / Q000494:pharmacology
D015780:Carbapenems; D005971:Glutamates; D007769:Lactams; D011994:Recombinant Proteins; D013845:Thienamycins; C093704:1-carbapen-2-em-3-carboxylic acid; D018698:Glutamic Acid; D008316:Malonyl Coenzyme A; C057794:glutamate-1-semialdehyde; D000105:Acetyl Coenzyme A; D002244:Carbon; D004228:Dithionitrobenzoic Acid; D019755:Carbon-Carbon Lyases; C484323:carboxymethylproline synthase; D004746:Enoyl-CoA Hydratase; C015537:thienamycin