Structural and Mechanistic Studies on Carboxymethylproline Synthase (CarB), a Unique Member of the Crotonase Superfamily Catalyzing the First Step in Carbapenem Biosynthesis

• Published in: The Journal of biological chemistry 2005-10, Vol.280 (41), p.34956-34965
• Main Authors: Sleeman, Mark C., Sorensen, John L., Batchelar, Edward T., McDonough, Michael A., Schofield, Christopher J.
• Format: Article
• Language: English
• Subjects: Aldehydes - chemistry; Anhydrides - chemistry; Binding Sites; Carbapenems - biosynthesis; Carbon-Carbon Lyases - chemistry; Carbon-Carbon Lyases - physiology; Catalysis; Chromatography, Liquid; Crystallography, X-Ray; Enoyl-CoA Hydratase - chemistry; Escherichia coli - metabolism; Esters - chemistry; Glutamates - chemistry; Glutamic Acid - chemistry; Histidine - chemistry; Hydrogen Bonding; Mass Spectrometry; Models, Chemical; Models, Molecular; Mutagenesis; Proline - chemistry; Protein Binding; Protein Conformation; Protein Structure, Secondary; Selenomethionine - chemistry; Substrate Specificity; Thiolester Hydrolases - chemistry; Trypsin - pharmacology
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M507196200

The first step in the biosynthesis of the medicinally important carbapenem family of β-lactam antibiotics is catalyzed by carboxymethylproline synthase (CarB), a unique member of the crotonase superfamily. CarB catalyzes formation of (2S,5S)-carboxymethylproline [(2S,5S)-t-CMP] from malonyl-CoA and l-glutamate semialdehyde. In addition to using a cosubstrate, CarB catalyzes C-C and C-N bond formation processes as well as an acyl-coenzyme A hydrolysis reaction. We describe the crystal structure of CarB in the presence and absence of acetyl-CoA at 2.24 Å and 3.15 Å resolution, respectively. The structures reveal that CarB contains a conserved oxy-anion hole probably required for decarboxylation of malonyl-CoA and stabilization of the resultant enolate. Comparison of the structures reveals that conformational changes (involving His229) in the cavity predicted to bind l-glutamate semialdehyde occur on (co)substrate binding. Mechanisms for the formation of the carboxymethylproline ring are discussed in the light of the structures and the accompanying studies using isotopically labeled substrates; cyclization via 1,4-addition is consistent with the observed labeling results (providing that hydrogen exchange at the C-6 position of carboxymethylproline does not occur). The side chain of Glu131 appears to be positioned to be involved in hydrolysis of the carboxymethylproline-CoA ester intermediate. Labeling experiments ruled out the possibility that hydrolysis proceeds via an anhydride in which water attacks a carbonyl derived from Glu131, as proposed for 3-hydroxyisobutyryl-CoA hydrolase. The structural work will aid in mutagenesis studies directed at altering the selectivity of CarB to provide intermediates for the production of clinically useful carbapenems.