Structural and mechanistic studies on carboxymethylproline synthase (CarB), a unique member of the crotonase superfamily catalyzing the first step in carbapenem biosynthesis.
The first step in the biosynthesis of the medicinally important carbapenem family of beta-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 A and 3.15 A 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 His(229)) 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 Glu(131) 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 Glu(131), 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.
Sleeman
Mark C
MC
Department of Chemistry, Chemistry Research Laboratory, University of Oxford, Mansfield Road, Oxford OX1 3TA, United Kingdom.
Sorensen
John L
JL
Batchelar
Edward T
ET
McDonough
Michael A
MA
Schofield
Christopher J
CJ
D000447:Aldehydes / Q000737:chemistry; D000812:Anhydrides / Q000737:chemistry; D001665:Binding Sites; D015780:Carbapenems / Q000096:biosynthesis*; D019755:Carbon-Carbon Lyases / Q000737:chemistry* / Q000502:physiology*; D002384:Catalysis; D002853:Chromatography, Liquid; D018360:Crystallography, X-Ray; D004746:Enoyl-CoA Hydratase / Q000737:chemistry*; D004926:Escherichia coli / Q000378:metabolism; D004952:Esters / Q000737:chemistry; D005971:Glutamates / Q000737:chemistry; D018698:Glutamic Acid / Q000737:chemistry; D006639:Histidine / Q000737:chemistry; D006860:Hydrogen Bonding; D013058:Mass Spectrometry; D008956:Models, Chemical; D008958:Models, Molecular; D016296:Mutagenesis; D011392:Proline / Q000737:chemistry; D011485:Protein Binding; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D012645:Selenomethionine / Q000737:chemistry; D013379:Substrate Specificity; D013869:Thiolester Hydrolases / Q000737:chemistry; D014357:Trypsin / Q000494:pharmacology
D000447:Aldehydes; D000812:Anhydrides; D015780:Carbapenems; D004952:Esters; D005971:Glutamates; D018698:Glutamic Acid; D006639:Histidine; C057794:glutamate-1-semialdehyde; D012645:Selenomethionine; D011392:Proline; D013869:Thiolester Hydrolases; C035908:3-hydroxyisobutyryl-CoA hydrolase; D014357:Trypsin; D019755:Carbon-Carbon Lyases; C484323:carboxymethylproline synthase; D004746:Enoyl-CoA Hydratase