2A7K

carboxymethylproline synthase (CarB) from pectobacterium carotovora, apo enzyme

Summary for 2A7K

• Entry DOI: 10.2210/pdb2a7k/pdb
• Descriptor: CarB (2 entities in total)
• Functional Keywords: carbapenem, carbapenam, crotonase, antibiotic, beta-lactam, biosynthetic protein
• Biological source: Pectobacterium carotovorum
• Total number of polymer chains: 9
• Total formula weight: 248474.48

Authors

Sleeman, M.C.,Sorensen, J.L.,Batchelar, E.T.,McDonough, M.A.,Schofield, C.J. (deposition date: 2005-07-05, release date: 2005-08-23, Last modification date: 2024-02-14)

Primary citation

Sleeman, M.C.,Sorensen, J.L.,Batchelar, E.T.,McDonough, M.A.,Schofield, C.J.
Structural and Mechanistic Studies on Carboxymethylproline Synthase (CarB), a Unique Member of the Crotonase Superfamily Catalyzing the First Step in Carbapenem Biosynthesis.
J.Biol.Chem., 280:34956-34965, 2005

PubMed Abstract: 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.

PubMed: 16096274
DOI: 10.1074/jbc.M507196200

Experimental method

X-RAY DIFFRACTION (2.24 Å)