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. 2005 Oct 14;280(41):34956-65. doi: 10.1074/jbc.M507196200. Epub 2005 Aug 11.

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • 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

Substances

  • Aldehydes
  • Anhydrides
  • Carbapenems
  • Esters
  • Glutamates
  • Glutamic Acid
  • Histidine
  • glutamate-1-semialdehyde
  • Selenomethionine
  • Proline
  • Thiolester Hydrolases
  • 3-hydroxyisobutyryl-CoA hydrolase
  • Trypsin
  • Carbon-Carbon Lyases
  • carboxymethylproline synthase
  • Enoyl-CoA Hydratase

Associated data

  • PDB/2A7K
  • PDB/2A81