Publications

• Crystal structure of carbapenam synthetase (CarA).

  Miller M.T., Gerratana B., Stapon A., Townsend C.A., Rosenzweig A.C.

  Carbapenam synthetase (CarA) is an ATP/Mg2+-dependent enzyme that catalyzes formation of the beta-lactam ring in (5R)-carbapenem-3-carboxylic acid biosynthesis. CarA is homologous to beta-lactam synthetase (beta-LS), which is involved in clavulanic acid biosynthesis. The catalytic cycles of CarA a ... >> More

  Carbapenam synthetase (CarA) is an ATP/Mg2+-dependent enzyme that catalyzes formation of the beta-lactam ring in (5R)-carbapenem-3-carboxylic acid biosynthesis. CarA is homologous to beta-lactam synthetase (beta-LS), which is involved in clavulanic acid biosynthesis. The catalytic cycles of CarA and beta-LS mediate substrate adenylation followed by beta-lactamization via a tetrahedral intermediate or transition state. Another member of this family of ATP/Mg2+-dependent enzymes, asparagine synthetase (AS-B), catalyzes intermolecular, rather than intramolecular, amide bond formation in asparagine biosynthesis. The crystal structures of apo-CarA and CarA complexed with the substrate (2S,5S)-5-carboxymethylproline (CMPr), ATP analog alpha,beta-methyleneadenosine 5'-triphosphate (AMP-CPP), and a single Mg2+ ion have been determined. CarA forms a tetramer. Each monomer resembles beta-LS and AS-B in overall fold, but key differences are observed. The N-terminal domain lacks the glutaminase active site found in AS-B, and an extended loop region not observed in beta-LS or AS-B is present. Comparison of the C-terminal synthetase active site to that in beta-LS reveals that the ATP binding site is highly conserved. By contrast, variations in the substrate binding pocket reflect the different substrates of the two enzymes. The Mg2+ coordination is also different. Several key residues in the active site are conserved between CarA and beta-LS, supporting proposed roles in beta-lactam formation. These data provide further insight into the structures of this class of enzymes and suggest that CarA might be a versatile target for protein engineering experiments aimed at developing improved production methods and new carbapenem antibiotics. << Less

  J. Biol. Chem. 278:40996-41002(2003) [PubMed] [EuropePMC]

• Inhibition and alternate substrate studies on the mechanism of carbapenam synthetase from Erwinia carotovora.

  Gerratana B., Stapon A., Townsend C.A.

  The Erwinia carotorova carA, carB, and carC gene products are essential for the biosynthesis of (5R)-carbapen-2-em-3-carboxylic acid, the simplest carbapenem beta-lactam antibiotic. CarA (hereafter named carbapenam synthetase) has been proposed to catalyze formation of (3S,5S)-carbapenam-3-carboxy ... >> More

  The Erwinia carotorova carA, carB, and carC gene products are essential for the biosynthesis of (5R)-carbapen-2-em-3-carboxylic acid, the simplest carbapenem beta-lactam antibiotic. CarA (hereafter named carbapenam synthetase) has been proposed to catalyze formation of (3S,5S)-carbapenam-3-carboxylic acid from (2S,5S)-5-carboxymethyl proline based on characterization of the products of fermentation experiments in Escherichia coli cells transformed with pET24a/carB and pET24a/carAB, and on sequence homology to beta-lactam synthetase, an enzyme that catalyzes formation of a monocyclic beta-lactam ring with concomitant ATP hydrolysis. In this study, we have purified recombinant carbapenam synthetase and shown in vitro that it catalyzes the ATP-dependent formation of (3S,5S)-carbapenam-3-carboxylic acid from (2S,5S)-5-carboxymethyl proline. The kinetic mechanism is Bi-Ter where ATP is the first substrate to bind followed by (2S,5S)-5-carboxymethyl proline and PPi is the last product released based on initial velocity, product and dead-end inhibition studies. The reactions catalyzed by carbapenam synthetase with different diastereomers of the natural substrate and with alternate alpha-amino diacid substrates were studied by HPLC, ESI mass spectrometry, and steady-state kinetic analysis. On the basis of these results, we have proposed a role for each moiety of (2S,5S)-5-carboxymethyl proline for binding to the active site of carbapenam synthetase. Coupled enzyme assays of AMP and pyrophosphate release in the reactions catalyzed by carbapenam synthetase with adipic and glutaric acid, which lack the alpha-amino group, in the presence and absence of hydroxylamine support the formation of an acyladenylate intermediate in the catalytic cycle. << Less

  Biochemistry 42:7836-7847(2003) [PubMed] [EuropePMC]

• A conserved tyrosyl-glutamyl catalytic dyad in evolutionarily linked enzymes: carbapenam synthetase and beta-lactam synthetase.

  Raber M.L., Arnett S.O., Townsend C.A.

  Beta-lactam-synthesizing enzymes carbapenam synthetase (CPS) and beta-lactam synthetase (beta-LS) are evolutionarily linked to a common ancestor, asparagine synthetase B (AS-B). These three relatives catalyze substrate acyl-adenylation and nucleophilic acyl substitution by either an external (AS-B ... >> More

  Beta-lactam-synthesizing enzymes carbapenam synthetase (CPS) and beta-lactam synthetase (beta-LS) are evolutionarily linked to a common ancestor, asparagine synthetase B (AS-B). These three relatives catalyze substrate acyl-adenylation and nucleophilic acyl substitution by either an external (AS-B) or internal (CPS, beta-LS) nitrogen source. Unlike AS-B, crystal structures of CPS and beta-LS revealed a putative Tyr-Glu dyad (CPS, Y345/E380; beta-LS, Y348/E382) proposed to deprotonate the respective internal nucleophile. CPS and beta-LS site-directed mutagenesis (Y345/8A, Y345/8F, E380/2D, E380/2Q, E380A) resulted in the reduction of their catalytic efficiency, with Y345A, E380A, and E382Q producing undetectable amounts of beta-lactam product. However, [(32)P]PP(i)-ATP exchange assays demonstrated Y345A and E380A undergo the first half-reaction, with the remaining active mutants showing decreased forward commitment to beta-lactam cyclization. pH-rate profiles of CPS and beta-LS supported the importance of a Tyr-Glu dyad in beta-lactam formation and suggested its reverse protonation in beta-LS. The kinetics of CPS double-site mutants reinforced the synergism of Tyr-Glu in catalysis. Furthermore, significant solvent isotope effects on k(cat) ((D)k(cat)) for Y345F (1.9) and Y348F (1.7) maintained the assignment of Y345/8 in proton transfer. A proton inventory on Y348F determined its (D)(k(cat)/K(m)) = 0.2 to arise from multiple reactant-state fractionation factors, presumably from water molecule(s) replacing the missing Tyr hydroxyl. The role of a CPS and beta-LS Tyr-Glu catalytic dyad was solidified by a significant decrease in mutant k(cat) viscosity dependence with respect to the wild-type enzymes. The evolutionary relation and potential for engineered biosynthesis were demonstrated by beta-LS acting as a carbapenam synthetase. << Less

  Biochemistry 48:4959-4971(2009) [PubMed] [EuropePMC]

• Rate-limiting steps and role of active site Lys443 in the mechanism of carbapenam synthetase.

  Arnett S.O., Gerratana B., Townsend C.A.

  Carbapenam synthetase (hereafter named CPS) catalyzes the formation of the beta-lactam ring in the biosynthetic pathway to (5R)-carbapen-2-em-3-carboxylate, the simplest of the carbapenem antibiotics. Kinetic studies showed remarkable tolerance to substrate stereochemistry in the turnover rate but ... >> More

  Carbapenam synthetase (hereafter named CPS) catalyzes the formation of the beta-lactam ring in the biosynthetic pathway to (5R)-carbapen-2-em-3-carboxylate, the simplest of the carbapenem antibiotics. Kinetic studies showed remarkable tolerance to substrate stereochemistry in the turnover rate but did not distinguish between chemistry and a nonchemical step such as product release or conformational change as being rate-determining. Also, X-ray structural studies and modest sequence homology to beta-lactam synthetase, an enzyme that catalyzes the formation of a monocyclic beta-lactam ring in a similar ATP/Mg2+-dependent reaction, implicate K443 as an essential residue for substrate binding and intermediate stabilization. In these experiments, we use pH-rate profiles, deuterium solvent isotope effects, and solvent viscosity measurements to examine the rate-limiting step in this complex overall process of substrate adenylation and intramolecular ring formation. Mutagenesis and chemical rescue demonstrate that K443 is the general acid visible in the pH-rate profile of the wild-type CPS-catalyzed reaction. On the basis of these results, we propose a mechanism in which the rate-limiting step is beta-lactam ring formation coupled to a protein conformational change and underscore the role of K443 throughout the reaction. << Less

  Biochemistry 46:9337-9345(2007) [PubMed] [EuropePMC]