Inhibition and Alternate Substrate Studies on the Mechanism of Carbapenam Synthetase from Erwinia carotovora

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Abstract
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 β-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 β-lactam synthetase, an enzyme that catalyzes formation of a monocyclic β-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 α-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 α-amino group, in the presence and absence of hydroxylamine support the formation of an acyladenylate intermediate in the catalytic cycle.