Purification and characterization of clavaminate synthase from Streptomyces clavuligerus: an unusual oxidative enzyme in natural product biosynthesis

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Abstract
A pivotal step in the biosynthetic pathway to the .beta.-lactamase inhibitor clavulanic acid is the conversion of proclavaminic acid to clavaminic acid in a reaction requiring Fe2+, .alpha.-ketoglutarate, and oxygen [Elson, S. W., Baggaley, K. H., Gillett, J., Holland, S., Nicholson, N. H., Slime, J. T., and Woroniecki, S. R. (1987) J. Chem. Soc., Chem. Commun., 1736-1738]. Clavaminate synthase, the enzyme that catalyzes this oxidative cyclization/desaturation, has been purified to homogeneity for clavulanic acid producing cells of Streptomyces clavuligerus (ATCC 27064). The enzyme behaved as a monomer during gel filtration and migrated with Mr 47,000 during denaturing gel electrophoresis. After ion-exchange FPLC two active forms of the protein were resolved that differed slightly in kinetic constants and apparent molecular weight. Kinetic comparisons with the four possible diastereomers of proclavaminate confirmed the absolute configuration of the substrate to be 2S,3R. The stoichiometry of the overall transformation was determined to be proclavaminate + 2(.alpha.-ketoglutarate) + 2O2 .fwdarw. clavaminate + 2(succinate) + 2CO2 + 2H2O. In the absence of proclavaminate a slow decarboxylation of .alpha.-ketoglutarate to succinate and CO2 was observed in an uncoupled reaction which resulted in enzyme inactivation. Steady-state kinetic studies were undertaken for an initial description of the enzyme''s catalytic cycle. The double-reciprocal plot with .alpha.-ketoglutarate as the variable substrate was linear; this supports the proposal that two stepwise oxidations of proclavaminate occur, each with the consumption of .alpha.-ketoglutarate and oxygen and the release of succinate, CO2, and H2O. The intersecting initial velocity plots obtained from pairwise variation of substrate concentrations were consistent with a sequential kinetic mechanism for the first oxidation. Similarities observed between clavaminate synthase and .alpha.-ketoglutarate-dependent dioxygenases argue for a common mechanism of oxygen activation. However, the nature of the interactions of the substrates in the active site of clavaminate synthase apparently redirects the conventional hydroxylase activity of dioxygenases to the construction of a strained bicyclic skeleton driven by the overall reduction of dioxygen.