Expression and Purification of Two Isozymes of Clavaminate Synthase and Initial Characterization of the Iron Binding Site

Abstract

Clavaminate synthase is an Fe(2+)-, O2-, and alpha-ketoglutarate-dependent oxygenase that catalyzes three transformations in the biosynthesis of the important beta-lactamase inhibitor clavulanic acid. The genes from Streptomyces clavuligerus encoding two isoenzymes of clavaminate synthase have been over-expressed in Escherichia coli to give soluble proteins whose reactions, kinetic properties, and molecular masses are in excellent agreement with the wild-type isozymes. Preliminary investigation of the active site of clavaminate synthase was undertaken using diethyl pyrocarbonate and N-ethylmaleimide. Each was inhibitory to catalytic activity. Protection from inactivation in the presence of these reagents by Fe2+, O2, and alpha-ketoglutaric acid was thwarted by the rapid self-inactivation of the enzyme in the absence of substrate. However, protection was achieved when Co2+, a potent competitive inhibitor of clavaminate synthase 2 with respect to Fe2+, was substituted. This is consistent with the presence of histidine and cysteine, respectively, at or near the active site and possibly involved in iron binding. In the course of constructing the expression vector, a simply applied general error analysis of the polymerase chain reaction was formulated to calculate the proportion of correctly replicated DNA and guide the design of experiments using this method.

Identification, cloning, sequencing, and overexpression of the gene encoding proclavaminate amidino hydrolase and characterization of protein function in clavulanic acid biosynthesis

“…Even pure compound 2 was unaffected by prolonged incubation with fresh enzyme when incubation was followed by either HPLC or the sensitive UV assay of the imidazole adduct expected from compound 4 or 6. However, coincubation of this fusion protein with recombinant CS2 (8) in the presence of compound 1 revealed the time-dependent formation of clavaminic acid (compound 4) as detected by reaction with imidazole and generation of the characteristic chromophore at 312 nm (6). The conversion of compound 1 to compound 4 shows that all four reactions can be successively catalyzed by these two enzymes under the same conditions and demonstrates the role of PAH in the overall biosynthetic pathway to clavulanic acid.…”

“…Crude E. coli extracts were assayed for PAH activity (32). These extracts readily converted compound 1 to compound 4 in the presence of CS2 as well as compound 2 to compound 3 in the absence of recombinant CS2 (8).…”

Biosynthetic Pathway for Mannopeptimycins, Lipoglycopeptide Antibiotics Active against Drug-Resistant Gram-Positive Pathogens

“…The mannopeptimycin cluster contains one oxygenase gene, mppO, with a deduced amino acid sequence displaying homology (46% similarity and 31% identity) with clavaminate synthase I (CSI) and CSII from Streptomyces clavuligerus (3). Clavaminate synthases are trifunctional ␣-ketoglutarate-dependent enzymes involved in clavulanic acid synthesis.…”

Purification and Characterization of the FeII- and α-Ketoglutarate-Dependent Xanthine Hydroxylase from Aspergillus nidulans

“…Zn II and Cu II are potent inhibitors of XanA, and several other metals also inhibit the enzyme (Figure 7). This situation resembles that known for related enzymes such as TauD where Co II and Ni II inhibition has been studied (41), clavaminate synthase where Co II inhibition was characterized (69), or TfdA where the Cu IIinhibited enzyme was analyzed (70). The inhibitory metal ions are likely to substitute for Fe II and presumably utilize the same set of amino acid side chain ligands.…”