Characterization of the Rhodobacter sphaeroides 5-aminolaevulinic acid synthase isoenzymes, HemA and HemT, isolated from recombinant Escherichia coli

The hemA and hemT genes encoding 5-aminolaevulinic acid synthase (ALAS) fro m the photosynthetic bacterium Rhodobacter sphaeroides, were cloned to allo w high expression in Escherichia coli. Both HemA and HemT appeared to be ac tive in vivo as plasmids carrying the respective genes complemented an E. c oli hemA strain (glutamyl-tRNA reductase deficient). The over-expressed iso enzymes were isolated and purified to homogeneity. Isolated HemA was solubl e and catalytically active whereas HemT was largely insoluble and failed to show any activity ex vivo. Pure HemA was recovered in yields of 5-7 mg.L-1 of starting bacterial culture and pure HemT at 10 mg.L-1. HemA has a final specific activity of 13 U.mg(-1) with 1 unit defined as 1 mu mol of 5-amin olaevulinic acid formed per hour at 37 degrees C. The K-m values for HemA a re 1.9 mM for glycine and 17 mu M for succinyl-CoA, with the enzyme showing a turnover number of 430 h(-1). In common with other ALASs the recombinant R. sphaeroides HemA requires pyr idoxal 5'-phosphate (PLP) as a cofactor for catalysis. Removal of this cofa ctor resulted in inactive apo-ALAS. Similarly, reduction of the HemA-PLP co mplex using sodium borohydride led to > 90% inactivation of the enzyme. Ult raviolet-visible spectroscopy with HemA suggested the presence of an aldimi ne linkage between the enzyme and pyridoxal 5'-phosphate that was not obser ved when HemT was incubated with the cofactor. HemA was found to be sensiti ve to reagents that modify histidine, arginine and cysteine amino acid resi dues and the enzyme was also highly sensitive to tryptic cleavage between A rg151 and Ser152 in the presence or absence of PLP and substrates. Antibodi es were raised to both HemA and HemT but the respective antisera were not o nly found to bind both enzymes but also to cross-react with mouse ALAS, ind icating that all of the proteins have conserved epitopes.