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) from the photosynthetic bacterium Rhodobacter sphaeroides, were cloned to allow high expression in Escherichia coli. Both HemA and HemT appeared to be active in vivo as plasmids carrying the respective genes complemented an E. coli hemA strain (glutamyl-tRNA reductase deficient). The over-expressed isoenzymes were isolated and purified to homogeneity. Isolated HemA was soluble 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 x L-1 of starting bacterial culture and pure HemT at 10 mg x L-1 x HemA has a final specific activity of 13 U x mg-1 with 1 unit defined as 1 micromol of 5-aminolaevulinic acid formed per hour at 37 degrees C. The Km values for HemA are 1.9 mM for glycine and 17 microM 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 pyridoxal 5'-phosphate (PLP) as a cofactor for catalysis. Removal of this cofactor resulted in inactive apo-ALAS. Similarly, reduction of the HemA-PLP complex using sodium borohydride led to > 90% inactivation of the enzyme. Ultraviolet-visible spectroscopy with HemA suggested the presence of an aldimine linkage between the enzyme and pyridoxal 5'-phosphate that was not observed when HemT was incubated with the cofactor. HemA was found to be sensitive to reagents that modify histidine, arginine and cysteine amino acid residues and the enzyme was also highly sensitive to tryptic cleavage between Arg151 and Ser152 in the presence or absence of PLP and substrates. Antibodies were raised to both HemA and HemT but the respective antisera were not only found to bind both enzymes but also to cross-react with mouse ALAS, indicating that all of the proteins have conserved epitopes.
Bolt
E L
EL
Department of Molecular Genetics, Institute of Ophthalmology, University College London, UK.
Kryszak
L
L
Zeilstra-Ryalls
J
J
Shoolingin-Jordan
P M
PM
Warren
M J
MJ
D000624:5-Aminolevulinate Synthetase / Q000037:antagonists & inhibitors / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D003001:Cloning, Molecular; D004047:Diethyl Pyrocarbonate / Q000494:pharmacology; D004852:Epoxy Compounds / Q000494:pharmacology; D004926:Escherichia coli / Q000235:genetics; D005033:Ethylmaleimide / Q000494:pharmacology; D007527:Isoenzymes / Q000235:genetics / Q000378:metabolism; D007700:Kinetics; D011387:Prokaryotic Cells / Q000201:enzymology; D017510:Protein Folding; D011732:Pyridoxal Phosphate / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D012242:Rhodobacter sphaeroides / Q000201:enzymology*; D013439:Sulfhydryl Reagents / Q000494:pharmacology
D004852:Epoxy Compounds; D007527:Isoenzymes; D011994:Recombinant Proteins; D013439:Sulfhydryl Reagents; D011732:Pyridoxal Phosphate; C007366:diepoxybutane; D000624:5-Aminolevulinate Synthetase; D004047:Diethyl Pyrocarbonate; D005033:Ethylmaleimide