title: Cloning, expression, and characterization of 5-aminolevulinic acid synthase from Rhodopseudomonas palustris KUGB306.
abstract: The hemA gene encoding 5-aminolevulinic acid synthase (ALAS) was cloned from the genomic DNA of photosynthetic bacterium Rhodopseudomonas palustris KUGB306. The deduced protein (ALAS) of this gene contained 409 amino acids. The hemA gene was subcloned into an expression vector pGEX-KG and the encoded protein was overexpressed as a fusion protein with glutathione-S-transferase (GST) in Escherichia coli BL21. The recombinant ALAS was purified and isolated free of the fusion partner (GST) by affinity purification on glutathione-Sepharose 4B resin and cleavage of the purified fusion protein by thrombin protease. The optimum pH and temperature of the recombinant ALAS was found to be at pH 7.5-8.0 and 35-40 degrees C, respectively. The Km value of the enzyme was 2.01 mM for glycine and 49.55 microM for succinyl-CoA. The enzyme activity was strongly inhibited by Pb2+, Fe2+, Co2+, Cu2+, and Zn2+ at 1 mM, but slightly affected by Mg2+ and K+. The recombinant ALAS required pyridoxal 5'-phosphate (PLP) as a cofactor for catalysis. Removal of this cofactor led to complete loss of the activity. Ultraviolet-visible spectroscopy with the ALAS suggested the presence of an aldimine linkage between the enzyme and PLP.
authors.affiliation: School of Life Sciences and Biotechnology, Korea University, Seoul 136-701, South Korea.
authors.forename: Han-Pil
authors.initials: HP
authors.lastname: Choi
authors.forename: Jeong-Woon
authors.initials: JW
authors.lastname: Hong
authors.forename: Ki-Hyeong
authors.initials: KH
authors.lastname: Rhee
authors.forename: Ha-Chin
authors.initials: HC
authors.lastname: Sung
mesh_terms: D000624:5-Aminolevulinate Synthetase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D000214:Acyl Coenzyme A / Q000378:metabolism; D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D003067:Coenzymes / Q000032:analysis; D004269:DNA, Bacterial / Q000737:chemistry / Q000302:isolation & purification; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D005998:Glycine / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D008670:Metals / Q000494:pharmacology; D008969:Molecular Sequence Data; D011732:Pyridoxal Phosphate / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D012241:Rhodopseudomonas / Q000201:enzymology* / Q000235:genetics; D017422:Sequence Analysis, DNA; D013057:Spectrum Analysis; D013379:Substrate Specificity; D013696:Temperature
chemical_list: D000214:Acyl Coenzyme A; D003067:Coenzymes; D004269:DNA, Bacterial; D004791:Enzyme Inhibitors; D008670:Metals; D011993:Recombinant Fusion Proteins; D011732:Pyridoxal Phosphate; C012046:succinyl-coenzyme A; D000624:5-Aminolevulinate Synthetase; D005998:Glycine