Cloning, expression, and characterization of 5-aminolevulinic acid synthase from Rhodopseudomonas palustris KUGB306.
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.
Choi
Han-Pil
HP
School of Life Sciences and Biotechnology, Korea University, Seoul 136-701, South Korea.
Hong
Jeong-Woon
JW
Rhee
Ki-Hyeong
KH
Sung
Ha-Chin
HC
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
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