Gene cloning of alpha-methylserine aldolase from Variovorax paradoxus and purification and characterization of the recombinant enzyme

• Published in: Bioscience, biotechnology, and biochemistry biotechnology, and biochemistry, 2008-10, Vol.72 (10), p.2580-2588
• Main Authors: Nozaki, H.(Ajinomoto Co. Inc., Kawasaki, Kanagawa (Japan). AminoScience Labs.), Kuroda, S, Watanabe, K, Yokozeki, K
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; BACTERIA; Biological and medical sciences; CLONACION MOLECULAR; CLONAGE MOLECULAIRE; Cloning, Molecular; Comamonadaceae - enzymology; Comamonadaceae - genetics; Conserved Sequence; Escherichia coli; EXPRESION GENICA; EXPRESSION DES GENES; Fructose-Bisphosphate Aldolase - chemistry; Fructose-Bisphosphate Aldolase - genetics; Fructose-Bisphosphate Aldolase - isolation & purification; Fructose-Bisphosphate Aldolase - metabolism; Fundamental and applied biological sciences. Psychology; GENE EXPRESSION; GENETIC ENGINEERING; GENIE GENETIQUE; Hydrogen-Ion Concentration; INGENIERIA GENETICA; LIASAS; LYASE; LYASES; MOLECULAR CLONING; Molecular Sequence Data; PROTEIN SYNTHESIS; PROTEINAS RECOMBINANTES; PROTEINE RECOMBINANTE; pyridoxal 5′-phosphate; RECOMBINANT PROTEINS; Recombinant Proteins - chemistry; Recombinant Proteins - genetics; Recombinant Proteins - isolation & purification; Recombinant Proteins - metabolism; Sequence Alignment; SERINA; SERINE; Serine - analogs & derivatives; Serine - metabolism; SINTESIS DE PROTEINAS; Spectrophotometry; Substrate Specificity; SYNTHESE PROTEIQUE; Temperature; Variovorax paradoxus; α-methyl-L-serine; α-methylserine aldolase
• ISSN: 0916-8451; 1347-6947
• DOI: 10.1271/bbb.80274

The alpha-methylserine aldolase gene from Variovorax paredoxus strains AJ110406, NBRC 15149, and NBRC15150 was cloned and expressed in Escherichia coli. Formaldehyde release activity from alpha-methyl-L-serine was detected in the cell-free extract of E. coli expressing the gene from three strains. The recombinant enzyme from V. paradoxus NBRC15150 was purified. The Vsub(max) and Ksub(m) of the enzyme for the formaldehyde release reaction from alpha-methyl-L-serine were 1.89 micromol/min/mg and 1.2 mM respectively. The enzyme was also capable of catalyzing the synthesis of alpha-methyl-L-serine and alpha-ethyl-L-serine from L-alanine and L-2-aminobutyric acid respectively, accompanied by hydroxymethyl transfer from formaldehyde. The purified enzyme also catalyzed alanine racemization. It contained 1 mole of pyridoxal 5'-phosphate per mol of the enzyme subunit, and exhibited a specific spectral peak at 429 nm. With L-alanine and L-2-aminobutyric acid as substrates, the specific peak, assumed to be a result of the formation of a quinonoid intermediate, increased at 498 nm and 500 nm respectively.