Gene cloning of alpha-methylserine aldolase from Variovorax paradoxus and purification and characterization of the recombinant enzyme.
The alpha-methylserine aldolase gene from Variovorax paradoxus strains AJ110406, NBRC15149, 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 Vmax and Km of the enzyme for the formaldehyde release reaction from alpha-methyl-L-serine were 1.89 micromol min(-1) mg(-1) 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.
Nozaki
Hiroyuki
H
AminoScience Laboratories, Ajinomoto Co., Inc., Kawasaki, Japan. hiroyuki_nozaki@ajinomoto.com
Kuroda
Shinji
S
Watanabe
Kunihiko
K
Yokozeki
Kenzo
K
D000595:Amino Acid Sequence; D003001:Cloning, Molecular; D042621:Comamonadaceae / Q000201:enzymology* / Q000235:genetics; D017124:Conserved Sequence; D005634:Fructose-Bisphosphate Aldolase / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D015870:Gene Expression; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D016415:Sequence Alignment; D012694:Serine / Q000031:analogs & derivatives* / Q000378:metabolism; D013053:Spectrophotometry; D013379:Substrate Specificity; D013696:Temperature
D011994:Recombinant Proteins; C035563:2-methylserine; D012694:Serine; D005634:Fructose-Bisphosphate Aldolase