A novel metal-activated pyridoxal enzyme with a unique primary structure, low specificity D-threonine aldolase from Arthrobacter sp. Strain DK-38. Molecular cloning and cofactor characterization.
The gene encoding low specificity D-threonine aldolase, catalyzing the interconversion of D-threonine/D-allo-threonine and glycine plus acetaldehyde, was cloned from the chromosomal DNA of Arthrobacter sp. strain DK-38. The gene contains an open reading frame consisting of 1,140 nucleotides corresponding to 379 amino acid residues. The enzyme was overproduced in recombinant Escherichia coli cells and purified to homogeneity by ammonium sulfate fractionation and three-column chromatography steps. The recombinant aldolase was identified as a pyridoxal enzyme with the capacity of binding 1 mol of pyridoxal 5'-phosphate per mol of subunit, and Lys59 of the enzyme was determined to be the cofactor binding site by chemical modification with NaBH4. In addition, Mn2+ ion was demonstrated to be an activator of the enzyme, although the purified enzyme contained no detectable metal ions. Equilibrium dialysis and atomic absorption studies revealed that the recombinant enzyme could bind 1 mol of Mn2+ ion per mol of subunit. Remarkably, the predicted amino acid sequence of the enzyme showed no significant similarity to those of the currently known pyridoxal 5'-phosphate-dependent enzymes, indicating that low specificity D-threonine aldolase is a new pyridoxal enzyme with a unique primary structure. Taken together, low specificity D-threonine aldolase from Arthrobacter sp. strain DK-38, with a unique primary structure, is a novel metal-activated pyridoxal enzyme.
Liu
J Q
JQ
Laboratory of Biocatalytic Chemistry, Biotechnology Research Center, Toyama Prefectural University, Kosugi Machi, Toyama 939-0398, Japan.
Dairi
T
T
Itoh
N
N
Kataoka
M
M
Shimizu
S
S
Yamada
H
H
D000595:Amino Acid Sequence; D001173:Arthrobacter / Q000201:enzymology*; D001483:Base Sequence; D002851:Chromatography, High Pressure Liquid; D003001:Cloning, Molecular; D004269:DNA, Bacterial; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation; D004926:Escherichia coli / Q000235:genetics; D012696:Glycine Hydroxymethyltransferase / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D008670:Metals / Q000378:metabolism*; D008969:Molecular Sequence Data; D011730:Pyridoxal / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity
D004269:DNA, Bacterial; D008670:Metals; D011730:Pyridoxal; D012696:Glycine Hydroxymethyltransferase