The GLY1 gene of Saccharomyces cerevisiae encodes a low-specific L-threonine aldolase that catalyzes cleavage of L-allo-threonine and L-threonine to glycine--expression of the gene in Escherichia coli and purification and characterization of the enzyme.
The GLY1 gene of Saccharomyces cerevisiae is required for the biosynthesis of glycine for cell growth [McNeil, J. B., McIntosh, E. V., Taylor, B. V., Zhang, F-R., Tang, S. & Bognar, A. L. (1994) J. Biol. Chem. 269, 9155-9165], but its gene product has not been identified. We have found that the GLY1 protein is similar in primary structure to L-allo-threonine aldolase of Aeromonas jandiae DK-39, which stereospecifically catalyzes the interconversion of L-allo-threonine and glycine. The GLY1 gene was amplified by PCR, with a designed ribosome-binding site, cloned into pUC118, and expressed in Escherichia coli cells. The enzyme was purified to homogeneity, as judged by polyacrylamide gel electrophoresis. The enzyme has a molecular mass of about 170 kDa and consists of four subunits identical in molecular mass. The enzyme contains 2 mol pyridoxal 5'-phosphate/4 mol of subunit as a cofactor, and its absorption spectrum exhibits maxima at 280 nm and 420 nm. The enzyme catalyzes the cleavage of not only L-allo-threonine to glycine but also L-threonine. We have termed the enzyme a low-specific L-threonine aldolase to distinguish it from L-allo-threonine aldolase.
Liu
J Q
JQ
Laboratory of Biocatalytic Chemistry, Biotechnology Research Centre, Toyama Prefectural University, Kosugi City, Japan.
Nagata
S
S
Dairi
T
T
Misono
H
H
Shimizu
S
S
Yamada
H
H
D000333:Aeromonas / Q000201:enzymology; D000446:Aldehyde-Lyases; D000595:Amino Acid Sequence; D001483:Base Sequence; D004926:Escherichia coli; D015971:Gene Expression Regulation, Enzymologic; D005998:Glycine / Q000378:metabolism*; D012696:Glycine Hydroxymethyltransferase / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D008970:Molecular Weight; D011487:Protein Conformation; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics; D029701:Saccharomyces cerevisiae Proteins; D016415:Sequence Alignment; D013054:Spectrophotometry, Atomic; D013237:Stereoisomerism; D013696:Temperature; D013912:Threonine / Q000378:metabolism*
D029701:Saccharomyces cerevisiae Proteins; D013912:Threonine; D012696:Glycine Hydroxymethyltransferase; D000446:Aldehyde-Lyases; C086382:GLY1 protein, S cerevisiae; D005998:Glycine