Cloning and expression of the gene for serine-glyoxylate aminotransferase from an obligate methylotroph Hyphomicrobium methylovorum GM2.
The gene encoding serine-glyoxylate aminotransferase, one of key enzymes for the assimilation of one-carbon compounds in methylotrophs, and its flanking regions were isolated from an obligate methylotrophic bacterium, Hyphomicrobium methylovorum GM2. Nucleotide sequencing of the recombinant plasmids revealed that the serine-glyoxylate aminotransferase gene encodes a 405-amino-acid protein with a calculated molecular mass of 43880 Da. The amino acid sequence of the enzyme showed identity to the sequences of serine-glyoxylate aminotransferase of Methylobacterium extorquens AM1 (57%), aspartate aminotransferase of Methanobacterium thermoformicicum (31%), human peroxisomal alanine-glyoxylate aminotransferase (27%), and serine-pyruvate aminotransferase of rat liver mitochondria (33%). The recombinant plasmid, which was constructed by ligation of the cloned gene and an expression vector pKK223-3, was introduced into Escherichia coli HB101. The recombinant enzyme was purified from transformed E. coli cells and analyzed by immunological and enzymological methods. The overexpressed enzyme was indistinguishable from the wild-type enzyme isolated from H. methylovorum GM2.
Hagishita
T
T
Department of Food and Nutrition, Faculty of Agriculture, Kinki University, Japan.
Yoshida
T
T
Izumi
Y
Y
Mitsunaga
T
T
D000595:Amino Acid Sequence; D001419:Bacteria / Q000201:enzymology*; D001426:Bacterial Proteins / Q000737:chemistry*; D001483:Base Sequence; D015139:Blotting, Southern; D015153:Blotting, Western; D003001:Cloning, Molecular; D017124:Conserved Sequence / Q000235:genetics; D002373:Cyclic AMP Receptor Protein / Q000737:chemistry; D017931:DNA Primers; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000302:isolation & purification; D015183:Restriction Mapping; D017386:Sequence Homology, Amino Acid; D012697:Serine Endopeptidases / Q000378:metabolism; D000637:Transaminases / Q000235:genetics*
D001426:Bacterial Proteins; D002373:Cyclic AMP Receptor Protein; D017931:DNA Primers; D010446:Peptide Fragments; D011994:Recombinant Proteins; D000637:Transaminases; C022636:serine-glyoxylate aminotransferase; D012697:Serine Endopeptidases; C036813:lysyl endopeptidase