Molecular cloning and expression of the gene for serine hydroxymethyltransferase from an obligate methylotroph Hyphomicrobium methylovorum GM2.
The gene encoding serine hydroxymethyltransferase (SHMT), one of the key enzymes of the one-carbon-compound assimilation of a methylotroph, Hyphomicrobium methylovorum GM2, and its flanking regions were isolated using a DNA fragment encoding Escherichia coli SHMT as a probe. Nucleotide sequencing of the recombinant plasmids revealed the SHMT gene codes for the 434-amino-acid protein with a calculated molecular mass of 46,068 Da. The amino-acid sequence of the enzyme showed identity to the sequences of the enzymes from E. coli (55%) and rabbit liver (44%). The recombinant plasmid, which was constructed by ligation of the cloned gene and an expression vector pKK223-3, was introduced to an SHMT-deficient E. coli mutant ME5427 (glyA-). The transformed E. coli cells expressed SHMT, which was immunologically and enzymologically indistinguishable from the enzyme isolated from H. methylovorum GM2.
Miyata
A
A
Department of Pharmacology, National Cardiovascular Center Research Institute, Osaka, Japan.
Yoshida
T
T
Yamaguchi
K
K
Yokoyama
C
C
Tanabe
T
T
Toh
H
H
Mitsunaga
T
T
Izumi
Y
Y
D000595:Amino Acid Sequence; D000818:Animals; D001419:Bacteria / Q000201:enzymology* / Q000235:genetics; D001483:Base Sequence; D015139:Blotting, Southern; D003001:Cloning, Molecular / Q000379:methods; D004269:DNA, Bacterial / Q000235:genetics / Q000302:isolation & purification; D004926:Escherichia coli / Q000235:genetics; D005798:Genes, Bacterial; D012696:Glycine Hydroxymethyltransferase / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D008930:Mitochondria, Liver / Q000201:enzymology; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides; D010802:Phylogeny; D011817:Rabbits; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D015183:Restriction Mapping; D017386:Sequence Homology, Amino Acid
D004269:DNA, Bacterial; D009838:Oligodeoxyribonucleotides; D011994:Recombinant Proteins; D012696:Glycine Hydroxymethyltransferase