Enzymatic and biochemical properties of a novel human serine dehydratase isoform.
A cDNA clone similar to human serine dehydratase (SDH) is deposited in the GenBank/EMBL databases, but its structural and functional bases remain unknown. Despite the occurrence of mRNA, the expected protein level was found to be low in cultured cells. To learn about physicochemical properties of the protein, we expressed the cDNA in Escherichia coli, and compared the expressed protein with that of a hepatic SDH. The purified protein showed l-serine and l-threonine dehydratase activity, demonstrating to be an isoform of SDH. However, their Km and Vmax constants were different in a range of two-order. Removal of Pro128 from the hepatic SDH consisting of 328 residues, which is missing in the corresponding position of the isoform consisting of 329 residues, significantly changed the Michaelis constants and Kd value for pyridoxal 5'-phosphate, whereas addition of a proline residue to the isoform was without effect. These findings suggest the difference in the structures of the active sites of the two enzymes. Another striking feature was that the expressed level of the isoform in E. coli was 7-fold lower than that of the hepatic SDH. Substitution of Val for Leu287 in the isoform dramatically increased the protein level. The high yield of the mutated isoform was also confirmed by the in vitro transcription and translation experiment. The poor expression of the isoform could be explained by the more stable secondary structure of the mRNA than that of the hepatic SDH mRNA. The present findings may provide a clue as to why the protein level in cultured cells is low.
Ogawa
Hirofumi
H
Department of Biochemistry, Toyama Medical and Pharmaceutical University Faculty of Medicine, Toyama 930-0194, Japan. hogawa@ms.toyama-mpu.ac.jp
Gomi
Tomoharu
T
Nishizawa
Mikio
M
Hayakawa
Yumiko
Y
Endo
Shunro
S
Hayashi
Kyoko
K
Ochiai
Hiroshi
H
Takusagawa
Fusao
F
Pitot
Henry C
HC
Mori
Hisashi
H
Sakurai
Hiroaki
H
Koizumi
Keiichi
K
Saiki
Ikuo
I
Oda
Hirofumi
H
Fujishita
Takashi
T
Miwa
Toshiro
T
Maruyama
Muneharu
M
Kobayashi
Masashi
M
D000595:Amino Acid Sequence; D000818:Animals; D045744:Cell Line, Tumor; D003001:Cloning, Molecular; D004926:Escherichia coli; D006801:Humans; D007527:Isoenzymes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012695:L-Serine Dehydratase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D008175:Lung Neoplasms / Q000201:enzymology; D008969:Molecular Sequence Data; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
D007527:Isoenzymes; D011994:Recombinant Proteins; D012695:L-Serine Dehydratase