Isolation, characterization and sequence analysis of a full-length cDNA clone encoding NADH-dependent hydroxypyruvate reductase from cucumber.
A full-length cDNA encoding NADH-dependent hydroxypyruvate reductase (HPR), a photorespiratory enzyme localized in leaf peroxisomes, was isolated from a lambda gt11 cDNA library made by reverse transcription of poly(A)+ RNA from cucumber cotyledons. In vitro transcription and translation of this clone yielded a major polypeptide which was identical in size, 43 kDA, to the product of in vitro translation of cotyledonary poly(A)+ RNA and subsequent immunoprecipitation with HPR antiserum. Escherichia coli cultures transformed with a plasmid construct containing the cDNA insert were induced to express HPR enzyme activity. RNA blot analysis showed that HPR transcript levels rise significantly in the first eight days of light-grown seedling development. This closely resembles the pattern seen for HPR-specific translatable mRNA. DNA blot analysis indicated that a single HPR gene is likely present per haploid genome. Nucleotide sequence analysis revealed an open reading frame of 1146 bases which encodes a polypeptide with a calculated molecular weight of 41.7 kDa. The derived amino acid sequence from this open reading frame is 26% identical and 50% similar to the amino acid sequence of the E. coli enzyme phosphoglycerate dehydrogenase, which catalyzes a similar reaction and functions in a related pathway. Statistical analyses show that this similarity is significant (z greater than 10). The derived amino acid sequence for HPR also contains the characteristics of an NAD-binding domain.
Greenler
J M
JM
Department of Botany, University of Wisconsin, Madison 53706.
Sloan
J S
JS
Schwartz
B W
BW
Becker
W M
WM
D000429:Alcohol Oxidoreductases / Q000235:genetics*; D000595:Amino Acid Sequence; D001483:Base Sequence; D002237:Carbohydrate Dehydrogenases / Q000235:genetics; D003001:Cloning, Molecular; D004247:DNA / Q000235:genetics*; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D050537:Hydroxypyruvate Reductase; D008969:Molecular Sequence Data; D050543:Phosphoglycerate Dehydrogenase; D063245:Plant Development; D010944:Plants / Q000235:genetics* / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D012689:Sequence Homology, Nucleic Acid
D012333:RNA, Messenger; D004247:DNA; D000429:Alcohol Oxidoreductases; D002237:Carbohydrate Dehydrogenases; D050537:Hydroxypyruvate Reductase; D050543:Phosphoglycerate Dehydrogenase