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Isolation, characterization and sequence analysis of a full-length cDNA clone encoding NADH-dependent hydroxypyruvate reductase from cucumber

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Abstract

A full-length cDNA encoding NADH-dependent hydroxypyruvate reductase (HPR), a photorespiratory enzyme localized in leaf peroxisomes, was isolated from a λgt11 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>10). The derived amino acid sequence for HPR also contains the characteristics of an NAD-binding domain.

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Authors and Affiliations

  1. Department of Botany and Genetics, University of Wisconsin, 53706, Madison, WI, USA

    John McC. Greenler, James S. Sloan, Brian W. Schwartz, Wayne M. Becker & Wayne M. Becker

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  1. John McC. Greenler
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  2. James S. Sloan
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  3. Brian W. Schwartz
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  4. Wayne M. Becker
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Greenler, J.M., Sloan, J.S., Schwartz, B.W. et al. Isolation, characterization and sequence analysis of a full-length cDNA clone encoding NADH-dependent hydroxypyruvate reductase from cucumber. Plant Mol Biol 13, 139–150 (1989). https://doi.org/10.1007/BF00016133

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  • DOI: https://doi.org/10.1007/BF00016133

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