Isolation, characterization and expression of the gene that encodes d-arabinitol dehydrogenase in candida tropicalis

Jeffrey S. Murray, M. Laurie Wong, C. Garrett Miyada, Arthur C. Switchenko, Thomas C. Goodman, Brian Wong

Research output: Contribution to journalArticle

9 Citations (Scopus)

Abstract

The gene (ARD) that encodes NAD-dependent d-arabinitol dehydrogenase (ArDH) in the pathogenic fungus Candida tropicalis (Ct) was cloned by transforming Escherichia coli (Ec) BW31M (araC) with a plasmid library of Ct genomic DNA and selecting for d-arabinitol-utilizing (d-arab+) clones. Plasmid DNA from a d-arab+ clone retransformed fresh Ec BW31M cells to d-arab+; these cells produced both ArDH catalytic activity and a 31-kDa protein recognized by antibodies to native Ct ArDH. The plasmid contained an 846-bp open reading frame (ORF) that encoded a deduced protein of 282 amino acids (aa) (30 748 Da). Four partial aa sequences from Ct ArDH were present in the deduced aa sequence, thus verifying that Ct ARD had been cloned. Ct ArDH was 95% identical to ArDH from Candida albicans (Ca), 85% identical to a xylitol dehydrogenase (XDH) from Pichia stipitis (Ps) and 20-25% identical to many other short-chain dehydrogenases. Ct ArDH, Ca ArDH and Ps XDH were typical short-chain dehydrogenases except that they lacked an N-terminal Gly that is conserved in other members of this family. Thus, these enzymes may represent a subclass of closely-related fungal pentitol dehydrogenases. Large amounts of recombinant ArDH (re-ArDH) were produced in Ec and purified by dye ligand affinity chromatography. The physical and catalytic properties of re-ArDH were similar to those of native Ct ArDH, and re-ArDH and native ArDH performed similarly in an automated enzymatic assay for d-arabinitol in human serum.

Original languageEnglish (US)
Pages (from-to)123-128
Number of pages6
JournalGene
Volume155
Issue number1
DOIs
StatePublished - Mar 21 1995
Externally publishedYes

Fingerprint

Candida tropicalis
Oxidoreductases
Gene Expression
D-Xylulose Reductase
Plasmids
Pichia
Escherichia coli
Candida albicans
Amino Acid Sequence
Clone Cells
DNA
Enzyme Assays
Affinity Chromatography
NAD

Keywords

  • Candidiasis
  • D-arabitol
  • diagnostic marker
  • pentitol
  • polyol

ASJC Scopus subject areas

  • Genetics

Cite this

Murray, J. S., Wong, M. L., Miyada, C. G., Switchenko, A. C., Goodman, T. C., & Wong, B. (1995). Isolation, characterization and expression of the gene that encodes d-arabinitol dehydrogenase in candida tropicalis. Gene, 155(1), 123-128. https://doi.org/10.1016/0378-1119(94)00900-D

Isolation, characterization and expression of the gene that encodes d-arabinitol dehydrogenase in candida tropicalis. / Murray, Jeffrey S.; Wong, M. Laurie; Miyada, C. Garrett; Switchenko, Arthur C.; Goodman, Thomas C.; Wong, Brian.

In: Gene, Vol. 155, No. 1, 21.03.1995, p. 123-128.

Research output: Contribution to journalArticle

Murray, JS, Wong, ML, Miyada, CG, Switchenko, AC, Goodman, TC & Wong, B 1995, 'Isolation, characterization and expression of the gene that encodes d-arabinitol dehydrogenase in candida tropicalis', Gene, vol. 155, no. 1, pp. 123-128. https://doi.org/10.1016/0378-1119(94)00900-D
Murray, Jeffrey S. ; Wong, M. Laurie ; Miyada, C. Garrett ; Switchenko, Arthur C. ; Goodman, Thomas C. ; Wong, Brian. / Isolation, characterization and expression of the gene that encodes d-arabinitol dehydrogenase in candida tropicalis. In: Gene. 1995 ; Vol. 155, No. 1. pp. 123-128.
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abstract = "The gene (ARD) that encodes NAD-dependent d-arabinitol dehydrogenase (ArDH) in the pathogenic fungus Candida tropicalis (Ct) was cloned by transforming Escherichia coli (Ec) BW31M (araC) with a plasmid library of Ct genomic DNA and selecting for d-arabinitol-utilizing (d-arab+) clones. Plasmid DNA from a d-arab+ clone retransformed fresh Ec BW31M cells to d-arab+; these cells produced both ArDH catalytic activity and a 31-kDa protein recognized by antibodies to native Ct ArDH. The plasmid contained an 846-bp open reading frame (ORF) that encoded a deduced protein of 282 amino acids (aa) (30 748 Da). Four partial aa sequences from Ct ArDH were present in the deduced aa sequence, thus verifying that Ct ARD had been cloned. Ct ArDH was 95{\%} identical to ArDH from Candida albicans (Ca), 85{\%} identical to a xylitol dehydrogenase (XDH) from Pichia stipitis (Ps) and 20-25{\%} identical to many other short-chain dehydrogenases. Ct ArDH, Ca ArDH and Ps XDH were typical short-chain dehydrogenases except that they lacked an N-terminal Gly that is conserved in other members of this family. Thus, these enzymes may represent a subclass of closely-related fungal pentitol dehydrogenases. Large amounts of recombinant ArDH (re-ArDH) were produced in Ec and purified by dye ligand affinity chromatography. The physical and catalytic properties of re-ArDH were similar to those of native Ct ArDH, and re-ArDH and native ArDH performed similarly in an automated enzymatic assay for d-arabinitol in human serum.",
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