Identification in the yeast Pichia stipitis of the first L-rhamnose-1-dehydrogenase gene

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Abstract

There are two distinctly different pathways for the catabolism of l-rhamnose in microorganisms. One pathway with phosphorylated intermediates was described in bacteria; here the enzymes and the corresponding gene sequences are known. The other pathway has no phosphorylated intermediates and has only been described in eukaryotic microorganisms. For this pathway, the enzyme activities have been described but not the corresponding gene sequences. The first enzyme in this catabolic pathway is the NAD-utilizing l-rhamnose 1-dehydrogenase. The enzyme was purified from the yeast Pichia stipitis, and the mass of its tryptic peptides was determined using MALDI-TOF MS. This enabled the identification of the corresponding gene, RHA1. It codes for a protein with 258 amino acids belonging to the protein family of short-chain alcohol dehydrogenases. The ORF was expressed in Saccharomyces cerevisiae. As the gene contained a CUG codon that codes for serine in P. stipitis but for leucine in S. cerevisiae, this codon has changed so that the same amino acid was expressed in S. cerevisiae. The heterologous protein showed the highest activity and affinity with l-rhamnose and a lower activity and affinity with l-mannose and l-lyxose. The enzyme was specific for NAD. A northern blot analysis revealed that transcription in P. stipitis is induced during growth on l-rhamnose but not on other carbon sources.

Original languageEnglish
Pages (from-to)2482-2488
JournalFEBS Journal
Volume275
Issue number10
DOIs
Publication statusPublished - 1 May 2008
MoE publication typeA1 Journal article-refereed

Fingerprint

Rhamnose
Pichia
Yeast
Oxidoreductases
Genes
Yeasts
Saccharomyces cerevisiae
Enzymes
Microorganisms
NAD
Codon
Amino Acids
Proteins
Alcohol Dehydrogenase
Enzyme activity
Transcription
Mannose
Leucine
Serine
Matrix-Assisted Laser Desorption-Ionization Mass Spectrometry

Keywords

  • L-rhamnonate
  • L-rhamnose catabolism
  • L-rhamnose dehydrogenase
  • MALDI-TOF MS
  • Pichia stipitis

Cite this

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title = "Identification in the yeast Pichia stipitis of the first L-rhamnose-1-dehydrogenase gene",
abstract = "There are two distinctly different pathways for the catabolism of l-rhamnose in microorganisms. One pathway with phosphorylated intermediates was described in bacteria; here the enzymes and the corresponding gene sequences are known. The other pathway has no phosphorylated intermediates and has only been described in eukaryotic microorganisms. For this pathway, the enzyme activities have been described but not the corresponding gene sequences. The first enzyme in this catabolic pathway is the NAD-utilizing l-rhamnose 1-dehydrogenase. The enzyme was purified from the yeast Pichia stipitis, and the mass of its tryptic peptides was determined using MALDI-TOF MS. This enabled the identification of the corresponding gene, RHA1. It codes for a protein with 258 amino acids belonging to the protein family of short-chain alcohol dehydrogenases. The ORF was expressed in Saccharomyces cerevisiae. As the gene contained a CUG codon that codes for serine in P. stipitis but for leucine in S. cerevisiae, this codon has changed so that the same amino acid was expressed in S. cerevisiae. The heterologous protein showed the highest activity and affinity with l-rhamnose and a lower activity and affinity with l-mannose and l-lyxose. The enzyme was specific for NAD. A northern blot analysis revealed that transcription in P. stipitis is induced during growth on l-rhamnose but not on other carbon sources.",
keywords = "L-rhamnonate, L-rhamnose catabolism, L-rhamnose dehydrogenase, MALDI-TOF MS, Pichia stipitis",
author = "Koivistoinen, {Outi M.} and Satu Hilditch and Voutilainen, {Sanni P.} and Harry Boer and Merja Penttil{\"a} and Peter Richard",
year = "2008",
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Identification in the yeast Pichia stipitis of the first L-rhamnose-1-dehydrogenase gene. / Koivistoinen, Outi M.; Hilditch, Satu; Voutilainen, Sanni P.; Boer, Harry; Penttilä, Merja; Richard, Peter (Corresponding Author).

In: FEBS Journal, Vol. 275, No. 10, 01.05.2008, p. 2482-2488.

Research output: Contribution to journalArticleScientificpeer-review

TY - JOUR

T1 - Identification in the yeast Pichia stipitis of the first L-rhamnose-1-dehydrogenase gene

AU - Koivistoinen, Outi M.

AU - Hilditch, Satu

AU - Voutilainen, Sanni P.

AU - Boer, Harry

AU - Penttilä, Merja

AU - Richard, Peter

PY - 2008/5/1

Y1 - 2008/5/1

N2 - There are two distinctly different pathways for the catabolism of l-rhamnose in microorganisms. One pathway with phosphorylated intermediates was described in bacteria; here the enzymes and the corresponding gene sequences are known. The other pathway has no phosphorylated intermediates and has only been described in eukaryotic microorganisms. For this pathway, the enzyme activities have been described but not the corresponding gene sequences. The first enzyme in this catabolic pathway is the NAD-utilizing l-rhamnose 1-dehydrogenase. The enzyme was purified from the yeast Pichia stipitis, and the mass of its tryptic peptides was determined using MALDI-TOF MS. This enabled the identification of the corresponding gene, RHA1. It codes for a protein with 258 amino acids belonging to the protein family of short-chain alcohol dehydrogenases. The ORF was expressed in Saccharomyces cerevisiae. As the gene contained a CUG codon that codes for serine in P. stipitis but for leucine in S. cerevisiae, this codon has changed so that the same amino acid was expressed in S. cerevisiae. The heterologous protein showed the highest activity and affinity with l-rhamnose and a lower activity and affinity with l-mannose and l-lyxose. The enzyme was specific for NAD. A northern blot analysis revealed that transcription in P. stipitis is induced during growth on l-rhamnose but not on other carbon sources.

AB - There are two distinctly different pathways for the catabolism of l-rhamnose in microorganisms. One pathway with phosphorylated intermediates was described in bacteria; here the enzymes and the corresponding gene sequences are known. The other pathway has no phosphorylated intermediates and has only been described in eukaryotic microorganisms. For this pathway, the enzyme activities have been described but not the corresponding gene sequences. The first enzyme in this catabolic pathway is the NAD-utilizing l-rhamnose 1-dehydrogenase. The enzyme was purified from the yeast Pichia stipitis, and the mass of its tryptic peptides was determined using MALDI-TOF MS. This enabled the identification of the corresponding gene, RHA1. It codes for a protein with 258 amino acids belonging to the protein family of short-chain alcohol dehydrogenases. The ORF was expressed in Saccharomyces cerevisiae. As the gene contained a CUG codon that codes for serine in P. stipitis but for leucine in S. cerevisiae, this codon has changed so that the same amino acid was expressed in S. cerevisiae. The heterologous protein showed the highest activity and affinity with l-rhamnose and a lower activity and affinity with l-mannose and l-lyxose. The enzyme was specific for NAD. A northern blot analysis revealed that transcription in P. stipitis is induced during growth on l-rhamnose but not on other carbon sources.

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KW - L-rhamnose catabolism

KW - L-rhamnose dehydrogenase

KW - MALDI-TOF MS

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