Identification in the yeast Pichia stipitis of the first L-rhamnose-1-dehydrogenase gene.
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.
Koivistoinen
Outi M
OM
VTT Technical Research Centre of Finland, Espoo, Finland.
Hilditch
Satu
S
Voutilainen
Sanni P
SP
Boer
Harry
H
Penttilä
Merja
M
Richard
Peter
P
D015971:Gene Expression Regulation, Enzymologic; D015966:Gene Expression Regulation, Fungal; D005800:Genes, Fungal; D015394:Molecular Structure; D016297:Mutagenesis, Site-Directed; D016366:Open Reading Frames; D010843:Pichia / Q000201:enzymology / Q000235:genetics; D012210:Rhamnose / Q000737:chemistry / Q000378:metabolism*; D013401:Sugar Alcohol Dehydrogenases / Q000235:genetics / Q000378:metabolism
D013401:Sugar Alcohol Dehydrogenases; D012210:Rhamnose