tailieunhanh - Báo cáo khoa học: Identification in the yeast Pichia stipitis of the first L-rhamnose-1-dehydrogenase gene

There are two distinctly different pathways for the catabolism ofl-rham-nose in microorganisms. One pathway with phosphorylated intermediates was described in bacteria; here the enzymes and the corresponding gene sequences are known. | ễFEBS Journal Identification in the yeast Pichia stipitis of the first L-rhamnose-1-dehydrogenase gene Outi M. Koivistoinen Satu Hilditch Sanni P. Voutilainen Harry Boer Merja Penttila and Peter Richard VTT TechnicalResearch Centre of Finland Espoo Finland Keywords L-rhamnonate L-rhamnose catabolism L-rhamnose dehydrogenase MALDI- TOF MS Pichia stipitis Correspondence P. Richard TechnicalResearch Centre of Finland Tietotie 2 Espoo PO Box 1000 02044 VTT Finland Fax 358 20 722 7071 Tel 358 20 722 7190 E-mail Received 4 January 2008 revised 10 March 2008 accepted 11 March 2008 doi There are two distinctly different pathways for the catabolism of L-rham-nose 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 .