Identification in the yeast Pichia stipitis of the first L-rhamnose-1-dehydrogenase gene
Identification in the yeast Pichia stipitis of the first L-rhamnose-1-dehydrogenase gene
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DOI：
10.1111/j.1742-4658.2008.06392.x
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发表时间：
2008-05-01
期刊：
影响因子：
5.4
通讯作者：
Richard, Peter
中科院分区：
文献类型：
--
作者：
Koivistoinen, Outi M.;Hilditch, Satu;Richard, Peter
L-鼠李糖在微生物中的分解代谢有两条截然不同的途径。在细菌中描述了一种具有磷酸化中间产物的途径；在这里，酶和相应的基因序列是已知的。另一种途径没有磷酸化的中间产物，只在真核微生物中被描述过。对于这一途径，已经描述了酶的活性，但没有相应的基因序列。这种分解代谢途径中的第一个酶是利用L-鼠李糖1-脱氢酶的NAD。从树干毕赤酵母中纯化了该酶，并用MALDI-TOF MS测定了其胰蛋白酶多肽的质量，从而对相应的基因RHA1进行了鉴定。它编码一种258个氨基酸的蛋白质，属于短链乙醇脱氢酶蛋白质家族。ORF在酿酒酵母中得到了表达。由于该基因包含一个CUG密码子，该密码子编码树枝杆菌中的丝氨酸，而在酿酒酵母中编码亮氨酸，因此该密码子发生了变化，使得相同的氨基酸在酿酒酵母中表达。异源蛋白与L-鼠李糖的活性和亲和力最高，而与L-甘露糖和L-赖糖的活性和亲和力较低。该酶是NAD所特有的。Northern印迹分析表明，树干拟青霉在L-鼠李糖的生长过程中被诱导转录，而在其他碳源上不被诱导。
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.