Cloning, expression, characterization, and nucleophile identification of family 3, Aspergillus niger β-glucosidase
Cloning, expression, characterization, and nucleophile identification of family 3, Aspergillus niger β-glucosidase
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DOI：
10.1074/jbc.275.7.4973
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发表时间：
2000-02-18
影响因子：
4.8
通讯作者：
Shoseyov, O
中科院分区：
文献类型：
--
作者：
Dan, S;Marton, I;Shoseyov, O
来自尼日尔曲霉（CMI. CC 324262）进行纯化，并测定N-末端序列和两个内部序列。从基因组文库中克隆Bgl Ⅰ基因组基因，并通过逆转录-聚合酶链反应克隆其cDNA。该cDNA在酿酒酵母和毕赤酵母中成功表达。序列分析显示，该基因编码一种92 kDa的酶，是糖苷酶家族3的成员。该酶催化的反应的H-1-NMR分析证实，与其它家族3糖苷酶一样，该酶水解时净保留异头构型。因此，通过捕获共价糖基酶中间体，α-脱氧-α-氟β-葡萄糖基氟化物使酶失活，动力学参数为k（i）= 4.5 min（-1），K-I = 35.4 mM。与亚麻苦苷孵育导致再活化，推测是通过转糖基化机制，证明了该中间体的催化能力。2-脱氧-2-氟葡萄糖基酶的消化和随后的分析的高压液相色谱洗脱液通过电喷雾电离三重四极杆质谱法在中性损失模式允许本地化的2-脱氧-2-氟葡萄糖基-肽。在子离子扫描模式下，通过串联质谱法对该标记肽进行序列测定，可将Asp-261鉴定为序列VMSDW中的催化亲核试剂。Asp-261在该家族中完全保守，与其关键作用一致，并与之前通过用conduritol B环氧化物标记在文氏曲霉酶中鉴定的天冬氨酸残基对齐。
The beta-glucosidase from Aspergillus niger (CMI. CC 324262) was purified, and an N-terminal sequence and two internal sequences were determined. BglI genomic gene and the cDNA were cloned from a genomic library and by reverse transcriptase-polymerase chain reaction, respectively. The cDNA was successfully expressed in Saccharomyces cerevisiae and Pichia pastoris. Sequence analysis revealed that the gene encodes a 92-kDa enzyme that is a member of glycosidase family 3. H-1-NMR analysis of the reaction catalyzed by this enzyme confirmed that, in common with other family 3 glycosidases, this enzyme hydrolyzes with net retention of anomeric configuration. Accordingly, the enzyme was inactivated by a-deoxy-a-fluoro beta-glucosyl fluoride, with kinetic parameters of k(i) = 4.5 min(-1), K-I = 35.4 mM, through the trapping of a covalent glycosyl enzyme intermediate, The catalytic competence of this intermediate was demonstrated by the fact that incubation with linamarin resulted in reactivation, presumably via a transglycosylation mechanism. Peptic digestion of the 2-deoxy-2-fluoroglucosyl enzyme and subsequent analysis of high pressure liquid chromatography eluates by electrospray ionization triple quadrupole mass spectrometry in the neutral loss mode allowed the localization of a 2-deoxy-2-fluoroglucosyl-peptide. Sequence determination of this labeled peptide by tandem mass spectrometry in the daughter ion scan mode permitted the identification of Asp-261 as the catalytic nucleophile within the sequence VMSDW, Asp-261 is fully conserved within this family, consistent with its key role, and aligns with the aspartic acid residue previously identified in the Aspergillus wentii enzyme by labeling with conduritol B epoxide.