Sequence of the lid affects activity and specificity of Candida rugosa lipase isoenzymes
Sequence of the lid affects activity and specificity of Candida rugosa lipase isoenzymes
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DOI：
10.1110/ps.0304003
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发表时间：
2003-10-01
期刊：
影响因子：
8
通讯作者：
Lotti, M
中科院分区：
文献类型：
--
作者：
Brocca, S;Secundo, F;Lotti, M
真菌Candida rugosa产生多种脂肪酶同工酶（CRL），其在底物特异性方面具有明显差异，特别是关于对脂肪酰基链长度的选择性。此外，同种型CRL 3显示出对胆固醇酯的高活性。脂肪酶同工酶共享超过80%的序列同一性，但在盖子的序列中存在分歧，盖子是一个调节活性位点进入的移动的环。在活性酶构象中，开盖参与底物结合位点并有助于底物识别。为了解决盖在CRL活性和特异性中的作用，我们取代了重组rCRL 1中同工酶CRL 3和CRL 4的盖序列，从而获得仅在这一段残基上不同的酶。交换CRL 3盖足以赋予CRL 1胆固醇酯酶活性。另一方面，未观察到链长特异性的特定偏移。嵌合蛋白在反应介质中对去污剂表现出不同的敏感性。
The fungus Candida rugosa produces multiple lipase isoenzymes (CRLs) with distinct differences in substrate specificity, in particular with regard to selectivity toward the fatty acyl chain length. Moreover, isoform CRL3 displays high activity towards cholesterol esters. Lipase isoenzymes share over 80% sequence identity but diverge in the sequence of the lid, a mobile loop that modulates access to the active site. In the active enzyme conformation, the open lid participates in the substrate-binding site and contributes to substrate recognition. To address the role of the lid in CRL activity and specificity, we substituted the lid sequences from isoenzymes CRL3 and CRL4 in recombinant rCRL1, thus obtaining enzymes differing only in this stretch of residues. Swapping the CRL3 lid was sufficient to confer to CRL1 cholesterol esterase activity. On the other hand, a specific shift in the chain-length specificity was not observed. Chimeric proteins displayed different sensitivity to detergents in the reaction medium.