...
  • 主题
高级搜索  >
历史搜索 清空历史
首页> 外文期刊>Biochemistry >Identification of residues essential for differential fatty acyl specificity of Geotrichum candidum lipases I and II
【24h】

Identification of residues essential for differential fatty acyl specificity of Geotrichum candidum lipases I and II

机译：鉴定对于念珠菌脂肪酶I和II的脂肪酰基特异性差异至关重要的残基

获取原文
获取原文并翻译 | 示例
           
页面导航
  • 摘要
  • 著录项
  • 相似文献
  • 相关主题

摘要

The fungus Geotrichum candidum produces two lipase isoenzymes, GCL I and GCL II, with distinct differences in substrate specificity despite their 86% identical primary structure. GCL I prefers ester substrates with long-chain cis (delta-9) unsaturated fatty acid moieties, whereas GCL II also accepts medium-length (C8-C14) acyl moieties in the substrate. To reveal structural elements responsible for differences in substrate differentiating ability of these isoenzymes, we designed, expressed, and characterized 12 recombinant lipase variants. Three chimeric lipases containing unique portions of the N-terminal and the C-terminal part of GCL I and GCL II, respectively, were constructed and enzymatically characterized. Activities were measured against mixed triglyceride-poly(dimethyl siloxane) particles. Our results indicate that residues within sequence positions 349-406 are essential for GCL I's high triolein/trioctanoin activity ratio of 20. The substitution of that segment in the specific GCL I to the corresponding residues in the nonspecific GCL II resulted in an enzyme with a triolein/trioctanoin activity ratio of 1.4, identical to that of GCL II. The reverse mutation in GCL II increased its specificity for triolein by a factor of 2, thus only in part restoring the high specificity seen with GCL I. In further experiments, the point mutations at the active site entrance of the GCL I, Leu358Phe and Ile357Ala/Leu358Phe, lowered the triolein/trioctanoin activity ratio from 20 to 4 and 2.5, respectively. The substitutions Cys379Phe/Ser380Tyr at the bottom of the active site cavity of GCL I decreased its specificity to a value of 3.6. Measurements of lipase activity with substrate particles composed of pure triglycerides or ethyl esters of oleic and octanoic acids resulted in qualitatively similar results as reported above. Our data reveal for the first time the identity of residues essential for the unusual substrate preference of GCL I and show that the anatomy, both at the entrance and the bottom of the active site cavity, plays a key role in substrate discrimination.
机译：念珠菌真菌产生两种脂肪酶同工酶，GCL I和GCL II，尽管它们的一级结构相同，但底物特异性却存在明显差异，尽管它们的86％相同。 GCL I首选具有长链顺式（δ-9）不饱和脂肪酸部分的酯底物，而GCL II也接受底物中的中等长度（C8-C14）酰基部分。为了揭示负责这些同工酶底物分化能力差异的结构元件，我们设计，表达和表征了12种重组脂肪酶变体。构建了三种分别包含GCL I和GCL II N末端和C末端独特部分的嵌合脂肪酶，并进行了酶学表征。测量了针对混合的甘油三酸酯-聚（二甲基硅氧烷）颗粒的活性。我们的结果表明，序列位置349-406中的残基对于GCL I的高油酸甘油三酯/三辛酸活性比为20是必不可少的。将特定GCL I中的该片段替换为非特异性GCL II中的相应残基会导致产生带有三油精/三辛酸活性比为1.4，与GCL II相同。 GCL II的反向突变将其对三油精的特异性提高了2倍，因此仅部分恢复了GCL I所见的高特异性。在进一步的实验中，GCL I，Leu358Phe和Ile357Ala活性位点入口处的点突变/ Leu358Phe将三油精/三辛酸的活性比分别从20降低到4和2.5。 GCL I活性位点腔底部的取代Cys379Phe / Ser380Tyr使其特异性降低至3.6。用由纯甘油三酸酯或油酸和辛酸的乙酯组成的底物颗粒测量脂肪酶活性，得到了与上述报道相似的定性结果。我们的数据首次揭示了对于GCL I异常的底物偏好而言必不可少的残基，并表明在活动部位腔的入口和底部的解剖结构在底物识别中起着关键作用。

著录项

相似文献

  • 外文文献
  • 中文文献
  • 专利
获取原文

客服邮箱：kefu@zhangqiaokeyan.com

京公网安备：11010802029741号 ICP备案号：京ICP备15016152号-6 六维联合信息科技 (北京) 有限公司©版权所有

  • 客服微信

  • 服务号