Identification and characterisation of the catalytic triad of the alkaliphilic thermotolerant PHA depolymerase PhaZ7 of Paucimonas lemoignei
Identification and characterisation of the catalytic triad of the alkaliphilic thermotolerant PHA depolymerase PhaZ7 of Paucimonas lemoignei
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DOI：
10.1016/s0378-1097(03)00425-7
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发表时间：
2003-07-15
影响因子：
2.1
通讯作者：
Jendrossek, D
中科院分区：
文献类型：
--
作者：
Braaz, R;Handrick, R;Jendrossek, D
最近发现的Paucimonas lemoignei的细胞外聚[（R）-3-羟基丁酸]（PHB）解聚酶PhaZ 7代表丝氨酸水解酶的新亚组（EC www.example.com）的第一个成员3.1.1.75，其与常规的PHB解聚酶没有显著的氨基酸相似性，脂肪酶或其它水解酶，除了潜在的脂肪酶盒样基序（Ala-His-Ser（136）-Met-Gly）和催化三联体和氧阴离子口袋氨基酸的潜在候选物。为了鉴定活性所必需的氨基酸，通过定点诱变产生phaZ 7的11个突变体，并在重组蛋白酶缺陷型枯草芽孢杆菌WB 800中表达。野生型解聚酶和11种突变蛋白中的10种（Ser（136）Cys除外）由B表达并有效分泌。枯草芽孢杆菌，如无细胞培养液蛋白质的蛋白质印迹所示。在具有以下取代之一的菌株中未检测到PHB解聚酶活性：His（47）Ala、Ser（136）Ala、Asp（242）Ala、Asp（242）Asn、His（306）Ala，表明这些氨基酸对活性的重要性。用Thr替换Ser（136）导致活性降低至野生型水平的约20%，并表明丝氨酸侧链的羟基对于活性是重要的，但可以部分地被苏氨酸的羟基官能团替换。Asp（256）改变为Ala或Asn或假定的丝氨酸水解酶五肽基序（Ala-His-Ser（136）-Met-Gly）与脂肪酶盒共有序列（Gly（134）-His-Ser（136）-Met-Gly）或与PHB解聚酶盒共有序列（Gly（134）-Leu（135）-Ser（136）-Met-Gly）的结合对PHB解聚酶活性没有显著影响，表明这些氨基酸或序列基序不是活性所必需的。综上所述，PHB解聚酶PhaZ 7是一种丝氨酸水解酶，具有催化三联体和由His（47）、Ser（136）、Asp（242）和His（306）组成的氧阴离子口袋。(C)2003年，欧洲微生物学会联合会。由Elsevier Science B. V.出版，版权所有。
The recently discovered extracellular poly[(R)-3-hydroxybutyrate] (PHB) depolymerase PhaZ7 of Paucimonas lemoignei represents the first member of a new subgroup (EC 3.1.1.75) of serine hydrolases with no significant amino acid similarities to conventional PHB depolymerases, lipases or other hydrolases except for a potential lipase box-like motif (Ala-His-Ser(136)-Met-Gly) and potential candidates for catalytic triad and oxyanion pocket amino acids. In order to identify amino acids essential for activity 11 mutants of phaZ7 were generated by site-directed mutagenesis and expressed in recombinant protease-deficient Bacillus subtilis WB800. The wild-type depolymerase and 10 of the 11 mutant proteins (except for Ser(136)Cys) were expressed and efficiently secreted by B. subtilis as shown by Western blots of cell-free culture fluid proteins. No PHB depolymerase activity was detected in strains harbouring one of the following substitutions: His(47)Ala, Ser(136)Ala, Asp(242)Ala, Asp(242)Asn, His(306)Ala, indicating the importance of these amino acids for activity. Replacement of Ser(136) by Thr resulted in a decrease of activity to about 20% of the wild-type level and suggested that the hydroxy group of the serine side chain is important for activity but can be partially replaced by the hydroxy function of threonine. Alterations of Asp(256) to Ala or Asn or of the putative serine hydrolase pentapeptide motif (Ala-His-Ser(136)-Met-Gly) to a lipase box consensus sequence (Gly(134)-His-Ser(136)-Met-Gly) or to the PHB depolymerase box consensus sequence (Gly(134)-Leu(135)-Ser(136)-Met-Gly) had no significant effect on PHB depolymerase activity, indicating that these amino acids or sequence motifs were not essential for activity. In conclusion, the PHB depolymerase PhaZ7 is a serine hydrolase with a catalytic triad and oxyanion pocket consisting of His(47), Ser(136), Asp(242) and His(306). (C) 2003 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.