Structure and Function of a Hypothetical Pseudomonas aeruginosa Protein PA1167 Classified into Family PL-7
Structure and Function of a Hypothetical Pseudomonas aeruginosa Protein PA1167 Classified into Family PL-7
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DOI：
10.1074/jbc.m402466200
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发表时间：
2004-07
影响因子：
4.8
通讯作者：
M. Yamasaki;Satoko Moriwaki;O. Miyake;W. Hashimoto;K. Murata;B. Mikami
中科院分区：
文献类型：
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作者：
M. Yamasaki;Satoko Moriwaki;O. Miyake;W. Hashimoto;K. Murata;B. Mikami
藻酸盐裂解酶的结构和功能分析对于澄清铜绿假单胞菌生物膜依赖性生态系统和开发细菌性疾病治疗剂具有重要意义。大多数海藻酸解酶根据其一级结构可分为多糖解酶（多糖解酶）家族5和-7。目前对PL-7家族酶的结构特性的研究还很有限。在PL-7家族中，在P. aeruginosa基因组中发现了一个编码与藻酸鞘单胞菌裂解酶A1-II同源的假设蛋白（PA1167）的基因。PA1167在大肠杆菌中过表达，裂解海藻酸盐中的糖苷键，释放不饱和糖，表明PA1167是一种海藻酸盐裂解酶，催化β-消除反应。该酶在pH 8.5和40°C条件下对异聚区域具有较好的内溶性作用，工作效率最高。而PA1167的比活性远弱于已知的海藻酸解聚酶AlgL，说明在P. aeruginosa中，AlgL在海藻酸解聚过程中起主要作用。除了这种比活性外，PA1167和AlgL在酶性质上也存在差异，如分子质量、最适pH、盐效应和底物特异性。在2.0 Å分辨率下测定了PL-7海藻酸解酶家族的第一个晶体结构。PA1167形成了一个由15条β链和3条α-螺旋组成的手套状β-三明治。PL-7家族的β-三明治PA1167与PL-5家族的α/α-桶状AlgL之间的结构差异可能是导致酶特性的原因。目前测定的多糖裂解酶的晶体结构表明，它们可归属于以平行β-螺旋结构、α/α-桶状结构和α/α-桶状+反平行β-片状结构为基本框架的3个折叠基团。PA1167是在多糖裂解酶中发现的第四个新的折叠结构。
Structural and functional analyses of alginate lyases are important in the clarification of the biofilm-dependent ecosystem in Pseudomonas aeruginosa and in the development of therapeutic agents for bacterial disease. Most alginate lyases are classified into polysaccharide lyase (PL) family-5 and -7 based on their primary structures. Family PL-7 enzymes are still poorly characterized especially in structural properties. Among family PL-7, a gene coding for a hypothetical protein (PA1167) homologous to Sphingomonas alginate lyase A1-II was found to be present in the P. aeruginosa genome. PA1167 overexpressed in Escherichia coli cleaved glycosidic bonds in alginate and released unsaturated saccharides, indicating that PA1167 is an alginate lyase catalyzing a β-elimination reaction. The enzyme acted preferably on heteropolymeric regions endolytically and worked most efficiently at pH 8.5 and 40 °C. The specific activity of PA1167, however, was much weaker than that of the known alginate lyase AlgL, suggesting that AlgL plays a main role in alginate depolymerization in P. aeruginosa. In addition to this specific activity, differences were found between PA1167 and AlgL in enzyme properties such as molecular mass, optimum pH, salt effect, and substrate specificity. The first crystal structure of the family PL-7 alginate lyase was determined at 2.0 Å resolution. PA1167 was found to form a glove-like β-sandwich composed of 15 β-strands and 3 α-helices. The structural difference between the β-sandwich PA1167 of family PL-7 and α/α-barrel AlgL of family PL-5 may be responsible for the enzyme characteristics. Crystal structures of polysaccharide lyases determined so far indicate that they can be assigned to three folding groups having parallel β-helix, α/α-barrel, and α/α-barrel + antiparallel β-sheet structures as basic frames. PA1167 is the fourth novel folding structure found among polysaccharide lyases.