Chaloupková Radka, Sýkorová Jana, Prokop Zbynek, Jesenská Andrea, Monincová Marta, Pavlová Martina, Tsuda Masataka, Nagata Yuji, Damborský Jirí
National Centre for Biomolecular Research, Masaryk University, Kotlarska 2, 611 37 Brno, Czech Republic.
J Biol Chem. 2003 Dec 26;278(52):52622-8. doi: 10.1074/jbc.M306762200. Epub 2003 Oct 2.
Structural comparison of three different haloalkane dehalogenases suggested that substrate specificity of these bacterial enzymes could be significantly influenced by the size and shape of their entrance tunnels. The surface residue leucine 177 positioned at the tunnel opening of the haloalkane dehalogenase from Sphingomonas paucimobilis UT26 was selected for modification based on structural and phylogenetic analysis; the residue partially blocks the entrance tunnel, and it is the most variable pocket residue in haloalkane dehalogenase-like proteins with nine substitutions in 14 proteins. Mutant genes coding for proteins carrying all possible substitutions in position 177 were constructed by site-directed mutagenesis and heterologously expressed in Escherichia coli. In total, 15 active protein variants were obtained, suggesting a relatively high tolerance of the site for the introduction of mutations. Purified protein variants were kinetically characterized by determination of specific activities with 12 halogenated substrates and steady-state kinetic parameters with two substrates. The effect of mutation on the enzyme activities varied dramatically with the structure of the substrates, suggesting that extrapolation of one substrate to another may be misleading and that a systematic characterization of the protein variants with a number of substrates is essential. Multivariate analysis of activity data revealed that catalytic activity of mutant enzymes generally increased with the introduction of small and nonpolar amino acid in position 177. This result is consistent with the phylogenetic analysis showing that glycine and alanine are the most commonly occurring amino acids in this position among haloalkane dehalogenases. The study demonstrates the advantages of using rational engineering to develop enzymes with modified catalytic properties and substrate specificities. The strategy of using site-directed mutagenesis to modify a specific entrance tunnel residue identified by structural and phylogenetic analyses, rather than combinatorial screening, generated a high percentage of viable mutants.
对三种不同的卤代烷脱卤酶进行结构比较表明，这些细菌酶的底物特异性可能会受到其入口通道大小和形状的显著影响。基于结构和系统发育分析，选择了位于少动鞘氨醇UT26卤代烷脱卤酶通道开口处的表面残基亮氨酸177进行修饰；该残基部分阻塞了入口通道，并且是卤代烷脱卤酶样蛋白中变化最大的口袋残基，在14种蛋白中有9种发生了取代。通过定点诱变构建了编码在第177位携带所有可能取代的蛋白质的突变基因，并在大肠杆菌中进行了异源表达。总共获得了15种活性蛋白变体，表明该位点对引入突变具有较高的耐受性。通过测定12种卤化底物的比活性和两种底物的稳态动力学参数，对纯化的蛋白变体进行了动力学表征。突变对酶活性的影响随底物结构的不同而有很大差异，这表明将一种底物外推到另一种底物可能会产生误导，对多种底物的蛋白变体进行系统表征至关重要。活性数据的多变量分析表明，突变酶的催化活性通常随着在第177位引入小的非极性氨基酸而增加。这一结果与系统发育分析一致，该分析表明甘氨酸和丙氨酸是卤代烷脱卤酶中该位置最常见的氨基酸。该研究证明了使用合理工程开发具有修饰催化特性和底物特异性的酶的优势。通过定点诱变修饰由结构和系统发育分析确定的特定入口通道残基的策略，而不是组合筛选，产生了高比例的可行突变体。
