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首页> 外文期刊>Biochemistry >Functional Annotation of Two New Carboxypeptidases from the Amidohydrolase Superfamily of Enzymes
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Functional Annotation of Two New Carboxypeptidases from the Amidohydrolase Superfamily of Enzymes

机译：酰胺水解酶超家族的两个新的羧肽酶的功能注释。

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Two proteins from the amidohydrolase superfamily of enzymes were cloned, expressed,nand purified to homogeneity. The first protein, Cc0300, was from Caulobacter crescentus CB-15 (Cc0300),nwhile the second one (Sgx9355e) was derived from an environmental DNA sequence originally isolatednfrom the Sargasso Sea (gi|44371129). The catalytic functions and the substrate profiles for the twonenzymes were determined with the aid of combinatorial dipeptide libraries. Both enzymes were shownnto catalyze the hydrolysis of L-Xaa-L-Xaa dipeptides in which the amino acid at the N-terminus wasnrelatively unimportant. These enzymes were specific for hydrophobic amino acids at the C-terminus. WithnCc0300, substrates terminating in isoleucine, leucine, phenylalanine, tyrosine, valine, methionine, andntryptophan were hydrolyzed. The same specificity was observed with Sgx9355e, but this protein wasnalso able to hydrolyze peptides terminating in threonine. Both enzymes were able to hydrolyze N-acetylnand N-formyl derivatives of the hydrophobic amino acids and tripeptides. The best substrates identifiednfor Cc0300 were L-Ala-L-Leu with kcat and kcat/Km values of 37 s-1 and 1.1 u0001 105M-1 s-1, respectively, andnN-formyl-L-Tyr with kcat and kcat/Km values of 33 s-1 and 3.9 u0001 105M-1 s-1, respectively. The best substratenidentified for Sgx9355e was L-Ala-L-Phe with kcat and kcat/Km values of 0.41 s-1 and 5.8 u0001 103 M-1 s-1.nThe three-dimensional structure of Sgx9355e was determined to a resolution of 2.33 A ° with L-methioninenbound in the active site. The R-carboxylate of the methionine is ion-paired to His-237 and alsonhydrogen bonded to the backbone amide groups of Val-201 and Leu-202. The R-amino group of thenbound methionine interacts with Asp-328. The structural determinants for substrate recognition werenidentified and compared with other enzymes in this superfamily that hydrolyze dipeptides with differentnspecificities.
机译：克隆，表达并纯化来自酰胺水解酶超家族的两种蛋白质，使其均质。第一个蛋白Cc0300来自新月形杆菌CB-15（Cc0300），而第二个蛋白（Sgx9355e）则来自最初从Sargasso Sea（gi | 44371129）分离出的环境DNA序列。借助于组合二肽文库确定了两种酶的催化功能和底物谱。两种酶均显示出催化L-Xaa-L-Xaa二肽水解的作用，其中N端的氨基酸无关紧要。这些酶对C端的疏水性氨基酸具有特异性。使用nCc0300，水解终止于异亮氨酸，亮氨酸，苯丙氨酸，酪氨酸，缬氨酸，蛋氨酸和色氨酸的底物。用Sgx9355e观察到相同的特异性，但是该蛋白也不能水解终止于苏氨酸的肽。两种酶都能够水解疏水性氨基酸和三肽的N-乙酰基和N-甲酰基衍生物。对于Cc0300鉴定出的最佳底物是L-Ala-L-Leu，其kcat和kcat / Km值分别为37 s-1和1.1 u0001 105M-1 s-1，以及nN-甲酰基-L-Tyr，其kcat和kcat / Km值分别为33 s-1和3.9 u0001 105M-1 s-1。确定Sgx9355e的最佳底物是L-Ala-L-Phe，kcat和kcat / Km值分别为0.41 s-1和5.8 u0001 103 M-1 s-1.n确定Sgx9355e的三维结构的分辨率为2.33在活动位点带有L-蛋氨酸的一个°。甲硫氨酸的R-羧酸盐与His-237离子配对，氢也与Val-201和Leu-202的骨架酰胺基键合。然后结合的蛋氨酸的R-氨基与Asp-328相互作用。确定了底物识别的结构决定因素，并与该超家族中水解具有不同特异性的二肽的其他酶进行了比较。

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