Torres-Bacete Jesús, Hormigo Daniel, Torres-Gúzman Raquel, Arroyo Miguel, Castillón María Pilar, García Luis José, Acebal Carmen, de la Mata Isabel
Appl Environ Microbiol. 2015 Feb;81(4):1225-33. doi: 10.1128/AEM.02352-14.
The pva gene from Streptomyces lavendulae ATCC 13664, encoding a novel penicillin V acylase (SlPVA), has been isolated and characterized. The gene encodes an inactive precursor protein containing a secretion signal peptide that is activated by two internal autoproteolytic cleavages that release a 25-amino-acid linker peptide and two large domains of 18.79 kDa (alpha-subunit) and 60.09 kDA (beta-subunit). Based on sequence alignments and the three-dimensional model of SlPVA, the enzyme contains a hydrophobicpocket involved in catalytic activity, including Serbeta1, Hisbeta23, Valbeta70, and Asnbeta272, which were confirmed by site-directed mutagenesis studies. The heterologous expression of pva in S. lividans led to the production of an extracellularly homogeneous heterodimeric enzyme at a 5-fold higher concentration (959 IU/liter) than in the original host and in a considerably shorter time. According to the catalytic properties of SlPVA, the enzyme must be classified as a new member of the Ntn-hydrolase superfamily, which belongs to a novel subfamily of acylases that recognize substrates with long hydrophobic acyl chains and have biotechnological applications in semisynthetic antifungal production.
已从淡紫色链霉菌ATCC 13664中分离并鉴定出编码新型青霉素V酰基转移酶（SlPVA）的pva基因。该基因编码一种无活性的前体蛋白，其包含一个分泌信号肽，该信号肽通过两次内部自蛋白水解切割而被激活，这两次切割释放出一个25个氨基酸的连接肽以及两个分别为18.79 kDa（α亚基）和60.09 kDa（β亚基）的大结构域。基于序列比对和SlPVA的三维模型，该酶含有一个参与催化活性的疏水口袋，包括Serβ1、Hisβ23、Valβ70和Asnβ272，这些通过定点诱变研究得到了证实。pva在变铅青链霉菌中的异源表达导致产生一种细胞外均一的异二聚体酶，其浓度（959 IU/升）比在原始宿主中高5倍，且所需时间大大缩短。根据SlPVA的催化特性，该酶必须被归类为Ntn水解酶超家族的一个新成员，该超家族属于酰基转移酶的一个新亚家族，其识别具有长疏水酰基链的底物，并在半合成抗真菌生产中具有生物技术应用。
