An Amidase Gene, ipaH, Is Responsible for the Initial Step in the Iprodione Degradation Pathway of Paenarthrobacter sp Strain YJN-5
An Amidase Gene, ipaH, Is Responsible for the Initial Step in the Iprodione Degradation Pathway of Paenarthrobacter sp Strain YJN-5
复制标题
酰胺酶基因 ipaH 负责类杆菌属异菌脲降解途径的第一步。
DOI：
10.1128/aem.01150-18
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发表时间：
2018-10-01
影响因子：
4.4
通讯作者：
Hong, Qing
中科院分区：
文献类型：
--
作者：
Yang, Zhangong;Jiang, Wankui;Hong, Qing
异菌酮是一种广泛使用的双甲酰胺类杀菌剂，在环境中经常检测到其残留。美国环境保护署已将扑灭酮归类为对小动物中等毒性，对人类可能致癌。异菌酮的细菌降解性已被广泛研究。先前的研究表明，其N-1酰胺键的水解是异菌酮典型的细菌降解途径的第一步，但涉及异菌酮降解的酶或基因尚未见报道。在这项研究中，一个新的ipah基因编码了一个酰胺酶，该酶负责在Paenarthrobacter sp.中参与异菌二酮的起始降解步骤。克隆了菌株YJN-5。此外，还对IPAH的性质和关键氨基酸位点进行了研究。这些发现加深了我们对扑菌酮微生物降解机理的理解。摘要异丙二酮[3-(3，5-二氯苯基)N-isopropyl-2，4-dioxoimidazolidine-1-carboxamide]]是一种高效广谱双甲酰胺类杀菌剂。已经报道了几种具有异丙双酮降解能力的细菌；然而，参与这一过程的酶和基因尚未被表征。在本研究中，一株异扑菌酮降解菌Paenarthrobacter sp.对菌株YJN-5进行了分离和鉴定。菌株YJN-5通过典型的途径降解扑二酮，其N-1酰胺键以N-(3，5-二氯苯基)-2，4-二氧基咪唑烷为起始步骤。用鸟枪法从YJN-5菌株中克隆了ipaH基因，该基因编码一种新的与该步骤有关的酰胺酶。IPAH与重氮慢生根瘤菌USDA 110中的吲哚乙酰胺水解酶(IAHH)同源性最高(40%)。IPAH在35℃、pH 7.5时酶活最高，不是金属酰胺酶。IPAH对扑菌丹的kcat和Km分别为22.42 S−1和7.33μM，催化效率(kcat/Km)为3.09μM−1 S−1。iPAH具有丝氨酸-丝氨酸-赖氨酸基序，该基序在酰胺酶信号家族成员中保守。在Ipah中，Lys82、Ser157和Ser181被丙氨酸取代，导致酶活性完全丧失。此外，菌株YJN-5M失去了降解异丙二酮的能力，这表明ipah是唯一负责异丙二酮初始降解步骤的基因。Ipah基因也可以从另一株已报道的异扑菌酮降解菌--微杆菌中扩增出来。YJN-G菌株两个IpaH在氨基酸水平上的序列相似性为98%，表明IpaH在不同的菌株中存在保守性。异丙二酮是一种广泛使用的双甲酰胺类杀菌剂，在环境中经常检测到其残留量。美国环境保护署已将扑灭酮归类为对小动物中等毒性，对人类可能致癌。异菌酮的细菌降解性已被广泛研究。先前的研究表明，其N-1酰胺键的水解是异菌酮典型的细菌降解途径的第一步，但涉及异菌酮降解的酶或基因尚未见报道。在这项研究中，一个新的ipah基因编码了一个酰胺酶，该酶负责在Paenarthrobacter sp.中参与异菌二酮的起始降解步骤。克隆了菌株YJN-5。此外，还对IPAH的性质和关键氨基酸位点进行了研究。这些发现加深了我们对扑菌酮微生物降解机理的理解。
Iprodione is a widely used dicarboxamide fungicide, and its residue has been frequently detected in the environment. The U.S. Environmental Protection Agency has classified iprodione as moderately toxic to small animals and a probable carcinogen to humans. Bacterial degradation of iprodione has been widely investigated. Previous studies demonstrate that hydrolysis of its N-1 amide bond is the initial step in the typical bacterial degradation pathway of iprodione; however, enzymes or genes involved in iprodione degradation have yet to be reported. In this study, a novel ipaH gene encoding an amidase responsible for the initial degradation step of iprodione in Paenarthrobacter sp. strain YJN-5 was cloned. In addition, the characteristics and key amino acid sites of IpaH were investigated. These findings enhance our understanding of the microbial degradation mechanism of iprodione. ABSTRACT Iprodione [3-(3,5-dichlorophenyl) N-isopropyl-2,4-dioxoimidazolidine-1-carboxamide] is a highly effective broad-spectrum dicarboxamide fungicide. Several bacteria with iprodione-degrading capabilities have been reported; however, the enzymes and genes involved in this process have not been characterized. In this study, an iprodione-degrading strain, Paenarthrobacter sp. strain YJN-5, was isolated and characterized. Strain YJN-5 degraded iprodione through the typical pathway, with hydrolysis of its N-1 amide bond to N-(3,5-dichlorophenyl)-2,4-dioxoimidazolidine as the initial step. The ipaH gene, encoding a novel amidase responsible for this step, was cloned from strain YJN-5 by the shotgun method. IpaH shares the highest similarity (40%) with an indoleacetamide hydrolase (IAHH) from Bradyrhizobium diazoefficiens USDA 110. IpaH displayed maximal enzymatic activity at 35°C and pH 7.5, and it was not a metalloamidase. The kcat and Km of IpaH against iprodione were 22.42 s−1 and 7.33 μM, respectively, and the catalytic efficiency value (kcat/Km) was 3.09 μM−1 s−1. IpaH has a Ser-Ser-Lys motif, which is conserved among members of the amidase signature family. The replacement of Lys82, Ser157, and Ser181 with alanine in IpaH led to the complete loss of enzymatic activity. Furthermore, strain YJN-5M lost the ability to degrade iprodione, suggesting that ipaH is the only gene responsible for the initial iprodione degradation step. The ipaH gene could also be amplified from another previously reported iprodione-degrading strain, Microbacterium sp. strain YJN-G. The sequence similarity between the two IpaHs at the amino acid level was 98%, indicating that conservation of IpaH exists in different strains. IMPORTANCE Iprodione is a widely used dicarboxamide fungicide, and its residue has been frequently detected in the environment. The U.S. Environmental Protection Agency has classified iprodione as moderately toxic to small animals and a probable carcinogen to humans. Bacterial degradation of iprodione has been widely investigated. Previous studies demonstrate that hydrolysis of its N-1 amide bond is the initial step in the typical bacterial degradation pathway of iprodione; however, enzymes or genes involved in iprodione degradation have yet to be reported. In this study, a novel ipaH gene encoding an amidase responsible for the initial degradation step of iprodione in Paenarthrobacter sp. strain YJN-5 was cloned. In addition, the characteristics and key amino acid sites of IpaH were investigated. These findings enhance our understanding of the microbial degradation mechanism of iprodione.