乙酰辅酶A合成酶家族中保守的色氨酸残基的生化功能（英文）  

The Role of a Conserved Tryptophan Residue in Acetyl-Coenzyme A Synthetase

作　　者：孟宇 MENG Yu(School of Natural Sciences, Kean University, 1000 Morris Ave, Union, NJ 07083, U S A)

机构地区：[1]肯恩大学自然科学学院生命科学系

出　　处：《中国生物化学与分子生物学报》2016年第11期1219-1226,共8页Chinese Journal of Biochemistry and Molecular Biology

基　　金：Supported by Zhejiang Province Natural Science Foundation(No.LY16H080008)~~

摘　　要：甲烷菌与甲烷八叠球菌是仅有的两种已知利用乙酸盐进行甲烷生成的菌属。稻田以及厌氧的废物分解物是甲烷生物生成的主要来源。甲烷菌在自然界广泛分布,相比甲烷八叠球菌,在低乙酸盐的环境中对乙酸盐仍有高亲和力。在甲烷生成第一步即将乙酸盐转化为乙酰辅酶A的过程中,与甲烷八叠球菌利用乙酸激酶与磷酸转乙酰酶激活途径不同,甲烷菌通过腺嘌呤形成乙酰辅酶A合成酶进行催化。在甲烷菌一属(Methanosaeta concilii)中,共发现5个乙酰辅酶A合成酶的编码基因,其中3种乙酰辅酶A合成酶的生化及酶活特性已被确定。该3种乙酰辅酶A合成酶均以乙酸盐为其最优底物。尽管在短链乙酰辅酶A合成酶家族中,发现酰基底物结合位点高度保守,但乙酰辅酶A合成酶家族的酰基底物范围极为广泛。本研究对甲烷菌中不同种乙酰辅酶A合成酶的酰基底物结合位点的关键氨基酸进行识别与比较,从而对乙酰辅酶A合成酶家族的酶活特性有更全面深入的了解。首先,我们对甲烷菌一属中乙酰辅酶A合成酶4进行生化性质测定。结果表明,该酶无催化一系列酰基底物为酰基辅酶A或其中间产物酰基腺苷酸的活性。通过序列对比发现,嗜热自养甲烷杆菌的乙酰辅酶A合成酶1中高度保守的416位色氨酸残基在甲烷菌一属的乙酰辅酶A合成酶4中被替换成528位苯丙氨酸残基。将甲烷菌一属的乙酰辅酶A合成酶4中的528位苯丙氨酸残基点突变为色氨酸残基后,进行酶学性质测定,未检测到该突变体具有乙酰辅酶A/乙酰腺苷酸合成活性。我们进一步对嗜热自养甲烷杆菌的乙酰辅酶A合成酶1中的416位色氨酸残基点突变为苯丙氨酸残基,酶活性质结果显示,突变酶对于乙酸盐以及丙酸盐作为底物时的活性未有明显差异。然而,以丙酸盐为底物时,释放丙酰腺苷酸中间产物。该结果表明,热自养甲烷杆菌的�Methanosaeta and Methanosarcina are the only two known genera that are able to utilize acetate as a substrate for methanogenesis. Methanosaeta are widely distributed in nature. Due to their high affinity for acetate, Methanosaeta prevail over Methanosarcina in the low acetate environments of rice paddies and anaerobic waste digesters, both of which are major sources of biogenic methane. Unlike Methanosarcina which utilizes the acetate kinase-phosphotransacetylase pathway to activate acetate to acetyl-CoA in the first step of the methanogenesis, Methanosaeta employs AMP-forming acetyl-CoA synthetase（Acs）. There are five Acs-encoding genes in total that have been identified in Methanosaeta concilii and three of which have been characterized previously to show substrate preference toward acetate. Although the acyl substrate binding pocket has been reported to be highly conserved in small- chain Acs, the archaeal Acs family has a wide range of acyl substrate utilization. In this study, we aimed to identify the key residues involved in the acyl substrate binding pockets among various Acs in Methanosaeta to further understand the enzymatic mechanism of Acs family. Here we firstly characterized the Ms. concilii Acsd-（ ACS4M~~ ）, which showed very little to no acyl-CoA synthetase/acyl-adenylate synthetase activities. According to sequence alignment, the Trp416 in Methanothermobacter thermautotrophicus Acsl （Acsl Mt） ＇ shown to determine the acyl substrate utilization, is highly conserved among other Acs＇ s but replaced by Phe528 in Acs4Mso. Characterization of the Phe528Trp variant in ACS4Msc still showed undetectable acyl-CoA synthetase/acyl-adenylate synthetase activity as compared to the wild type enzyme. Furthermore, the Trp416Phe variant in Acsl Mt showed similar kinetic parameters for acyl-CoA synthetase activity with either acetate or propionate; however, this variant released propionyl-adenylate as a free product, suggesting that AcslM, has different interactions with acetate and propionate. The b

关 键 词：关键词乙酰辅酶A合成酶 甲烷八叠球菌 甲烷菌 酶活动力学 酰基底物 

分 类 号：Q936[生物学—微生物学]