Gardner Jeffrey G, Escalante-Semerena Jorge C
Department of Bacteriology, University of Wisconsin, Madison, Wisconsin 53706-1567, USA.
J Bacteriol. 2008 Jul;190(14):5132-6. doi: 10.1128/JB.00340-08. Epub 2008 May 16.
The acuABC genes of Bacillus subtilis comprise a putative posttranslational modification system. The AcuA protein is a member of the Gcn5-related N-acetyltransferase (GNAT) superfamily, the AcuC protein is a class I histone deacetylase, and the role of the AcuB protein is not known. AcuA controls the activity of acetyl coenzyme A synthetase (AcsA; EC 6.2.1.1) in this bacterium by acetylating residue Lys549. Here we report the kinetic analysis of wild-type and variant AcuA proteins. We contrived a genetic scheme for the identification of AcuA residues critical for activity. Changes at residues H177 and G187 completely inactivated AcuA and led to its rapid turnover. Changes at residues R42 and T169 were less severe. In vitro assay conditions were optimized, and an effective means of inactivating the enzyme was found. The basic kinetic parameters of wild-type and variant AcuA proteins were obtained and compared to those of eukaryotic GNATs. Insights into how the isolated mutations may exert their deleterious effect were investigated by using the crystal structure of an AcuA homolog.
枯草芽孢杆菌的acuABC基因组成了一个假定的翻译后修饰系统。AcuA蛋白是Gcn5相关N - 乙酰转移酶（GNAT）超家族的成员，AcuC蛋白是I类组蛋白脱乙酰酶，而AcuB蛋白的作用尚不清楚。AcuA通过乙酰化残基Lys549来控制该细菌中乙酰辅酶A合成酶（AcsA；EC 6.2.1.1）的活性。在此，我们报告了野生型和变体AcuA蛋白的动力学分析。我们设计了一种遗传方案来鉴定对活性至关重要的AcuA残基。H177和G187残基的变化使AcuA完全失活，并导致其快速周转。R42和T169残基的变化影响较小。优化了体外测定条件，并找到了一种使该酶失活的有效方法。获得了野生型和变体AcuA蛋白的基本动力学参数，并与真核GNATs的参数进行了比较。通过使用AcuA同源物的晶体结构，研究了单个突变可能如何发挥其有害作用。
