Francklyn C, Adams J, Augustine J
Department of Biochemistry, University of Vermont College of Medicine, Health Sciences Complex, Burlington, VT, 05405, USA. franck@emba/uvm.edu
J Mol Biol. 1998 Jul 31;280(5):847-58. doi: 10.1006/jmbi.1998.1902.
The expression of histidine biosynthetic genes in enteric bacteria is regulated by an attenuation mechanism in which the level of histidyl-tRNA serves as a key sensor of the intracellular histidine pool. Among the early observations that led to the formation of this model for Salmonella typhimurium were the identification of mutants in the gene (hisS) encoding histidyl-tRNA synthetase. We report here the detailed biochemical characterization of five of these S. typhimurium bradytrophic mutants isolated by selection for resistance to histidine analogs, including identification of the deduced amino acid substitutions and determination of the resulting effects on the kinetics of adenylation and aminoacylation. Using the crystal structure of the closely related Escherichia coli histidyl-tRNA synthetase (HisRS) as a guide, two mutants were mapped to a highly conserved proline residue in motif 2 (P117S, P117Q), and were correlated with a fivefold decrease in the kcat for the pyrophosphate exchange reaction, as well as a tenfold increase in the Km for tRNA in the aminoacylation reaction. Another mutant substitution (A302T) mapped to a residue adjacent to the histidine binding pocket, leading to a tenfold increase in Km for histidine in the pyrophosphate exchange reaction. The remaining two mutants (S167F, N254T) substitute residues in or directly adjacent to the hinge region, which joins the insertion domain between motif 2 and motif 3 to the catalytic core, and cause the Km for tRNA to increase four- to tenfold. The kinetic analysis of these mutants establishes a direct link between critical interactions within the active site of HisRS and regulation of histidine biosynthesis, and provides further evidence for the importance of local conformational changes during the catalytic cycle.
肠道细菌中组氨酸生物合成基因的表达受衰减机制调控，其中组氨酰 - tRNA的水平是细胞内组氨酸库的关键传感器。在导致鼠伤寒沙门氏菌这一模型形成的早期观察结果中，有对编码组氨酰 - tRNA合成酶的基因（hisS）中突变体的鉴定。我们在此报告通过选择对组氨酸类似物具有抗性而分离出的五个鼠伤寒沙门氏菌生长缓慢突变体的详细生化特性，包括推导的氨基酸取代的鉴定以及对腺苷酸化和氨酰化动力学产生的影响的测定。以密切相关的大肠杆菌组氨酰 - tRNA合成酶（HisRS）的晶体结构为指导，两个突变体被定位到基序2中一个高度保守的脯氨酸残基上（P117S，P117Q），并与焦磷酸交换反应的催化常数（kcat）降低五倍以及氨酰化反应中tRNA的米氏常数（Km）增加十倍相关。另一个突变取代（A302T）定位到与组氨酸结合口袋相邻的一个残基上，导致焦磷酸交换反应中组氨酸的Km增加十倍。其余两个突变体（S167F，N254T）取代了在铰链区或紧邻铰链区的残基，该铰链区将基序2和基序3之间的插入结构域与催化核心连接起来，并导致tRNA的Km增加四到十倍。这些突变体的动力学分析在HisRS活性位点内的关键相互作用与组氨酸生物合成的调控之间建立了直接联系，并为催化循环过程中局部构象变化的重要性提供了进一步的证据。
