Viola Ronald E, Yerman Lyudmyla, Fowler Janet M, Arvidson Cindy G, Brubaker Robert R
Department of Chemistry, University of Toledo, 2801 W. Bancroft Street, Toledo, OH 43606, USA.
Department of Microbiology and Molecular Genetics, Michigan State University, 2215 Biomedical Physical Sciences, East Lansing, MI 48824, USA.
Microbiology (Reading). 2008 May;154(Pt 5):1271-1280. doi: 10.1099/mic.0.2007/015529-0.
It is established that cells of Yersinia pestis, the causative agent of bubonic plague, excrete l-aspartic acid at the expense of exogenous l-glutamic acid during expression of the low-calcium response. Results of enzymic analysis provided here suggest that a previously defined deficiency of aspartase (AspA) accounts for this phenomenon rather than an elevated oxaloacetate pool. The only known distinction between most sequenced isolates of aspA from Y. pestis and the active gene in Yersinia pseudotuberculosis (the immediate progenitor of Y. pestis) is a single base transversion (G.C-->T.A) causing replacement of leucine (encoded by UUG) for valine (encoded by GUG) at amino acid position 363. The gene from Y. pestis KIM possesses a unique second transversion (G.C-->T.A) at amino acid 146 causing substitution of aspartic acid (encoded by GAU) with tyrosine (encoded by UAU). We show in this study that Y. pestis expresses aspA as cross-reacting immunological material (CRIM). Functional and inactive aspA of Y. pseudotuberculosis PB1 and Y. pestis KIM, respectively, were then cloned and expressed in AspA-deficient Escherichia coli. After purification to near homogeneity, the products were subjected to biochemical analysis and found to exhibit similar secondary, tertiary and quaternary (tetrameric) structures as well as comparable Michaelis constants for l-aspartic acid. However, the k(cat) of the Y. pestis CRIM of strain KIM is only about 0.1 % of that determined for the active AspA of Y. pseudotuberculosis. Return of valine for leucine at position 363 of the Y. pestis enzyme restored normal turnover (k(cat) 86+/-2 s(-1)) provided that the amino acid substitution at position 146 was also reversed. These observations have important implications for understanding the nature of the stringent low-calcium response of Y. pestis and its role in promoting acute disease.
已确定，腺鼠疫病原体鼠疫耶尔森菌的细胞在低钙反应表达过程中，以消耗外源性L-谷氨酸为代价排泄L-天冬氨酸。本文提供的酶分析结果表明，先前确定的天冬氨酸酶（AspA）缺陷是造成这种现象的原因，而非草酰乙酸池升高。鼠疫耶尔森菌大多数测序分离株的aspA与假结核耶尔森菌（鼠疫耶尔森菌的直接祖先）中的活性基因之间唯一已知的区别是一个单碱基颠换（G.C→T.A），导致第363位氨基酸处的亮氨酸（由UUG编码）被缬氨酸（由GUG编码）取代。鼠疫耶尔森菌KIM株的基因在第146位氨基酸处有一个独特的第二个颠换（G.C→T.A），导致天冬氨酸（由GAU编码）被酪氨酸（由UAU编码）取代。我们在本研究中表明，鼠疫耶尔森菌将aspA表达为交叉反应免疫物质（CRIM）。然后分别克隆假结核耶尔森菌PB1和鼠疫耶尔森菌KIM的功能性和无活性aspA，并在缺乏AspA的大肠杆菌中表达。纯化至接近均一性后，对产物进行生化分析，发现它们具有相似的二级、三级和四级（四聚体）结构，以及对L-天冬氨酸相当的米氏常数。然而，KIM株鼠疫耶尔森菌CRIM的k(cat)仅约为假结核耶尔森菌活性AspA的k(cat)的0.1%。如果第146位氨基酸的取代也被逆转，鼠疫耶尔森菌酶第363位的缬氨酸换回亮氨酸可恢复正常周转（k(cat) 86±2 s(-1)）。这些观察结果对于理解鼠疫耶尔森菌严格的低钙反应的本质及其在促进急性疾病中的作用具有重要意义。
