A missense mutation causes aspartase deficiency in Yersinia pestis

Abstract

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 Kcat 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 (Kcat 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.