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A missense mutation causes aspartase deficiency in Yersinia pestis

¹ Department of Chemistry, University of Toledo, 2801 W. Bancroft Street, Toledo, OH 43606, USA
² Department of Microbiology and Molecular Genetics, Michigan State University, 2215 Biomedical Physical Sciences, East Lansing, MI 48824, USA
³ Department of Microbiology, The University of Chicago, 920 E. 58th Street, Chicago, IL 60637, USA

Correspondence
Robert R. Brubaker
t-rbruba{at}bsd.uchicago.edu

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·CT·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·CT·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.

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