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Journal of Bacteriology, May 2006, p. 3589-3599, Vol. 188, No. 10
0021-9193/06/$08.00+0     doi:10.1128/JB.188.10.3589-3599.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Structural and Functional Analysis of Rv3214 from Mycobacterium tuberculosis, a Protein with Conflicting Functional Annotations, Leads to Its Characterization as a Phosphatase

Centre for Molecular Biodiscovery and School of Biological Sciences, University of Auckland, 3A Symonds St., Private Bag 92019, Auckland, New Zealand

Received 14 December 2005/ Accepted 8 March 2006

The availability of complete genome sequences has highlighted the problems of functional annotation of the many gene products that have only limited sequence similarity with proteins of known function. The predicted protein encoded by open reading frame Rv3214 from the Mycobacterium tuberculosis H37Rv genome was originally annotated as EntD through sequence similarity with the Escherichia coli EntD, a 4'-phosphopantetheinyl transferase implicated in siderophore biosynthesis. An alternative annotation, based on slightly higher sequence identity, grouped Rv3214 with proteins of the cofactor-dependent phosphoglycerate mutase (dPGM) family. The crystal structure of this protein has been solved by single-wavelength anomalous dispersion methods and refined at 2.07-Å resolution (R = 0.229; R_free = 0.245). The protein is dimeric, with a monomer fold corresponding to the classical dPGM /ß structure, albeit with some variations. Closer comparisons of structure and sequence indicate that it most closely corresponds with a broad-spectrum phosphatase subfamily within the dPGM superfamily. This functional annotation has been confirmed by biochemical assays which show negligible mutase activity but acid phosphatase activity with a pH optimum of 5.4 and suggests that Rv3214 may be important for mycobacterial phosphate metabolism in vivo. Despite its weak sequence similarity with the 4'-phosphopantetheinyl transferases (EntD homologues), there is little evidence to support this function.