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Identification of amino acids and domains required for catalytic activity of DPPR synthase, a cell wall biosynthetic enzyme of Mycobacterium tuberculosis

Stefan Berg^1,

¹ Department of Microbiology, Immunology, and Pathology, Colorado State University, Fort Collins, CO 80523, USA
² Beijing Tuberculosis and Thoracic Tumor Institute, Beijing 101149, China
³ Department of Plant Systems Biology, Flanders Interuniversity Institute for Biotechnology, Ghent University, Technologiepark 927, B-9052 Ghent, Belgium
⁴ University of Georgia, Complex Carbohydrate Research Center, 315 Riverbend Road, Athens, GA 30602, USA

Correspondence
Michael R. McNeil
mmcneil{at}colostate.edu

Decaprenylphosphoryl-D-arabinose (DPA) has been shown to be the donor of the essential D-arabinofuranosyl residues found in the cell wall of Mycobacterium tuberculosis. DPA is formed from phosphoribose diphosphate in a four-step process. The first step is the nucleophilic replacement of the diphosphate group with decaprenyl phosphate. This reaction is catalysed by the integral membrane protein 5-phospho--D-ribose-1-diphosphate : decaprenyl-phosphate 5-phosphoribosyltransferase (DPPR synthase). The enzyme is essential for growth and thereby an important target candidate for the development of new tuberculosis drugs. Although membrane proteins are an important subset of targets for current antibacterial agents, details about the structures and the active sites of such proteins are often not readily available by X-ray crystallography. To begin a different approach to the issue, homologues from Mycobacterium smegmatis and Corynebacterium glutamicum were expressed in Escherichia coli and shown to be active DPPR synthases. This was followed by bioinformatic analyses of the aligned sequences and then by site-directed mutagenesis of amino acids identified as likely to be important for activity. The results suggested that the enzymic synthesis of decaprenyl-phosphate 5-phosphoribose (DPPR) occurs on the cytoplasmic side of the plasma membrane. Amino acid substitutions showed that the predicted cytoplasmic N-terminal region and two cytoplasmic loops are involved in substrate binding and/or catalysis along with parts of some adjoining inner membrane regions. The enzyme lacks the classical phosphoribose diphosphate (pRpp) binding site found in nucleic acid precursor enzymes of both prokaryotes and eukaryotes but instead contains a conserved NDxxD motif required for enzymic activity. Thus, it is plausible that this DPPR synthase has a pRpp binding site that is different from that of the classical eukaryotic enzymes, and further work to develop inhibitors against this enzyme is thereby encouraged.

Present address: TB Research Group, Veterinary Laboratories Agency, Weybridge, New Haw, Addlestone, Surrey KT15 3NB, UK.