Abstract
Inosine 5'-monophosphate dehydrogenase (IMPDH) is the rate-limiting enzyme in de novo guanine nucleotide biosynthesis. IMPDH converts IMP to xanthosine 5'-monophosphate with concomitant conversion of NAD+ to NADH. All IMPDHs characterized to date contain a 130-residue "subdomain" that extends from an N-terminal loop of the alpha/beta barrel domain. The role of this subdomain is unknown. An IMPDH homolog has been cloned from Borrelia burgdorferi, the causative agent of Lyme disease (Margolis, N., Hogan, D., Tilly, K., and Rosa, P. A. (1994) J. Bacteriol. 176, 6427-6432). This homolog has replaced the subdomain with a 50-residue segment of unrelated sequence. We have expressed and characterized the B. burgdorferi IMPDH homolog. This protein has IMPDH activity, which unequivocally demonstrates that the subdomain is not required for catalytic activity. The monovalent cation and dinucleotide binding sites of B. burgdorferi IMPDH are significantly different from those of human IMPDH. Therefore, these sites are targets for the design of specific inhibitors for B. burgdorferi IMPDH. Such inhibitors might be new treatments for Lyme disease.
Highlights
We have demonstrated that the guaB homolog of B. burgdorferi encodes IMPDH
We show that the monovalent cation and dinucleotide binding sites of B. burgdorferi IMPDH differ significantly from mammalian IMPDHs
Assays were performed in 100 mM KCl, 1 mM dithiothreitol, 50 mM Tris, pH 8.0, 37 °C
Summary
Vol 272, No 35, Issue of August 29, pp. 21977–21981, 1997 Printed in U.S.A. Expression, Purification, and Characterization of Inosine 5-Monophosphate Dehydrogenase from Borrelia burgdorferi*. All IMPDHs characterized to date contain a 130-residue “subdomain” that extends from an N-terminal loop of the ␣/ barrel domain. The role of this subdomain is unknown. Inosine 5Ј-monophosphate dehydrogenase catalyzes the conversion of IMP to XMP1 with the concomitant reduction of NAD to NADH This reaction is the rate-limiting step in guanine nucleotide biosynthesis, and is a target for numerous chemotherapeutic agents (1). The genes encoding GMP synthase (guaA) and IMPDH (guaB) are located on a 26-kb circular plasmid (cp26) in B. burgdorferi (15). The subdomain is present in all of the IMPDHs characterized to date, the subdomain of the guaB1 homolog from Mycobacterium leprae is missing 90 residues Details of the purification are described under “Experimental Procedures.” Enzyme was assayed in 250 M IMP, 500 M NAD, 100 mM KCl, 1 mM dithiothreitol, 3 mM EDTA, 50 mM Tris, pH 8.0, 25 °C
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