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FEBS J. 2014 Sep;281(18):4123-37. doi: 10.1111/febs.12896. Epub 2014 Jul 23.

Structural enzymology and inhibition of the bi-functional folate pathway enzyme HPPK-DHPS from the biowarfare agent Francisella tularensis.

Author information

1
Macromolecular Crystallography Laboratory, National Cancer Institute, Frederick, MD, USA.

Abstract

Two valid targets for antibiotic development, 6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase (HPPK) and dihydropteroate synthase (DHPS), catalyze consecutive reactions in folate biosynthesis. In Francisella tularensis (Ft), these two activities are contained in a single protein, FtHPPK-DHPS. Although Pemble et al. (PLoS One 5, e14165) determined the structure of FtHPPK-DHPS, they were unable to measure the kinetic parameters of the enzyme. In this study, we elucidated the binding and inhibitory activities of two HPPK inhibitors (HP-18 and HP-26) against FtHPPK-DHPS, determined the structure of FtHPPK-DHPS in complex with HP-26, and measured the kinetic parameters for the dual enzymatic activities of FtHPPK-DHPS. The biochemical analyses showed that HP-18 and HP-26 have significant isozyme selectivity, and that FtHPPK-DHPS is unique in that the catalytic efficiency of its DHPS activity is only 1/260,000 of that of Escherichia coli DHPS. Sequence and structural analyses suggest that HP-26 is an excellent lead for developing therapeutic agents for tularemia, and that the very low DHPS activity is due, at least in part, to the lack of a key residue that interacts with the substrate p-aminobenzoic acid (pABA). A BLAST search of the genomes of ten F. tularensis strains indicated that the bacterium contains a single FtHPPK-DHPS. The marginal DHPS activity and the single copy existence of FtHPPK-DHPS in F. tularensis make this bacterium more vulnerable to DHPS inhibitors. Current sulfa drugs are ineffective against tularemia; new inhibitors targeting the unique pABA-binding pocket may be effective and less subject to resistance because any mutations introducing resistance may make the marginal DHPS activity unable to support the growth of F. tularensis.

DATABASE:

The coordinates and structure factors have been deposited in the Protein Data Bank under accession code 4PZV.

KEYWORDS:

DHPS; Francisella tularensis; HPPK; antibiotic; folate

PMID:
24975935
PMCID:
PMC5600157
DOI:
10.1111/febs.12896
[Indexed for MEDLINE]
Free PMC Article

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