title: Structural enzymology and inhibition of the bi-functional folate pathway enzyme HPPK-DHPS from the biowarfare agent Francisella tularensis.
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.
  
  
  METHODS
  The coordinates and structure factors have been deposited in the Protein Data Bank under accession code 4PZV.
authors: Macromolecular Crystallography Laboratory, National Cancer Institute, Frederick, MD, USA.
authors: Gary X
authors: GX
authors: Shaw
authors: Yue
authors: Y
authors: Li
authors: Genbin
authors: G
authors: Shi
authors: Yan
authors: Y
authors: Wu
authors: Scott
authors: S
authors: Cherry
authors: Danielle
authors: D
authors: Needle
authors: Di
authors: D
authors: Zhang
authors: Joseph E
authors: JE
authors: Tropea
authors: David S
authors: DS
authors: Waugh
authors: Honggao
authors: H
authors: Yan
authors: Xinhua
authors: X
authors: Ji
mesh_terms: D000595:Amino Acid Sequence; D000900:Anti-Bacterial Agents / Q000737:chemistry*; D001426:Bacterial Proteins / Q000737:chemistry*; D054045:Biological Warfare Agents; D020134:Catalytic Domain; D018360:Crystallography, X-Ray; D004791:Enzyme Inhibitors / Q000737:chemistry*; D005492:Folic Acid / Q000096:biosynthesis; D005604:Francisella tularensis / Q000201:enzymology*; D006860:Hydrogen Bonding; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009097:Multienzyme Complexes / Q000037:antagonists & inhibitors / Q000737:chemistry*; D011485:Protein Binding; D017433:Protein Structure, Secondary
chemical_list: C105176:6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase-7,8-dihydropteroate synthase; D000900:Anti-Bacterial Agents; D001426:Bacterial Proteins; D054045:Biological Warfare Agents; D004791:Enzyme Inhibitors; D009097:Multienzyme Complexes; D005492:Folic Acid
keywords: DHPS; Francisella tularensis; HPPK; antibiotic; folate