Direct Staudinger-Phosphonite Reaction Provides Methylphosphonamidates as Inhibitors of CE4 De-N-acetylases.

De-N-acetylases of ß-(1→6)-D-N-acetylglucosamine polymers (PNAG) and ß-(1→4)-D-N-acetylglucosamine residues in peptidoglycan are attractive targets for antimicrobial agents. PNAG de-N-acetylases are necessary for biofilm formation in numerous pathogenic bacteria. Peptidoglycan de-N-acetylation facilitates bacterial evasion of innate immune defenses. To target these enzymes, transition-state analogue inhibitors containing a methylphosphonamidate have been synthesized through a direct Staudinger-phosphonite reaction. The inhibitors were tested on purified PgaB, a PNAG de-N-acetylase from Escherichia coli, and PgdA, a peptidoglycan de-N-acetylase from Streptococcus pneumonia. Herein, we describe the most potent inhibitor of peptidoglycan de-N-acetylases reported to date (Ki =80 µM). The minimal inhibition of PgaB observed provides insight into key structural and functional differences in these enzymes that will need to be considered during the development of future inhibitors.

Relationship between Ni(II) and Zn(II) coordination and nucleotide binding by the Helicobacter pylori [NiFe]-hydrogenase and urease maturation factor HypB.

The pathogen Helicobacter pylori requires two nickel-containing enzymes, urease and [NiFe]-hydrogenase, for efficient colonization of the human gastric mucosa. These enzymes possess complex metallocenters that are assembled by teams of proteins in multistep pathways. One essential accessory protein is the GTPase HypB, which is required for Ni(II) delivery to [NiFe]-hydrogenase and participates in urease maturation. Ni(II) or Zn(II) binding to a site embedded in the GTPase domain of HypB modulates the enzymatic activity, suggesting a mechanism of regulation. In this study, biochemical and structural analyses of H. pylori HypB (HpHypB) revealed an intricate link between nucleotide and metal binding. HpHypB nickel coordination, stoichiometry, and affinity were modulated by GTP and GDP, an effect not observed for zinc, and biochemical evidence suggests that His-107 coordination to nickel toggles on and off in a nucleotide-dependent manner. These results are consistent with the crystal structure of HpHypB loaded with Ni(II), GDP, and Pi, which reveals a nickel site distinct from that of zinc-loaded Methanocaldococcus jannaschii HypB as well as subtle changes to the protein structure. Furthermore, Cys-142, a metal ligand from the Switch II GTPase motif, was identified as a key component of the signal transduction between metal binding and the enzymatic activity. Finally, potassium accelerated the enzymatic activity of HpHypB but had no effect on the other biochemical properties of the protein. Altogether, this molecular level information about HpHypB provides insight into its cellular function and illuminates a possible mechanism of metal ion discrimination.