Structural Insights into Substrate Selectivity and Activity of Bacterial Polyphosphate Kinases

Abstract

Polyphosphate (polyP) kinases are widely conserved enzymes with importance in basic bacterial metabolism and virulence in many pathogens. However, the molecular mechanisms of their substrate specificity and catalysis remain unknown. Here, we present the results of comprehensive biochemical and structural studies of three polyP kinases from different bacteria, which belong to different clusters of the PPK2 class III family. Purified PPK2 proteins catalyzed polyP-dependent phosphorylation of AMP, ADP, GMP, and GDP to corresponding nucleoside diphosphates and triphosphates. Crystal structures of these proteins in complex with substrates, products, Mg 2+ , and inhibitors revealed the binding sites for the nucleotide and polyP substrates overlapping at the Walker A and B loops. The Walker A loop is involved in the binding of polyP and the Mg 2+ ion, whereas the Walker B loop coordinates the nucleotide phosphate groups. Structure-based site-directed mutagenesis of CHU0107 from Cytophaga hutchinsonii demonstrated the critical role of several conserved residues from the PPK2 core and lid domains, which are involved in the coordination of both substrates and two Mg 2+ ions. In addition, a two-times higher activity was observed following deletion of the C-terminal tail in the CHU0107 mutant protein L285Stop. Crystal structures of PPK2 in complex with three aryl phosphonate inhibitors indicated the presence of at least two binding pockets for inhibitors located close to the Walker A loop and the catalytic residues Lys81 and Arg208. Our findings provide a molecular framework for understanding the molecular mechanisms of PPK2 kinases and have implications for future drug design and biocatalytic applications.

Several Polyphosphate Kinase 2 Enzymes Catalyse the Production of Adenosine 5′‐Polyphosphates

“…PPK2 enzymes are highly efficient biocatalysts in cofactor regeneration systems for adenosine 5′‐triphosphate (ATP)‐dependent processes . The PPK2 family can be phylogenetically divided into three classes (Scheme ) . It has been postulated that PPK2‐I enzymes catalyse the phosphorylation of nucleoside 5′‐diphosphates and PPK2‐II enzymes that of nucleoside 5′‐monophosphates, whereas PPK2‐III enzymes are able to catalyse both phosphorylation steps (Scheme ).…”

Bisphosphonic acids and related compounds as inhibitors of nucleotide‐ and polyphosphate‐processing enzymes: A PPK1 and PPK2 case study

“…Depending on the type and localization of the functionalities, the tested organophosphorus compounds were formally divided into seven categories: α‐hydroxyphosphonic acids 8a and 8b , α‐hydroxybis‐ H ‐phosphinic acids 9a–e , α‐hydroxybisphosphonic acids 10a–c , α‐aminobisphosphonic acids 11a–n , β‐aminobisphosphonic acids 12a–d , α‐amino[ P ‐(2‐phosphonoethyl]phosphinic acids 13a and 13b and α‐phosphonomethyl‐ or α‐2,2‐bisphosphonoethyl‐substituted cinnamic acids, 14a and 14b . Some of the studied compounds were preliminarily tested for inhibition of the PPK2 class II enzyme PA3455 from Pseudomonas aeruginosa and PPK2 class III from Cytophaga hutchinsonii (Nocek et al., ). Here, we increase the set of inhibitors by introducing new derivatives and a new polyacidic scaffold and compare the activity with the kinase PPK1 catalyzing the reverse phosphate transfer reaction.…”

“…More potent inhibitors were identified among bisphosphonates and other polyacids. As evidenced by the crystallographic studies, the two phosphonic groups of such compounds had the ability to occupy the positions of P1 and P2 phosphates and to follow the mode of PP recognition by PPK2 (Nocek et al., ). This mainly involved accommodation in the shallow cavity formed by a number of positively charged Lys and Arg residues present near the Walker A motif.…”

“…Contrarily, highly hydrophobic 11f comprising a p ‐benzylphenyl side chain was bound exclusively by PPK2 ( IC 50 = 62 μM) and did not inhibit PPK1. The enzyme‐ 11f interactions mode revealed by the crystal structure defined an additional hydrophobic pocket that accommodates the distal phenyl ring (Nocek et al., ). For aromatic β‐aminobisphosphonic, the situation is similar to the α‐amino counterparts.…”

“…A G‐quadruplex aptamer was selective for binding to Mycobacterium tuberculosis MTB PPK2 (a noncompetitive mechanism) with a nanomolar dissociation constant determined by isothermal titration calorimetry. Recently, we published the crystal structures of complexes that revealed the detailed binding mode of substrates, products, metal ions, and inhibitors with PPK2 class III (Nocek et al., ). In addition to these studies here, we present a systematic kinetic characterization of an extensive series of bisphosphonic and related acids as inhibitors of both polyphosphate kinases, PPK1 and PPK2, as a case study showing the general possibility of constructing inhibitors of enzymes related to oligo‐ or polyphosphate metabolism.…”