Comparative biochemical characterization of bacterial exopolyphosphatase proteins of diverse origins

Abstract

Inorganic polyphosphate (Poly-P) is a linear biopolymer made up of tens to hundreds of phosphate units linked by ‘high-energy’ phosphoanhydride bonds. This biopolymer is ubiquitously found in living organisms. In many species of pathogenic bacteria, poly-P has been shown to be involved in physiological processes that enable the formation of virulent and persistence infections.

Bacteria encode several conserved enzyme families to regulate intracellular poly-P levels. Members of the exopolyphosphatase-guanosine pentaphosphate phosphohydrolase (PPX-GppA) family are thought to be primarily responsible for the hydrolysis of poly-P in bacterial cells. PPX-GppA enzymes degrade poly-P from their termini, releasing phosphate units. Although PPX-GppA homologues are encoded within most bacteria, including many pathogens, they exhibit considerable variation in their respective size and sequence composition. Consequently, their respective biochemical activities and biological activities remain to be firmly established.

The first part of this thesis describes an investigation into the biochemical activities of a range of short (ca. 300-350aa) and long (ca. 500aa) PPX-GppA protein homologues from diverse bacterial species. The substrate preferences and reaction products formed by each of the respective recombinant PPX-GppA proteins were determined via chromatographic approaches. Particular emphasis was placed upon their reactivities towards nucleoside 5’-triphosphate substrates, and guanosine 5’-triphosphate, 3’-diphosphate (pppGpp). Results indicated that most ‘long’ PPX-GppA proteins possess efficient poly-P and pppGpp hydrolyzing activities, but many ‘short’ PPX-GppA homologues do not.

The second part outlines the characterization of a PPX-GppA homologue (Msmeg_5413) from Mycobacterium smegmatis. Results showed that the Msmeg_5413 protein only hydrolyzed short chain poly-P molecules of less than ca. 15 phosphate units. Mutant forms of this enzyme: N16A, E121A, D144A, E151A, T219A and R273A were created and studied. With the exception of R273A, the exopolyphosphatase and nucleotide hydrolysis activities of the mutants were significantly lower than those of the wild type protein. However, the hydrolytic activities of R273A were significantly increased. Structure-based analyses suggest this residue may act as a ‘gatekeeper’ of catalytic activities.

In the third part, the activities of the PPX-GppA protein from the oral pathogen Porphyromonas gingivalis (PG-PPX) were characterized. Unlike Msmeg_5413, the PG-PPX protein preferentially-digested long-chain poly-P. Although PG-PPX did not hydrolyze pppGpp, its exopolyphosphatase activities were inhibited by this ‘alarmone’. Furthermore, the poly-P hydrolysis activities of PG-PPX were also inhibited by AMP and GMP. The E118A mutant of PG-PPX lost most of its poly-P and nucleotide hydrolysis activities, and the R255A mutant exhibited increased nucleotide hydrolytic activities.

The last part of this thesis describes the biochemical activities of the PPX protein from Pseudomonas aeruginosa (PA-PPX). A truncation mutant, comprising the N-terminal (hydrolytic) domain of PA-PPX (PA-PPX1-318), was constructed and studied in parallel. Results indicated the PA-PPX1-318protein retained all the hydrolytic activities of the full-length protein, albeit with reduced rates or catalytic efficiencies.

In summary, my studies reveal that bacterial PPX-GppA proteins possess a range of catalytic activities that may be difficult or impossible to predict, based on sequence homology or (predicted) structure. This indicates that PPX-GppA proteins may play a variety of biochemical and biological roles within their respective host cells.