Submitted to: Journal of Bacteriology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 11/13/2003
Publication Date: 1/2/2004
Citation: Richards, G.P., Parveen, S., Hammer, C., Garfield, M. 2004. Characterization of lysyl aminopeptidase activity from phosphoglucose isomerase of vibrio vulnificus. Journal of Bacteriology. 1700(2):219-229. Interpretive Summary: Vibrio vulnificus is a marine bacterium which causes sickness and death to oyster consumers. It has a number of proteins which enhance its invasiveness to humans. In searching for proteins which cause V. vulnificus to spread more readily, we identified a well-known protein with an unusual enzyme activity. It is known as phosphoglucose isomerase (PGI) and is involved in the breakdown of sugar (glucose) within bacteria and animal cells. It is also associated with the growth and spread of cells including cancer cells. This PGI has an unusual enzyme activity not previously recognized. The activity removes the amino acid lysine from the end of certain proteins. This process may inactivate some proteins involved in cellular defenses and could lead to enhanced spread of the bacterium in infected individuals. The purpose of this paper is to describe the purification of PGI and the biochemical characteristics of the enzyme. Among the biochemical characteristics evaluated were the size and charge of the protein; the optimal temperature of the enzyme activity; the effects of enzyme inhibitors on the activity of PGI; the presence of similar enzyme activity in other Vibrio bacteria and in seven non-Vibrio bacteria; the DNA sequence of the PGI gene, and the amino acid sequence of the PGI protein. Results showed a high level of similarity between the PGI of V. vunificus and the PGI of V. cholerae (87%), E. coli (77%) and humans (63%). Together, these findings should facilitate studies on the role of PGI in bacterial invasiveness, and as a potential cause of human diseases.
Technical Abstract: Phosphoglucose isomerase (PGI) is a multifunctional enzyme involved in glycolysis and gluconeogenesis and, in mammalian cells, serves as neuroleukin, autocrine motility factor, and differentiation and maturation factor. We identified another function of PGI as a novel lysyl aminopeptidase (LAP). PGI with LAP activity (PGI-LAP+) was purified to apparent homogeneity from Vibrio vulnificus. Mass spectrometry revealed that PGI-LAP+ is a heterodimer consisting of 23.4- and 60.8-kDa subunits. Only the heterodimer displayed LAP activity. The stoichiometry of a putative catalytic triad accounting for the LAP activity is discussed in a companion manuscript (Richards, G. P. J. Bacteriol. Submitted). PGI-LAP+ has a pI around 6.0 and high specificity toward the synthetic, fluorogenic substrate L-Lysyl-7-amino-4-methylcoumarin. Enzyme activity is optimal at pH 8.0, is 63.7% higher at 37°C than at 21°C, and is strongly inhibited by classical serine protease inhibitors, but not by soybean trypsin inhibitor. LAP activity was also identified in V. parahaemolyticus and V. cholerae, but was absent from seven non-Vibrio pathogens. LAP activity did not directly correlate with the virulence of V. vulnificus as determined by mouse bioassays. The pgi gene from V. vulnificus contains 1653 bp coding for a 550 amino acid protein. Cloned and expressed PGI formed a homodimer with isomerase activity, but not LAP activity. The finding of LAP activity associated with heterodimeric PGI should foster a broad search for putative substrates in an effort to elucidate the role of PGI-LAP+ in bacteria and to investigate the possible roles of PGI-LAP+ in the pathophysiology of diseases.