Submitted to: Biochimica et Biophysica Acta
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: May 14, 2004
Publication Date: August 2, 2004
Citation: Richards, G.P. 2004. Characterization of a lysyl aminopeptidase activity associated with phosphoglucose isomerase of vibrio vulnificus. Biochimica et Biophysica Acta. V.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 rapidly, 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 show a high level of similarity between the PGI of V. vulnificus 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, functions as neuroleukin, autocrine motility factor, and differentiation and maturation factor. We isolated and characterized PGI with a novel lysyl aminopeptidase (LysAP) activity (PGI-LysAP) from Vibrio vulnificus. Mass spectrometry revealed that PGI-LysAP is a heterodimer consisting of 23.4- and 60.8-kDa subunits. Only the heterodimer displayed LysAP activity. PGI-LysAP has a pI around 6.0 and high specificity toward the synthetic, fluorogenic substrate L-lysyl-7-amino-4-methylcoumarin. LysAP activity is optimal at pH 8.0, is 64% higher at 37oC than at 21oC, does not directly correlate with virulence, and is strongly inhibited by serine protease and metalloprotease inhibitors. PGI-LysAP was also identified in V. parahaemolyticus and V. cholerae, but was absent from non-Vibrio human pathogens. Sequencing of the pgi gene revealed 1653 bp coding for a 550 amino acid protein. Cloned and expressed PGI formed a homodimer with isomerase activity, but not LysAP activity. The finding of LysAP activity associated with heterodimeric PGI should foster a broad search for putative substrates in an effort to elucidate the role of PGI-LysAP in bacteria and its roles in the pathophysiology of diseases.