부산대학교 도서관

To link to full-text access for this article, visit this link: http://dx.doi.org/10.1016/j.gene.2009.04.002 Byline: Sin-Lui Yeung (a), Chiwai Cheng (a), Thomas K.O. Lui (b), Jimmy S.H. Tsang (a), Wing-Tat Chan (b), Boon L. Lim (a) Keywords: Burkholderia; Phosphatase; Protein phosphatase; Purple acid phosphatase; Purple alkaline phosphatase; Twin-arginine signal peptide Abbreviations: BcPAP, Burkholderia cenocepacia J2315 PAP-like protein; rBcPAP, recombinant BcPAP; GST, glutathione S-transferase; ICP-MS, inductively coupled mass spectrometry; LB, Luria-Bertani; PAP, purple acid phosphatase; PEP, phosphoenolpyruvate; Pi, orthophosphate; p-NPP, p-nitrophenyl phosphate; PP, protein phosphatase Abstract: Purple acid phosphatases (PAP) are a group of dimetallic phosphohydrolase first identified in eukaryotes. Bioinformatics analysis revealed 57 prokaryotic PAP-like sequences in the genomes of 43 bacteria and 4 cyanobacteria species. A putative PAP gene (BcPAP) from the bacteria Burkholderia cenocepacia J2315 was chosen for further studies. Synteny analysis showed that this gene is present as an independent gene in most of the members of the genus Burkholderia. The predicted 561 a.a. polypeptide of BcPAP was found to harbour all the conserved motifs of the eukaryotic PAPs and an N-terminal twin-arginine translocation signal. Expression and biochemical characterization of BcPAP in Escherichia coli revealed that this enzyme has a relatively narrow substrate spectrum, preferably towards phosphotyrosine, phosphoserine and phosphoenolpyruvate. Interestingly, this enzyme was found to have a pH optimum at 8.5, rather than an acidic optima exhibited by eukaryotic PAPs. BcPAP contains a dimetallic ion centre composed of Fe and Zn, and site-directed mutagenesis confirmed that BcPAP utilizes the invariant residues for metal-ligation and catalysis. The enzyme is secreted by the wild type bacteria and its expression is regulated by the availability of orthophosphate. Our findings suggest that not all members in the PAP family have acidic pH optimum and broad substrate specificity. Author Affiliation: (a) School of Biological Sciences, University of Hong Kong, Hong Kong, China (b) Department of Chemistry, University of Hong Kong, Hong Kong, China Article History: Received 26 February 2009; Revised 31 March 2009; Accepted 7 April 2009 Article Note: (miscellaneous) Received by Dr. D.L. Court.