Functional analysis of the Burkholderia cenocepacia J2315 BceAJ protein with phosphomannose isomerase and GDP-D-mannose pyrophosphorylase activities.

IBB-Institute for Biotechnology and Bioengineering, Centre for Biological and Chemical Engineering, Instituto Superior Técnico, Lisbon, Portugal.
Applied Microbiology and Biotechnology (Impact Factor: 3.69). 07/2008; 80(6):1015-22. DOI: 10.1007/s00253-008-1612-1
Source: PubMed

ABSTRACT The bceA(J) gene from the cystic fibrosis isolate Burkholderia cenocepacia J2315 encodes a 56-kDa bifunctional protein, with phosphomannose isomerase (PMI) and guanosine diphosphate (GDP)-mannose pyrophosphorylase (GMP) activities, a new member of the poorly characterised type II PMI class of proteins. Due to the lack of homology between the type II PMIs and the human PMI, this class of proteins are being regarded as interesting potential targets to develop new antimicrobials. The BceA(J) protein conserves the four typical motifs of type II PMIs: the pyrophosphorylase signature, the GMP active site, the PMI active site and the zinc-binding motif. After overproduction of BceA(J) by Escherichia coli as a histidine tag derivative, the protein was purified to homogeneity by affinity chromatography. The GMP activity is dependent on the presence of Mg(2+) or Ca(2+) as cofactors, while the PMI activity uses a broader range of divalent ions, in the order of activation Mg(2+) > Ca(2+) > Mn(2+) > Co(2+) > Ni(2+). The kinetic parameters K(m), V(max) and K(cat)/K(m) for the PMI and GMP activities were determined. Results suggest that the enzyme favours the formation of GDP-mannose instead of mannose catabolism, thus channelling precursors to the formation of glycoconjugates.

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    ABSTRACT: Burkholderia cenocepacia J2315 is a highly virulent and epidemic clinical isolate of the B. cepacia complex (Bcc), a group of bacteria that have emerged as important pathogens to cystic fibrosis patients. This bacterium, together with all Bcc strains and a few other prokaryotes, is unusual for encoding in its genome two distinct and functional Hfq-like proteins. In this work, we show results indicating that the 188-amino-acid Hfq2 protein is required for the full virulence and stress resistance of B. cenocepacia J2315, despite the presence on its genome of the functional 79-amino-acid Hfq protein encoded by the hfq gene. Similar to other Hfq proteins, Hfq2 is able to bind RNA. However, Hfq2 is unique in its ability to apparently form trimers in vitro. Maximal transcription of hfq was observed in B. cenocepacia J2315 cells in the early exponential phase of growth. In contrast, hfq2 transcription reached maximal levels in cells in the stationary phase, depending on the CepR quorum-sensing regulator. These results suggest that tight regulation of the expression of these two RNA chaperones is required to maximize the fitness and virulence of this bacterium. In addition, the ability of Hfq2 to bind DNA, not observed for Hfq, suggests that Hfq2 might play additional roles besides acting as an RNA chaperone.
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    PLoS ONE 01/2011; 6(10):e25514. · 3.53 Impact Factor
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