Functional analysis of the Burkholderia cenocepacia J2315 BceAJ protein with phosphomannose isomerase and GDP-D-mannose pyrophosphorylase activities.
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.
Sousa
Sílvia A
SA
IBB-Institute for Biotechnology and Bioengineering, Centre for Biological and Chemical Engineering, Instituto Superior Técnico, Lisbon, Portugal.
Moreira
Leonilde M
LM
Leitão
Jorge H
JH
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D019117:Burkholderia / Q000201:enzymology*; D002413:Cations, Divalent / Q000494:pharmacology; D002846:Chromatography, Affinity; D003001:Cloning, Molecular; D003067:Coenzymes / Q000494:pharmacology; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D023061:Gene Order; D006801:Humans; D007700:Kinetics; D008359:Mannose-6-Phosphate Isomerase / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D008670:Metals / Q000494:pharmacology; D008969:Molecular Sequence Data; D009713:Nucleotidyltransferases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D016415:Sequence Alignment
D001426:Bacterial Proteins; D002413:Cations, Divalent; D003067:Coenzymes; D008670:Metals; D011993:Recombinant Fusion Proteins; D009713:Nucleotidyltransferases; C026917:mannose 1-phosphate guanylyltransferase; D008359:Mannose-6-Phosphate Isomerase