The reaction mechanism of type I phosphomannose isomerases: new information from inhibition and polarizable molecular mechanics studies.

Type I phosphomannose isomerases (PMIs) are zinc-dependent metalloenzymes involved in the reversible isomerization of D-mannose 6-phosphate (M6P) and D-fructose 6-phosphate (F6P). 5-Phospho-D-arabinonohydroxamic acid (5PAH), an inhibitor endowed with nanomolar affinity for yeast (Type I) and Pseudomonas aeruginosa (Type II) PMIs (Roux et al., Biochemistry 2004; 43:2926-2934), strongly inhibits human (Type I) PMI (for which we report an improved expression and purification procedure), as well as Escherichia coli (Type I) PMI. Its K(i) value of 41 nM for human PMI is the lowest value ever reported for an inhibitor of PMI. 5-Phospho-D-arabinonhydrazide, a neutral analogue of the reaction intermediate 1,2-cis-enediol, is about 15 times less efficient at inhibiting both enzymes, in accord with the anionic nature of the postulated high-energy reaction intermediate. Using the polarizable molecular mechanics, sum of interactions between fragments ab initio computed (SIBFA) procedure, computed structures of the complexes between Candida albicans (Type I) PMI and the cyclic substrate ß-D-mannopyranose 6-phosphate (ß-M6P) and between the enzyme and the high-energy intermediate analogue inhibitor 5PAH are reported. Their analysis allows us to identify clearly the nature of each individual active site amino acid and to formulate a hypothesis for the overall mechanism of the reaction catalyzed by Type I PMIs, that is, the ring-opening and isomerization steps, respectively. Following enzyme-catalyzed ring-opening of ß-M6P by zinc-coordinated water and Gln111 ligands, Lys136 is identified as the probable catalytic base involved in proton transfer between the two carbon atoms C1 and C2 of the substrate D-mannose 6-phosphate.

Carbohydrate-deficient glycoprotein syndrome 1b: a new answer to an old diagnostic dilemma.

A patient with carbohydrate-deficient glycoprotein syndrome type 1b (CDGS1b) is reported. The patient presented at 5 months of age with failure to thrive, prolonged diarrhoea, hepatomegaly and elevated serum liver transaminases. Liver biopsy showed steatosis. A low serum albumin and elevated serum liver transaminases persisted throughout childhood during which he had repeated infectious illnesses. From the age of 10 years he had oesophageal and duodenal ulceration together with recurrent bacterial cholangitis. Liver biopsy demonstrated hepatic fibrosis. CDGS1b was suspected, supported by the finding of a protein-losing enteropathy and finally confirmed by showing a reduced phosphomannoseisomerase activity. This case illustrates a rare condition with a wide range of presentations.

Pattern of changes in the activity of enzymes of GDP-D-mannuronic acid synthesis and in the level of transcription of algA, algC and algD genes accompanying the loss and emergence of mucoidy in Pseudomonas aeruginosa.

The low activity levels of the four GDP-D-mannuronic acid-forming enzymes, even in highly alginate-producing strains of Pseudomonas aeruginosa, have made it difficult to compare enzyme activities accompanying the loss/acquisition of mucoidy. Using optimized conditions, we compared the specific activity of these enzymes in three different mucoid P. aeruginosa cystic fibrosis isolates, in their nonmucoid spontaneous variants, and in mucoid variants that emerged during extended incubation of these nonmucoid forms in acetamide broth. A correlation was established between the promptness of emergence of the mucoid forms and the differing sensitivity to nutrient-limitation-induced death of the nonmucoid compared with the isogenic mucoid population. Consistent with the undetectable levels of algD mRNA in nonmucoid forms and with the concept that the step catalyzed by the algD-encoded GDP-mannose dehydrogenase (GMD) is a key step in control of the alginate pathway, GMD activity was undetectable or showed negligible values in nonmucoid variants and correlated with alginate production. However, phosphomannose isomerase (PMI), phosphomannomutase (PMM), and GDP-mannose pyrophosphorylase (GMP) activities in the nonmucoid forms were only slightly (40-70%) below the values in the mucoid forms. Nevertheless, no transcripts homologous to algA (encoding a bifunctional enzyme that possesses both PMI and GMP activities) were detected in the nonmucoid form, and the levels of algC (encoding PMM) transcripts, although detectable in the nonmucoid variants, were, in general, much higher in the mucoid forms. These apparently intriguing observations were cleared up by the identification of two algA functional homologues in P. aeruginosa, recently reported by others, and by the identification of one algC homologue, in contig225 of the PAO1 genome sequence, defining a polypeptide with a deduced amino acid sequence that showed significant homology with that of enzymes of the phosphohexomutase family found in databases. Results are also consistent with the requirement of PMI, GMP and PMM activities for the supply of GDP-D-mannose to (at least) A-band lipopolysaccharide synthesis, while GMD channels this precursor into the alginate pathway.

Selenomethionine labelling of phosphomannose isomerase changes its kinetic properties.

Phosphomannose isomerase (PMI) is an essential enzyme in the early steps of the protein glycosylation pathway in both prokaryotes and eukaryotes. Lack of the enzyme is lethal for fungal organisms and it is thus a potential fungicidal target. To facilitate the solution of the three-dimensional structure of the enzyme from the pathogen Candida albicans, we have produced the recombinant selenomethionine-labelled enzyme (SeMet-PMI). DL41, a methionine auxotroph Escherichia coli strain, was transformed with a PMI expression plasmid and grown on an enriched selenomethionine-containing medium to high-cell densities. The SeMet-PMI protein has been purified and found by amino acid analysis to have its methionine residues replaced by selenomethionine residues. Electrospray mass spectroscopy showed a major species of 49,063 +/- 10 Da for SeMet-PMI compared to 48,735 +/- 6 Da for the normal recombinant enzyme, accounting for the incorporation of seven selenomethionine residues. SeMet-PMI crystallised isomorphously with the normal PMI protein and the crystals diffract to 0.23 nm. Kinetic characterisation of SeMet-PMI showed that its Km for the substrate mannose-6-phosphate was fourfold higher than that of its methionine-containing counterpart. The inhibition constant for zinc ions was also increased by a similar factor. However, the Vmax was unaltered. These results suggested that one or more methionine residues must be in close proximity to the substrate-binding pocket in the active site, rendering substrate access more difficult compared to the normal enzyme. This hypothesis was confirmed by the finding of four methionine residues lying along one wall of the active site.

Cloning and heterologous expression of the Candida albicans gene PMI 1 encoding phosphomannose isomerase.

Using a DNA fragment derived from the Saccharomyces cerevisiae phosphomannose isomerase (PMI) structural gene as a probe against a random ordered array library of genomic DNA from the pathogenic fungus Candida albicans, we have cloned the C. albicans PMI 1 gene. This gene, which is unique in the C. albicans genome, can functionally complement PMI-deficient mutants of both S. cerevisiae and Escherichia coli. The DNA sequence of the PMI 1 gene predicts a protein with 64.1% identity to PMI from S. cerevisiae. Sequential gene disruption of PMI 1 produces a strain with an auxotrophic requirement for D-mannose. The heterologous expression of the PMI 1 gene at levels up to 45% of total cell protein in E. coli leads to partitioning of the enzyme between the soluble and particulate fractions. The protein produced in the soluble fraction is indistinguishable in kinetic properties from the material isolated from C. albicans cells.

Identification of amino acid residues involved in the activity of phosphomannose isomerase-guanosine 5'-diphospho-D-mannose pyrophosphorylase. A bifunctional enzyme in the alginate biosynthetic pathway of Pseudomonas aeruginosa.

Phosphomannose isomerase-guanosine 5'-diphospho-D-mannose pyrophosphorylase (PMI-GMP), which is encoded by the algA gene, catalyzes two noncontiguous steps in the alginate biosynthetic pathway of Pseudomonas aeruginosa; the isomerization of D-fructose 6-phosphate to D-mannose 6-phosphate and the synthesis of GDP-D-mannose and PPi from GTP and D-mannose 1-phosphate. Amino acids that are required for the GMP enzyme activity were identified through site-directed mutagenesis of the algA gene. Mutation of Lys-175 to arginine, glutamine, or glutamate produced an enzyme whose Km for D-mannose 1-phosphate was 470-3,200-fold greater than that measured for the wild type enzyme. In addition, these mutant enzymes had a lower Vmax for the GMP activity as compared with the wild type PMI-GMP. These results indicate that Lys-175 is primarily involved in the binding of the substrate D-mannose 1-phosphate, although it is likely that other residues are required for the specificity of binding. Mutation of Arg-19 to glutamine, histidine, or leucine resulted in a 2-fold lower Vmax for the GMP enzyme activity and a 4-7-fold increase in the Km for GTP as compared with the wild type enzyme. Thus, it appears that Arg-19 functions in the binding of GTP. In addition, chymotryptic digestion of PMI-GMP showed that the carboxyl terminus is critical for PMI activity but not for GMP activity. Taken together, these results support the hypothesis that the bifunctional PMI-GMP protein is composed of two independent enzymatic domains.

Purification, cDNA cloning and heterologous expression of human phosphomannose isomerase.

Phosphomannose isomerase catalyses the interconversion of fructose-6-P and mannose-6-P and has a critical role in the supply of D-mannose derivatives required for many eukaryotic glycosylation reactions. Three classes of enzymes possessing phosphomannose-isomerase activity have been identified in bacteria and lower eukaryotes. We have purified human phosphomannose isomerase to homogeneity from placental tissue. Protein sequence information obtained from internal fragments of the protein was used to design degenerate oligonucleotides which were used to amplify a fragment of a human phosphomannose-isomerase cDNA. A full-length cDNA was isolated from a human testes lambda gt11 library using this fragment as a probe. The cDNA encoded a protein with significant sequence identity to fungal and some bacterial phosphomannose isomerases but was unrelated to those from other bacteria. Based on amino acid sequence identity we propose a classification system for enzymes with phosphomannose-isomerase activity. The cDNA, under the control of the GAL1 promoter, was expressed in a Saccharomyces cerevisiae strain from which the native gene encoding phosphomannose isomerase had been deleted. The human enzyme was found to be able to functionally substitute for the yeast enzyme. Phosphomannose-isomerase mRNA was found in all human tissues tested but was more highly expressed in heart, brain and skeletal muscle. The cDNA was expressed in Escherichia coli permitting the isolation of pure recombinant protein which will be used for kinetic and structural studies.

Overproduction and assay of Pseudomonas aeruginosa phosphomannose isomerase.

Phosphomannose isomerase activity was undetectable in extracts of mucoid (alginate-producing) Pseudomonas aeruginosa. When a P. aeruginosa gene previously shown to complement an alginate-negative mutant was overexpressed under the control of the tac promoter in the broad-host-range controlled-expression vector pMMB22, phosphomannose isomerase activity could be measured in extracts of P. aeruginosa and in a manA (phosphomannose isomerase-negative) mutant of Escherichia coli. P. aeruginosa extracts containing induced levels of enzyme were shown to interconvert fructose 6-phosphate and mannose 6-phosphate. A 56,000-dalton polypeptide was visualized on sodium dodecyl sulfate-polyacrylamide gels after induction in both hosts. When RNA-DNA dot- blot hybridization analysis was used, transcription of algA, the gene coding for P. aeruginosa phosphomannose isomerase, was not measurable from the chromosomes of either mucoid or nonmucoid P. aeruginosa. However, a high level of algA transcription was detected after expression of algA under tac promoter control in pMMB22.