Phosphomannose Isomerase Activity Research Articles

PMI-controlled mannose metabolism and glycosylation determines tissue tolerance and virus fitness

Host survival depends on the elimination of virus and mitigation of tissue damage. Herein, we report the modulation of D-mannose flux rewires the virus-triggered immunometabolic response cascade and reduces tissue damage. Safe and inexpensive D-mannose can compete with glucose for the same transporter and hexokinase. Such competitions suppress glycolysis, reduce mitochondrial reactive-oxygen-species and succinate-mediated hypoxia-inducible factor-1α, and thus reduce virus-induced proinflammatory cytokine production. The combinatorial treatment by D-mannose and antiviral monotherapy exhibits in vivo synergy despite delayed antiviral treatment in mouse model of virus infections. Phosphomannose isomerase (PMI) knockout cells are viable, whereas addition of D-mannose to the PMI knockout cells blocks cell proliferation, indicating that PMI activity determines the beneficial effect of D-mannose. PMI inhibition suppress a panel of virus replication via affecting host and viral surface protein glycosylation. However, D-mannose does not suppress PMI activity or virus fitness. Taken together, PMI-centered therapeutic strategy clears virus infection while D-mannose treatment reprograms glycolysis for control of collateral damage.

Anthocyanin extract from Lycium ruthenicum enhanced production of biomass and polysaccharides during submerged fermentation of Agaricus bitorquis (Qu\xe9l.) Sacc. Chaidam

Agaricus bitorquis (Quél.) Sacc. Chaidam (ABSC) is a wild edible fungus uniquely found in the Tibet Plateau. ABSC is rich in polysaccharides that are considered biologically active. This study aimed to determine the feasibility of enhancing exopolysaccharide (EPS) production by ABSC in shake flask culture by supplementing the fermentation medium with anthocyanin extract. Different concentrations of Lycium ruthenicum Murr. (LRM) anthocyanin crude extract were tested on ABSC fermentation. The activity of phosphoglucose isomerase (PGI), phosphoglucose mutase (PGM), and phosphomannose isomerase (PMI), enzymes presumably involved in EPS synthesis by ABSC, was determined. ABSC transcriptomic profile in response to the presence of anthocyanins during fermentation was also investigated. LRM anthocyanin crude extract (0.06 mg/mL) was most effective in increasing EPS content and mycelial biomass (by 208.10% and 105.30%, respectively, P < 0.01). The activity of PGI, PGM, and PMI was increased in a medium where LRM anthocyanin extract and its main components (proanthocyanidins and petunia anthocyanin) were added. RNA-Seq analysis showed that 349 genes of ABSC were differentially expressed during fermentation in the medium containing anthocyanin extract of LRM; 93 genes were up-regulated and 256 genes down-regulated. From gene ontology enrichment analysis, differentially expressed genes were mostly assigned to carbohydrate metabolism and signal transduction categories. Collectively, LRM anthocyanins extract positively affected EPS production and mycelial biomass during ABSC fermentation. Our study provides a novel strategy for improving EPS production and mycelial growth during ABSC liquid submerged fermentation.

Leukocyte Phosphomannomutase and Phosphomannose Isomerase Activity in an Indian Cohort.

Advances in molecular sequencing technology has increased the diagnostic yield for Congenital disorder of glycosylation (CDG). However, novel variants or those of uncertain significance (vus) often pose a challenge and in such cases confirmed diagnosis can be warranted through enzyme analysis of these defects. We thus, aimed to optimize leukocyte-based enzyme assays for first two enzymes involved in N-glycosylation pathway i.e. Phosphomannomutase (PMM) and Phosphomannose isomerase (MPI). Study population comprised of 50 healthy non-alcoholic adults and 20 pediatric controls. Leukocyte enzyme activity was measured by monitoring the conversion of NADP to NADPH at 340nm. The conditions were optimized and precision was assessed for both low and normal activity leukocyte controls. Enzyme activities for PMM and MPI in healthy individuals were measured in the range 1.6-3.9 and 7-20nmol/min/mg protein respectively and did not vary with age and gender. The precision for both PMM and MPI showed %CV of 19.9 and 19.8 respectively. The enzyme activity in leukocyte pellet was found to be stable for up to 9months when stored at -80°C. The enzyme assays are optimized for PMM and MPI and can be used for evaluation of CDG patients in India.

Characterization of an algal phosphomannose isomerase gene and its application as a selectable marker for genetic manipulation of tomato

Establishing a transgenic plant largely relies on a selectable marker gene that can confer antibiotic or herbicide resistance to plant cells. The existence of such selectable marker genes in genetically modified foods has long been criticized. Plant cells generally exhibit too low an activity of phosphomannose isomerase (PMI) to grow with mannose as a sole carbon source. In this study, we characterized PMI from the green microalga Chlorococcum sp. and assessed its feasibility as a selectable marker for plant biotechnology. Chlorococcum sp. PMI (ChlPMI) was shown to be closely related to higher plants but more distant to bacterial counterparts. Overexpression of ChlPMI in tomato induced callus and shoot formation in media containing mannose (6 g/L) and had an average transformation rate of 3.9%. Based on this transformation system, a polycistronic gene cluster containing crtB, HpBHY, CrBKT and SlLCYB (BBBB) was co-expressed in a different tomato cultivar. Six putative transformants were achieved with a transformation rate of 1.4%, which produced significant amounts of astaxanthin due to the expression of the BBBB genes. Taken together, these findings indicate that we have established an additional tool for plant biotechnology that may be suitable for genetically modifying foods safely.

Experimental validation of in silico model-predicted isocitrate dehydrogenase and phosphomannose isomerase from Dehalococcoides mccartyi.

Gene sequences annotated as proteins of unknown or non-specific function and hypothetical proteins account for a large fraction of most genomes. In the strictly anaerobic and organohalide respiring Dehalococcoides mccartyi, this lack of annotation plagues almost half the genome. Using a combination of bioinformatics analyses and genome-wide metabolic modelling, new or more specific annotations were proposed for about 80 of these poorly annotated genes in previous investigations of D. mccartyi metabolism. Herein, we report the experimental validation of the proposed reannotations for two such genes (KB1_0495 and KB1_0553) from D. mccartyi strains in the KB-1 community. KB1_0495 or DmIDH was originally annotated as an NAD(+)-dependent isocitrate dehydrogenase, but biochemical assays revealed its activity primarily with NADP(+) as a cofactor. KB1_0553, also denoted as DmPMI, was originally annotated as a hypothetical protein/sugar isomerase domain protein. We previously proposed that it was a bifunctional phosphoglucose isomerase/phosphomannose isomerase, but only phosphomannose isomerase activity was identified and confirmed experimentally. Further bioinformatics analyses of these two protein sequences suggest their affiliation to potentially novel enzyme families within their respective larger enzyme super families.

Identification and characterization of a thermostable bifunctional enzyme with phosphomannose isomerase and sugar-1-phosphate nucleotidylyltransferase activities from a hyperthermophilic archaeon, Pyrococcus horikoshii OT3.

Mannosylglycerate is known as a compatible solute, and plays important roles for salinity adaptation and high temperature stability of microorganisms. In the gene cluster for the mannosylglycerate biosynthetic pathway predicted from the genomic data of Pyrococcus horikoshii OT3, the PH0925 protein was found as a putative bifunctional enzyme with phosphomannose isomerase (PMI) and mannose-1-phosphate guanylyltransferase (Man-1-P GTase) activities, which can synthesize GDP-mannose when accompanied by a phosphomannomutase/phosphoglucomutase (PMM/PGM) enzyme (PH0923). The recombinant PH0925 protein, expressed in E. coli, exhibited both expected PMI and Man-1-P GTase activities, as well as absolute thermostability; 95°C was the optimum reaction temperature. According to the guanylyltransferase activity (GTase) of the PH0925 protein, it was found that the protein can catalyze glucose-1-phosphate (Glc-1-P) and glucosamine-1-phosphate (GlcN-1-P) in addition to Man-1-P. The analyses of C-terminus-truncated forms of the PH0925 protein indicated that sugar-1-phosphate nucleotidylyltransferase (Sugar-1-P NTase) activity was located in the region from the N-terminus to the 345th residue, and that the C-terminal 114 residue region of the PH0925 protein inhibited the Man-1-P GTase activity. Conversely, the PMI activity was abolished by deletion of the C-terminal 14 residues. This is the first report of a thermostable enzyme with both PMI and multiple Sugar-1-P NTase activities.

Effects of culture conditions on monosaccharide composition of Ganoderma lucidum exopolysaccharide and on activities of related enzymes

We investigated the relationship between monosaccharide composition of Ganoderma lucidum exopolysaccharide (EPS) and activities of EPS synthesis enzymes under various culture temperatures and initial pH values. The mole percentages of three major EPS monosaccharides, glucose, galactose and mannose, varied depending on culture conditions and the resulting EPS displayed differing anti-tumor activities. In nine tested enzymes, higher enzyme activities were correlated with higher temperature and lower initial pH. Altered mole percentages of galactose and mannose under various culture conditions were associated with activities of α-phosphoglucomutase (PGM) and phosphoglucose isomerase (PGI), respectively, and that of mannose was also associated with phosphomannose isomerase (PMI) activity only under various pH. Our findings suggest that mole percentages of G. lucidum EPS monosaccharides can be manipulated by changes of culture conditions that affect enzyme activities, and that novel fermentation strategies based on this approach may enhance production and biological activity of EPS.

Asymptomatic phosphomannose isomerase deficiency (MPI-CDG) initially mistaken for excessive alcohol consumption

Case reportIn a routine company health check-up, a 32-year-old woman presented a highly elevated serum level of carbohydrate-deficient transferrin (CDT), a biomarker for excessive alcohol consumption. The test result (~17% disialotransferrin, reference interval <2.0%; ~3% asialotransferrin, reference 0%) was confirmed by analysis of a second sample, while another alcohol biomarker, phosphatidylethanol (PEth) in whole-blood, was negative. This suggested that her elevated CDT may be unrelated to heavy drinking. The abnormal “type-1” transferrin glycoform pattern indicated a defect in N-glycan assembly occurring in congenital disorders of glycosylation (CDG), a family of rare inherited metabolic disorders. Probing for the underlying enzyme defect(s) using cultured skin fibroblasts demonstrated normal activity of phosphomannomutase, whereas the activity of phosphomannose isomerase (MPI) was reduced (0.64mU/mg protein, reference 2.1–6.9), pointing to CDG of the MPI subtype (formerly called CDG-Ib). The diagnosis was confirmed by sequence analysis of the MPI gene revealing a homozygous missense mutation (c.656G>A) causing replacement of arginine by glutamine (p.R219Q). However, the woman had never experienced any clinical manifestations associated with MPI-CDG. Both parents, being distant relatives, were heterozygous mutation carriers with normal CDT values. Two of three siblings were not affected, whereas one brother was also homozygous for c.656G>A and had a highly elevated CDT and no clinical symptoms. ConclusionThe finding of MPI-CDG adults without clinical manifestations suggests that this type of the disorder may be underdiagnosed. If asymptomatic MPI-CDG subjects undergo CDT screening, their highly elevated test results may be wrongly interpreted as caused by excessive alcohol consumption.

Dolichol kinase deficiency (DOLK-CDG) with a purely neurological presentation caused by a novel mutation

A 4-month old boy presented with multiple epileptic seizure types including West syndrome. Screening for infectious and structural etiologies showed normal results. A metabolic investigation was undertaken to investigate the cause of his neurological disease.Screening for congenital disorders of glycosylation (CDG) by HPLC analysis of serum carbohydrate-deficient transferrin (CDT) showed a type 1 pattern with 18% disialotransferrin (reference<2%) and 2% asialotransferrin (reference 0). An undiagnosed 10-year old sister with a similar clinical history with infantile spasms at age 4months, intellectual disability and an autism spectrum disorder, also showed a type 1 CDT pattern. Both siblings lacked dysmorphic features and extra-cerebral symptoms. The boy had cytotoxic edema of the thalamus and mesencephalon on MRI at age 7months, whereas the girl had normal MRI at age 8months. Phosphomannomutase (PMM) and phosphomannose isomerase (MPI) activities in cultured fibroblasts were normal, excluding PMM2-CDG and MPI-CDG. Fibroblast lipid-linked oligosaccharide analysis was also normal, suggesting an early defect in glycan assembly. Sequence analysis of the dolichol kinase gene revealed a homozygous new missense mutation (p.M1?; c.2T>C) in both siblings.In conclusion, two siblings were demonstrated to suffer from DOLK-CDG (MIM 610768) and to be homozygous for a new mutation. They presented with West syndrome and so far show a purely neurological phenotype.

Clinical and Molecular Features of Patients with Congenital Disorders of Glycosylation in Brazil

Introduction: Congenital Disorders of Glycosylation are a group of genetic disorders due to abnormal glycosylation of glycoproteins and glycolipids. Based on isoelectric focusing of plasma transferrin results, CDG are classified in two groups: CDG-I and CDG-II. While the diagnosis of PMM2-CDG (formerly CDG-Ia) and PMI-CDG (formerly CDG-Ib) is made by demonstration of the enzyme deficiency or by gene sequencing, the diagnosis of the other CDG is not easily performed. Psychomotor delay/mental retardation, hypotonia, seizures, ataxia, cerebellar atrophy, strabismus, inverted nipples, lipodystrophy, and stroke-like episodes characterize PMM2-CDG, by far the most common CDG. There is almost no information available in the literature on the frequency of CDG in patients with psychomotor delay/ mental retardation. Patients and methods: We performed transferrin isoelectric focusing in 2619 patients who had psychomotor delay/mental retardation associated with other symptoms suggestive of CDG. Determination of leukocyte phosphomannomutase and phosphomannoseisomerase activities and PMM2 gene sequencing was performed in selected patients. Results: We found 32 affected patients (26 CDG-I and 6-CDG-II). CDG-I group: The most prevalent PMM2- CDG clinical symptoms were those expected. We identified two novel mutations: p.G79V and p.R21W. Non-PMM2, non-PMI-CDG showed more frequently coagulopathy, hypotonia, cerebellar atrophy, and cryptorchidism/micropenis. Early deaths were found exclusively in this group. Ataxia, strabismus, elevated blood FSH and LH levels were more frequent in PMM2-CDG patients. CDG-II group: four out of six patients presented cutis laxa, seizures, large fontanel, facial dysmorphism, and non-lissencephalic cortical dysplasia. Hip luxation was present in three patients, and hydronephrosis in one. The other two patients had heterogeneous features. Conclusions: We determined the frequency of CDG in a selected Brazilian cohort with symptoms suggestive of CDG as 1.2%(CDG-I ~ 1.0% and CDG-II ~ 0.2%), and identified two novel mutations in the PMM2 gene.

The de novo and salvage pathways of GDP‐mannose biosynthesis are both sufficient for the growth of bloodstream‐form Trypanosoma brucei

SummaryThe sugar nucleotide GDP-mannose is essential for Trypanosoma brucei. Phosphomannose isomerase occupies a key position on the de novo pathway to GDP-mannose from glucose, just before intersection with the salvage pathway from free mannose. We identified the parasite phosphomannose isomerase gene, confirmed that it encodes phosphomannose isomerase activity and localized the endogenous enzyme to the glycosome. We also created a bloodstream-form conditional null mutant of phosphomannose isomerase to assess the relative roles of the de novo and salvage pathways of GDP-mannose biosynthesis. Phosphomannose isomerase was found to be essential for parasite growth. However, supplementation of the medium with low concentrations of mannose, including that found in human plasma, relieved this dependence. Therefore, we do not consider phosphomannose isomerase to be a viable drug target. We further established culture conditions where we can control glucose and mannose concentrations and perform steady-state [U-13C]-d-glucose labelling. Analysis of the isotopic sugar composition of the parasites variant surface glycoprotein synthesized in cells incubated in 5 mM [U-13C]-d-glucose in the presence and absence of unlabelled mannose showed that, under physiological conditions, about 80% of GDP-mannose synthesis comes from the de novo pathway and 20% from the salvage pathway.

EFFICIENCY OF PMI/MANNOSE AS A SELECTABLE MARKER FOR TRANSFORMATION OF KALANCHOE

Mannose is an unsuitable carbon source for many plants. Various factors were studied to evaluate the efficacy of mannose in the selection process for production of transgenic plants using Agrobacterium tumefaciens-mediated transformation of leaf explants of Kalanchoe blossfeldiana. To establish a non-antibiotic selection system that utilizes the phosphomannose-isomerase (PMI) gene from E. coli, we determined the optimum mannose concentration for selecting transformed cells. Shoot organogenesis in K. blossfeldiana leaf explants was suppressed effectively on medium supplemented with mannose, or mannose with glucose, which indicated that Kalanchoe cells had little or no PMI activity and could not utilize mannose as a carbon source. The optimum concentration of mannose was found to eliminate non-transgenic shoots. Transformed plants were confirmed by GUS assay. Transformation efficiency ranged up to ca. 40%. The PMI/mannose system provides an efficient method for selecting transgenic plants without antibiotics or herbicides.

How to find and diagnose a CDG due to defective N‐glycosylation

The group of Congenital Disorders of Glycosylation (CDG) is expanding rapidly since the first clinical description of the N-glycosylation defect PMM2-CDG (CDG-Ia) in 1980 (Jaeken et al. 1980). Since then, more than 50 defects have been identified in protein N-glycosylation (some of them also associated with an O-glycosylation defect), in protein O-glycosylation only and in lipid glycosylation (GPI anchor and glycosphingolipid synthesis). Here, we provide a simple approach to the clinical, biochemical and genetic diagnosis of CDG due to a N-glycosylation defect (including combined N- and O-glycosylation defects).

Ontogeny of d-Mannose Transport and Metabolism in Rat Small Intestine

Oral mannose therapy is used to treat congenital disorders of glycosylation caused by a deficiency in phosphomannose isomerase. The segmental distribution and ontogenic regulation of D-mannose transport, phosphomannose isomerase, and phosphomannose mutase is investigated in the small intestine of fetuses, newborn, suckling, 1-month-old, and adult rats. The small intestine transports D-mannose by both Na(+)-dependent and Na(+)-independent transport mechanisms. The activities of both systems normalized to intestinal weight peak at birth and thereafter they decreased. In all the ages tested, the activity of the Na(+)-independent mechanism was higher than that of the Na(+)/mannose transport system. At birth, the Na(+)-independent D-mannose transport in the ileum was significantly higher than that in jejunum. Phosphomannose isomerase activity and mRNA levels increased at 1 month, and the values in the ileum were lower than in jejunum. Phosphomannose mutase activity in jejunum increased during the early stages of life, and it decreased at 1 month old, as does the amount of mannose incorporated into glycoproteins, whereas in the ileum, they were not affected by age. The phosphomannose isomerase/phosphomannose mutase activity ratio decreased at birth and during the suckling period, and increased at 1 month old. In conclusion, intestinal D-mannose transport activity and metabolism were affected by ontogeny and intestinal segment.

A novel mutation and first report of dilated cardiomyopathy in ALG6-CDG (CDG-Ic): a case report

Congenital disorders of glycosylation (CDG) are an expanding group of inherited metabolic diseases with multisystem involvement. ALG6-CDG (CDGIc) is an endoplasmatic reticulum defect in N-glycan assembly. It is usually milder than PMM2-CDG (CDG-Ia) and so is its natural course. It is characterized by psychomotor retardation, seizures, ataxia, and hypotonia. In contrast to PMM2-CDG (CDGIa), there is no cerebellar hypoplasia. Cardiomyopathy has been reported in a few CDG types and in a number of patients with unexplained CDG. We report an 11 year old Saudi boy with severe psychomotor retardation, seizures, strabismus, inverted nipples, dilated cardiomyopathy, and a type 1 pattern of serum transferrin isoelectrofocusing. Phosphomannomutase and phosphomannose isomerase activities were normal in fibroblasts. Full gene sequencing of the ALG6 gene revealed a novel mutation namely c.482A>G (p.Y161C) and heterozygosity in the parents. This report highlights the importance to consider CDG in the differential diagnosis of unexplained cardiomyopathy.

Nonglucose Carbohydrates and Infant Nutrition and Metabolism

Nonglucose Carbohydrates and Infant Nutrition and Metabolism

Cloning and characterization of phosphomannose isomerase from sphingomonas chungbukensis DJ77

Phosphomannose isomerase (PMI) catalyzes the interconversion of fructose-6-phosphate and mannose-6-phosphate in the extracellular polysaccharide (EPS) synthesis pathway. The gene encoding PMI in Sphingomonas chungbukensis DJ77 was cloned and expressed in E. coli. The pmi gene is 1,410 nucleotides long and the deduced amino acid sequence shares high homology with other bifunctional proteins that possess both PMI and GDP-mannose pyrophosphorylase (GMP) activities. The sequence analysis of PMI revealed two domains with three conserved motifs: a GMP domain at the N-terminus and a PMI domain at the C-terminus. Enzyme assays using the PMI protein confirmed its bifunctional activity. Both activities required divalent metal ions such as Co(2+), Ca(2+), Mg(2+), Ni(2+) or Zn(2+). Of these ions, Co(2+) was found to be the most effective activator of PMI. GDP-D-mannose was found to inhibit the PMI activity, suggesting feedback regulation of this pathway.

Characterization of the Aspergillus fumigatus phosphomannose isomerase Pmi1 and its impact on cell wall synthesis and morphogenesis

Phosphomannose isomerase (PMI) is an enzyme catalysing the interconversion of mannose 6-phosphate (Man-6-P) and fructose 6-phosphate (Fru-6-P). The reaction catalysed by PMI is the first committed step in the synthesis of mannose-containing sugar chains and provides a link between glucose metabolism and mannosylation. In this study, the pmi1 gene was identified to encode PMI in the human fungal pathogen Aspergillus fumigatus. Characterization of A. fumigatus Pmi1 expressed in Escherichia coli revealed that this PMI mainly catalysed the conversion of Fru-6-P to Man-6-P and that its binding affinity for Man-6-P was similar to that of yeast PMIs, but different to those of PMIs from bacteria or animals. Loss of pmi1 was lethal unless Man was provided in the growth medium. However, a Deltapmi1 mutant cell showed a significantly reduced growth rate at a high concentration of Man. Biochemical analysis revealed that both inadequate and replete Man led to an accumulation of intracellular Man-6-P and a reduction in the amount of alpha-glucan in the cell wall. Uncoupling of the link between energy production and glycosylation by deletion of the pmi1 gene led to phenotypes such as defects in cell wall integrity, abnormal morphology and reduced conidiation. Our results reveal that PMI activity is essential for viability and plays a central regulatory role in both cell wall synthesis and energy production in A. fumigatus.

Congenital disorder of glycosylation type Ia in a Malaysian family: Clinical outcome and description of a novel PMM2 mutation

There are few reports of congenital disorders of glycosylation (CDGs) in the Asian population, although they have been reported worldwide. We identified a Malaysian infant female at 2 days of life with CDG type Ia. The diagnosis was suspected on the basis of inverted nipples and abnormal fat distribution. She had cerebellar hypoplasia and developed coagulopathy, hypothyroidism and severe pericardial effusion and died at 7 months of life. The diagnosis was supported by abnormal serum transferrin isoform pattern that showed elevated levels of the disialotransferrin isoform and trace levels of the asialotransferrin isoform. Enzyme testing of peripheral leukocytes showed decreased level of phosphomannomutase (PMM) activity (0.6 nmol/min per mg protein, normal range 1.6-6.2) and a normal level of phosphomannose isomerase activity (19 nmol/min per mg protein, normal range 12-25), indicating a diagnosis of CDG type Ia. Mutation study of the PMM2 gene showed the patient was heterozygous for both the common p.R141H (c.422T>A) mutation and a novel sequence change in exon 7, c.618C>A. The latter change is predicted to result in the replacement of the highly conserved phenylalanine residue at position 206 with a leucine residue (p.F206L) and occurs in the same codon as the previously reported p.F206S mutation. Analysis of 100 control chromosomes has shown that the p.F206L sequence change is not present, making it highly likely that this change is functionally important. To the best of our knowledge, this is the first report of CDG in the Malay population. Prenatal diagnosis was successfully performed in a subsequent pregnancy for this family.

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.