Identification of the pgmG gene, encoding a bifunctional protein with phosphoglucomutase and phosphomannomutase activities, in the gellan gum-producing strain Sphingomonas paucimobilis ATCC 31461

The pgmG gene of Sphingomonas paucimobilis ATCC 31461, the industrial gellan gum-producing strain, was cloned and sequenced. It encodes a 50,059-Da polypeptide that has phosphoglucomutase (PGM) and phosphomannomutase (PMM) activities and is 37 to 59% identical to other bifunctional proteins with PGM and PMM activities from gram-negative species, including Pseudomonas aeruginosa AlgC. Purified PgmG protein showed a marked preference for glucose-1-phosphate (G1P); the catalytic efficiency was about 50-fold higher for G1P than it was for mannose-1-phosphate (M1P). The estimated apparent K(m) values for G1P and M1P were high, 0.33 and 1.27 mM, respectively. The pgmG gene allowed the recovery of alginate biosynthetic ability in a P. aeruginosa mutant with a defective algC gene. This result indicates that PgmG protein can convert mannose-6-phosphate into M1P in the initial steps of alginate biosynthesis and, together with other results, suggests that PgmG may convert glucose-6-phosphate into G1P in the gellan pathway.     

Cloning, expression and characterization of a phosphoglucomutase/phosphomannomutase from sphingan-producing Sphingomonas sanxanigenens

Several strains of the genus Sphingomonas produce sphingans, extracellular polysaccharides used as thickeners, emulsifiers and gelling agents. The pgmG gene from Sphingomonas sanxanigenens, which encodes a bifunctional protein with phosphoglucomutase and phosphomannomutase activities, was cloned and sequenced. The predicted amino acid sequence of the PgmG protein possessed 460 amino acids and a calculated molecular mass of 49.8 kDa, and it was 80 % identical to PGM/PMM from S. elodea. We overexpressed pgmG in Escherichia coli, and the purified protein displayed a K _m of 0.2 mM and a V _max of 1.3 μmol min^?1 mg^?1 with glucose 1-phosphate as substrate. The catalytic efficiency (K _cat/K _m) of PgmG was about 15-fold higher for glucose 1-phosphate than for mannose 1-phosphate. Overexpression of pgmG in S. sanxanigenens resulted in a 17 ± 0.3 % increase in sphingan production to ~12.5 g l^?1.     

Characterization of a thermostable enzyme with phosphomannomutase/phosphoglucomutase activities from the hyperthermophilic archaeon Pyrococcus horikoshii OT3

The phosphomannomutase/phosphoglucomutase (PMM/PGM) enzyme catalyzes reversibly the intra-molecular phosphoryl interconverting reaction of mannose-6-phosphate and mannose-1-phosphate or glucose-6-phosphate and glucose-1-phosphate. Glucose-6-phosphate and glucose-1-phosphate are known to be utilized for energy metabolism and cell surface construction, respectively. PMM/PGM has been isolated from many microorganisms. By performing similarity searches using existing PMM/PGM sequences, the homologous ORFs PH0923 and PH1210 were identified from the genomic data of Pyrococcus horikoshii OT3. Since PH0923 appears to be part of an operon consisting of four carbohydrate metabolic enzymes, PH0923 was selected as the first target for the investigation of PMM/PGM activity in P. horikoshii OT3. The coding region of PH0923 was cloned and the purified recombinant protein was utilized for an examination of its biochemical properties. The enzyme retained half its initial activity after treatment at 95 degrees C for 90 min. Detailed analyses of activities showed that this protein is capable of utilizing a variety of metal ions that are not utilized by previously characterized PMM/PGM proteins. A mutated protein with an alanine residue replacing the active site serine residue indicated that this residue plays an important but non-essential role in PMM/PGM activity.     

Purification and characterization of phosphomannomutase/phosphoglucomutase from Pseudomonas aeruginosa involved in biosynthesis of both alginate and lipopolysaccharide.

The algC gene from Pseudomonas aeruginosa has been shown to encode phosphomannomutase (PMM), an essential enzyme for biosynthesis of alginate and lipopolysaccharide (LPS). This gene was overexpressed under control of the tac promoter, and the enzyme was purified and its substrate specificity and metal ion effects were characterized. The enzyme was determined to be a monomer with a molecular mass of 50 kDa. The enzyme catalyzed the interconversion of mannose 1-phosphate (M1P) and mannose 6-phosphate, as well as that of glucose 1-phosphate (G1P) and glucose 6-phosphate. The apparent Km values for M1P and G1P were 17 and 22 microM, respectively. On the basis of Kcat/Km ratio, the catalytic efficiency for G1P was about twofold higher than that for M1P. PMM also catalyzed the conversion of ribose 1-phosphate and 2-deoxyglucose 6-phosphate to their corresponding isomers, although activities were much lower. Purified PMM/phosphoglucomutase (PGM) required Mg2+ for maximum activity; Mn2+ was the only other divalent metal that showed some activation. The presence of other divalent metals in addition to Mg2+ in the reaction inhibited the enzymatic activity. PMM and PGM activities could not be detected in nonmucoid algC mutant strain 8858 and in LPS-rough algC mutant strain AK1012, while they were present in the wild-type strains as well as in algC-complemented mutant strains. This evidence suggests that AlgC functions as PMM and PGM in vivo, converting phosphomannose and phosphoglucose in the biosynthesis of both alginate and LPS.     

Cloning and characterization of phosphoglucomutase and phosphomannomutase derived from Sphingomonas chungbukensis DJ77

The enzymes phosphoglucomutase (PGM) and phosphomannomutase (PMM) play an important role in the synthesis of extracellular polysaccharide. By colony hybridization of the fosmid library of Sphingomonas chungbukensis DJ77, an open reading frame (ORF-1) of 1,626 nucleotides, whose predicted product is highly homologous with other PGM proteins from several bacterial species, was identified. An additional open reading frame (ORF-2) of 1,437 nucleotides was identified, and its encoded protein shows a high level of similarity with the PGM/PMM protein family. The two genes were cloned into a bacterial expression vector pET-15b (+) and expressed in Escherichia coli as fusion proteins with (His)(6)-tag. Both recombinant proteins (designated as SP-1 and SP-2 for ORF-1 and ORF-2, respectively) exhibited PGM and PMM activities. The molecular masses of subunits of SP-1 and SP-2 were estimated to be around 58 and 51 kDa from SDS-PAGE, respectively. However, molecular masses of SP-1 and SP-2 in their native condition were determined to be approximately 59.5 and 105.4 kDa, according to non-denaturing PAGE, respectively. The SP-1 protein has a preference for glucose-1-phosphate rather than mannose-1-phosphate, while the preferred substrate of SP-2 is mannose-1-phosphate. Thus, the existence of two proteins with bifunctional PGM/PMM activities was first found S. chungbukensis DJ77.     

Molecular and functional analysis of phosphomannomutase (PMM) from higher plants and genetic evidence for the involvement of PMM in ascorbic acid biosynthesis in Arabidopsis and Nicotiana benthamiana

Phosphomannomutase (PMM) catalyzes the interconversion of mannose-6-phosphate and mannose-1-phosphate. However, systematic molecular and functional investigations on PMM from higher plants have hitherto not been reported. In this work, PMM cDNAs were isolated from Arabidopsis, Nicotiana benthamiana, soybean, tomato, rice and wheat. Amino acid sequence comparisons indicated that plant PMM proteins exhibited significant identity to their fungal and mammalian orthologs. In line with the similarity in primary structure, plant PMM complemented the sec53-6 temperature sensitive mutant of Saccharomyces cerevisiae. Histidine-tagged Arabidopsis PMM (AtPMM) purified from Escherichia coli converted mannose-1-phosphate into mannose-6-phosphate and glucose-1-phosphate into glucose-6-phosphate, with the former reaction being more efficient than the latter one. In Arabidopsis and N. benthamiana, PMM was constitutively expressed in both vegetative and reproductive organs. Reducing the PMM expression level through virus-induced gene silencing caused a substantial decrease in ascorbic acid (AsA) content in N. benthamiana leaves. Conversely, raising the PMM expression level in N. benthamiana using viral-vector-mediated ectopic expression led to a 20-50% increase in AsA content. Consistent with this finding, transgenic expression of an AtPMM-GFP fusion protein in Arabidopsis also increased AsA content by 25-33%. Collectively, this study improves our understanding on the molecular and functional properties of plant PMM and provides genetic evidence on the involvement of PMM in the biosynthesis of AsA in Arabidopsis and N. benthamiana plants.     

A zebrafish model of PMM2-CDG reveals altered neurogenesis and a substrate-accumulation mechanism for N-linked glycosylation deficiency

Congenital disorder of glycosylation (PMM2-CDG) results from mutations in pmm2, which encodes the phosphomannomutase (Pmm) that converts mannose-6-phosphate (M6P) to mannose-1-phosphate (M1P). Patients have wide-spectrum clinical abnormalities associated with impaired protein N-glycosylation. Although it has been widely proposed that Pmm2 deficiency depletes M1P, a precursor of GDP-mannose, and consequently suppresses lipid-linked oligosaccharide (LLO) levels needed for N-glycosylation, these deficiencies have not been demonstrated in patients or any animal model. Here we report a morpholino-based PMM2-CDG model in zebrafish. Morphant embryos had developmental abnormalities consistent with PMM2-CDG patients, including craniofacial defects and impaired motility associated with altered motor neurogenesis within the spinal cord. Significantly, global N-linked glycosylation and LLO levels were reduced in pmm2 morphants. Although M1P and GDP-mannose were below reliable detection/quantification limits, Pmm2 depletion unexpectedly caused accumulation of M6P, shown earlier to promote LLO cleavage in vitro. In pmm2 morphants, the free glycan by-products of LLO cleavage increased nearly twofold. Suppression of the M6P-synthesizing enzyme mannose phosphate isomerase within the pmm2 background normalized M6P levels and certain aspects of the craniofacial phenotype and abrogated pmm2-dependent LLO cleavage. In summary, we report the first zebrafish model of PMM2-CDG and uncover novel cellular insights not possible with other systems, including an M6P accumulation mechanism for underglycosylation.     

Overexpression of Mycobacterium tuberculosis manB, a phosphomannomutase that increases phosphatidylinositol mannoside biosynthesis in Mycobacterium smegmatis and mycobacterial association with human macrophages

Mycobacterium tuberculosis (M. tb) pathogenesis involves the interaction between the mycobacterial cell envelope and host macrophage, a process mediated, in part, by binding of the mannose caps of M. tb lipoarabinomannan (ManLAM) to the macrophage mannose receptor (MR). A presumed critical step in the biosynthesis of ManLAM, and other mannose-containing glycoconjugates, is the conversion of mannose-6-phosphate to mannose-1-phosphate, by a phosphomannomutase (PMM), to produce GDP-mannose, the primary mannose-donor in mycobacteria. We have identified four M. tb H37Rv genes with similarity to known PMMs. Using in vivo complementation of PMM and phosphoglucomutase (PGM) deficient strains of Pseudomonas aeruginosa, and an in vitro enzyme assay, we have identified both PMM and PGM activity from one of these genes, Rv3257c (MtmanB). MtmanB overexpression in M. smegmatis produced increased levels of LAM, lipomannan, and phosphatidylinositol mannosides (PIMs) compared with control strains and led to a 13.3 +/- 3.9-fold greater association of mycobacteria with human macrophages, in a mannan-inhibitable fashion. This increased association was mediated by the overproduction of higher order PIMs that possess mannose cap structures. We conclude that MtmanB encodes a functional PMM involved in the biosynthesis of mannosylated lipoglycans that participate in the association of mycobacteria with macrophage phagocytic receptors.     

Evolutionary History and Functional Diversification of Phosphomannomutase Genes

Phosphomannomutases (PMMs) catalyze the interconversion of mannose-6-phosphate to mannose-1-phosphate. In humans, two PMM enzymes exist—PMM1 and PMM2; yet, they have different functional specificities. PMM2 presents PMM activity, and its deficiency causes a Congenital Disorder of Glycosylation (PMM2-CDG). On the other hand, PMM1 can also act as glucose-1,6-bisphosphatase in the brain after stimulation with inosine monophosphate and thus far has not been implicated in any human disease. This study aims to refine the evolutionary time frame at which gene duplication gave rise to PMM1 and PMM2, and to identify the most likely amino acid positions underlying the proteins’ different functions. The phylogenetic analysis using available protein sequences, allowed us to establish that duplication occurred early in vertebrate evolution. In order to understand the molecular basis underlying the functional divergence, conserved and most likely functional divergence-related sites were identified, through the analysis of site-specific evolutionary rates. This analysis indicates that most of the sites known to be important in the homodimer formation and in the catalytic activity are conserved in both proteins. Among those potentially related to functional divergence, two positions (183 and 186 in human PMM1) emerge as the most interesting ones. The residues at these positions have different side-chain conformations in the protein structure in the unbound and bound states, and are highly but differently conserved in PMM1 and in PMM2 proteins. Altogether, these results provide new data into the evolutionary history of PMM1 and PMM2 duplicates and highlight the most probable sites that evolved to distinct functional specificities.     

Chemical shift assignments of domain 4 from the phosphohexomutase from Pseudomonas aeruginosa suggest that freeing perturbs its coevolved domain interface

A domain needed for the catalytic efficiency of an enzyme model of simple processivity and domain–domain interactions has been characterized by NMR. This domain 4 from phosphomannomutase/phosphoglucomutase (PMM/PGM) closes upon glucose phosphate and mannose phosphate ligands in the active site, and can modestly reconstitute activity of enzyme truncated to domains 1–3. This enzyme supports biosynthesis of the saccharide-derived virulence factors (rhamnolipids, lipopolysaccharides, and alginate) of the opportunistic bacterial pathogen Pseudomonas aeruginosa. ¹H, ¹³C, and ¹⁵N NMR chemical shift assignments of domain 4 of PMM/PGM suggest preservation and independence of its structure when separated from domains 1–3. The face of domain 4 that packs with domain 3 is perturbed in NMR spectra without disrupting this fold. The perturbed residues overlap both the most highly coevolved positions in the interface and residues lining a cavity at the domain interface.     

Structure of Leishmania mexicana phosphomannomutase highlights similarities with human isoforms

Phosphomannomutase (PMM) catalyses the conversion of mannose-6-phosphate to mannose-1-phosphate, an essential step in mannose activation and the biosynthesis of glycoconjugates in all eukaryotes. Deletion of PMM from Leishmania mexicana results in loss of virulence, suggesting that PMM is a promising drug target for the development of anti-leishmanial inhibitors. We report the crystallization and structure determination to 2.1 A of L. mexicana PMM alone and in complex with glucose-1,6-bisphosphate to 2.9 A. PMM is a member of the haloacid dehalogenase (HAD) family, but has a novel dimeric structure and a distinct cap domain of unique topology. Although the structure is novel within the HAD family, the leishmanial enzyme shows a high degree of similarity with its human isoforms. We have generated L. major PMM knockouts, which are avirulent. We expressed the human pmm2 gene in the Leishmania PMM knockout, but despite the similarity between Leishmania and human PMM, expression of the human gene did not restore virulence. Similarities in the structure of the parasite enzyme and its human isoforms suggest that the development of parasite-selective inhibitors will not be an easy task.     

Photosynthate metabolism in the source leaves of n(2)-fixing soybean plants

Soybean plants (Glycine max [L.] Merr. cv Williams), which were symbiotic with Bradyrhizobium japonicum, and which grew well upon reduced nitrogen supplied solely through N₂ fixation processes, often exhibited excess accumulation of starch and sucrose and diminished soluble protein in their source leaves. Nitrate and ammonia, when supplied to the nodulated roots of N₂-fixing plants, mediated a reduction of foliar starch accumulation and a corresponding increase in soluble protein in the source leaves. This provided an opportunity to examine the potential metabolic adjustments by which NO₃⁻ and NH₄⁺ (N) sufficiency or deficiency exerted an influence upon soybean leaf starch synthesis. When compared with soybean plants supplied with N, elevated starch accumulation was focused in leaf palisade parenchyma tissue of N₂-fixing plants. Foliar activities of starch synthesis pathway enzymes including fructose-1,6-bisphosphate phosphatase, phosphohexoisomerase, phosphoglucomutase (PGM), as well as adenosine diphosphate glucose pyrophosphorylase (in some leaves) exhibited highest activities in leaf extracts of N₂-fixing plants when expressed on a leaf protein basis. This was interpreted to mean that there was an adaptation of these enzyme activities in the leaves of N₂-fixing plants, and this contributed to an increase in starch accumulation. Another major causal factor associated with increased starch accumulation was the elevation in foliar levels of fructose-6-phosphate, glucose-6-phosphate, and glucose-1-phosphate (G1P), which had risen to chloroplast concentrations considerably in excess of the K_m values for their respective target enzymes associated with starch synthesis, e.g. elevated G1P with respect to adenosine diphosphate glucose pyrophosphorylase (ADPG-PPiase) binding sites. The cofactor glucose-1,6-bisphosphate (G1,6BP) was found to be obligate for maximal PGM activity in soybean leaf extracts of N₂-fixing as well as N-supplemented plants, and G1,6BP levels in N₂-fixing plant leaves was twice that of levels in N-supplied treatments. However the concentration of chloroplastic G1,6BP in illuminated leaves was computed to be saturating with respect to PGM in both N₂-fixing and N-supplemented plants. This suggested that the higher level of this cofactor in N₂-fixing plant leaves did not confer any higher PGM activation and was not a factor in higher starch synthesis rates. Relative to plants supplied with NO₃⁻ and NH₄⁺, the source leaf glycerate-3-phosphate (3-PGA) and orthophosphate (Pi) concentrations in leaves of N₂-fixing plants were two to four times higher. Although Pi is a physiological competitive inhibitor of leaf chloroplast ADPG-PPiase, and hence, starch synthesis, elevated chloroplast 3-PGA levels in N₂-fixing plant leaves apparently prevented interference of Pi with ADPG-PPiase catalysis and starch synthesis.     

Loss of endoplasmic reticulum membrane integrity: an image analysis of the glucose-6-phosphatase system in human hepatocyte.

Histochemical and cytochemical methods induce a loss of endoplasmic reticulum (ER) membrane integrity in hepatocytes. In order to evaluate the degree of ER membrane integrity, glucose-6-phosphatase (G6P-A) was localized in light and electron microscopy using glucose-6-phosphate (G6P) and mannose-6-phosphate (M6P) as substrates. In case of ER membrane alteration, M6P diffuses inside the ER and is hydrolysed by a non-specific phosphohydrolase. G6P and M6P hydrolysis was quantified with image analysis methods. In light microscopy, the ratio of reaction of M6P hydrolysis/G6P hydrolysis gave 75% of non specific reaction. In electron microscopic study this ratio was about 30%. These results showed that enzyme localization methods in electron microscopy produced less ER membrane alteration than light microscopic methods.     

Reduction of the Cytosolic Phosphoglucomutase in Arabidopsis Reveals Impact on Plant Growth,Seed and Root Development,and Carbohydrate Partitioning

Phosphoglucomutase (PGM) catalyses the interconversion of glucose 1-phosphate (G1P) and glucose 6-phosphate (G6P) and exists as plastidial (pPGM) and cytosolic (cPGM) isoforms. The plastidial isoform is essential for transitory starch synthesis in chloroplasts of leaves, whereas the cytosolic counterpart is essential for glucose phosphate partitioning and, therefore, for syntheses of sucrose and cell wall components. In Arabidopsis two cytosolic isoforms (PGM2 and PGM3) exist. Both PGM2 and PGM3 are redundant in function as single mutants reveal only small or no alterations compared to wild type with respect to plant primary metabolism. So far, there are no reports of Arabidopsis plants lacking the entire cPGM or total PGM activity, respectively. Therefore, amiRNA transgenic plants were generated and used for analyses of various parameters such as growth, development, and starch metabolism. The lack of the entire cPGM activity resulted in a strongly reduced growth revealed by decreased rosette fresh weight, shorter roots, and reduced seed production compared to wild type. By contrast content of starch, sucrose, maltose and cell wall components were significantly increased. The lack of both cPGM and pPGM activities in Arabidopsis resulted in dwarf growth, prematurely die off, and inability to develop a functional inflorescence. The combined results are discussed in comparison to potato, the only described mutant with lack of total PGM activity.     

Genetic validation of Aspergillus fumigatus phosphoglucomutase as a viable therapeutic target in invasive aspergillosis

Aspergillus fumigatus is the causative agent of invasive aspergillosis, an infection with mortality rates of up to 50%. The glucan-rich cell wall of A. fumigatus is a protective structure that is absent from human cells and is a potential target for antifungal treatments. Glucan is synthesized from the donor uridine diphosphate glucose, with the conversion of glucose-6-phosphate to glucose-1-phosphate by the enzyme phosphoglucomutase (PGM) representing a key step in its biosynthesis. Here, we explore the possibility of selectively targeting A. fumigatus PGM (AfPGM) as an antifungal treatment strategy. Using a promoter replacement strategy, we constructed a conditional pgm mutant and revealed that pgm is required for A. fumigatus growth and cell wall integrity. In addition, using a fragment screen, we identified the thiol-reactive compound isothiazolone fragment of PGM as targeting a cysteine residue not conserved in the human ortholog. Furthermore, through scaffold exploration, we synthesized a para-aryl derivative (ISFP10) and demonstrated that it inhibits AfPGM with an IC₅₀ of 2 μM and exhibits 50-fold selectivity over the human enzyme. Taken together, our data provide genetic validation of PGM as a therapeutic target and suggest new avenues for inhibiting AfPGM using covalent inhibitors that could serve as tools for chemical validation.     

One-pot enzymatic synthesis of deoxy-thymidine-diphosphate (TDP)-2-deoxy-α-d-glucose using phosphomannomutase

Production of deoxy-thymidine-diphosphate (TDP)-sugars as substrates of glycosyltransferases, has been one of main hurdles for combinatorial antibiotic biosynthesis, which combines sugar moiety with aglycon of various antibiotics. Here, we report the one-pot enzymatic synthesis of TDP-2-deoxy-glucose employing high efficient TMP kinase (TMK; E.C. 2.7.2.12), acetate kinase (ACK; E.C. 2.7.1.21), and TDP-glucose synthase (TGS; E.C. 2.7.7.24) with phosphomannomutase (PMM; E.C. 5.4.2.8). In this study, replacing phosphoglucomutase (PGM; E.C. 5.4.2) by PMM from Escherichia coli gave four times higher specific activity on 2-deoxy-6-phosphate glucose, suggesting that the activity on 2-deoxy-glucose-6-phosphate was mainly affected by PMM activity, not PGM activity. Using an in vitro system starting from TMP and 2-deoxy-glucose-6-phosphate glucose, TDP-2-deoxy-glucose (63% yield) was successfully synthesized. Considering low productivity of NDP-sugars from cheap starting materials, this paper showed how production of NDP-sugars could be enhanced by controlling mutase activity.