Identification of thepgmGGene, Encoding a Bifunctional Protein with Phosphoglucomutase and Phosphomannomutase Activities, in the Gellan Gum-Producing StrainSphingomonas paucimobilisATCC 31461

Abstract

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.Bacterial strains of the new genus Sphingomonas (47) are relatively ubiquitous in soil, water, and sediments, have broad catabolic capabilities (12,17,33,35), and produce at least eight extracellular acid heteropolysaccharides that have similar but not identical structures (9, 31). These polysaccharides, the sphingans (after the genus), exhibit properties which make them candidates for food and industrial applications, such as thermoreversible gel formation and solution viscosity (9, 34). The industrial strain Sphingomonas paucimobilis ATCC 31461 (formerly Pseudomonas elodea) synthesizes in high yields from different carbon sources and from cheese whey a new gelling agent, gellan gum (15,23,36). The commercial utility of gellan (9, 34) has been a stimulus for the study of its biosynthesis.The cloning and functional analysis of genes essential for gellan synthesis are indispensable in attempting the genetic and environmental manipulation of its biosynthetic pathway in order to develop new polysaccharides with distinct structural and physical properties. Among the gellan biosynthetic enzymes (29), phosphoglucomutase (PGM; EC 5.4.2.2) plays a pivotal role, being an ideal target for metabolic engineering. Indeed, PGM catalyzes the interconversion of D-glucose-6-phosphate (G6P) and D-glucose-1-phosphate (G1P), representing a branch point in carbohydrate metabolism. G6P enters catabolic processes to yield energy and reducing power, whereas G1P is the precursor of sugar nucleotides that are used by the cells in the synthesis of various polysaccharides. In gellan gum biosynthesis, G1P is required for the synthesis of three sugar nucleotides, UDP-D-glucose, dTDP-

Among Multiple Phosphomannomutase Gene Orthologues, Only One Gene Encodes a Protein with Phosphoglucomutase and Phosphomannomutase Activities in Thermococcus kodakaraensis

“…The three larger proteins displayed high levels of similarity with one another (TK2185 and TK1108, 44% identical; TK1777 and TK2185, 43% identical; TK1108 and TK1777, 38% identical) and also displayed equivalent levels of similarity with the biochemically characterized PGM from E. coli (TK1777, 24%; TK2185, 24%; TK1108, 25%). Two motifs conserved in various phosphosugar mutases (3,19), TXSHNP containing the active site serine residue and DXDXDR involved in metal binding, were also conserved among these three proteins (Fig. 1).…”

Presence of a Novel Phosphopentomutase and a 2-Deoxyribose 5-Phosphate Aldolase Reveals a Metabolic Link between Pentoses and Central Carbon Metabolism in the Hyperthermophilic Archaeon Thermococcus kodakaraensis

“…This implies that some of the proteins within COG1109 that have previously been annotated as phosphomannomutases or related phosphohexomutases in various genome sequences may indeed be PPMs. Sequence alignment of various phosphohexomutases and the structure of phosphoglucomutase from rabbits (7) reveal three conserved sequence motifs that are necessary for enzyme activity (32). The sequence TXSHNP contains the active-site serine residue, the DXDXDR motif is involved in metal binding, and the sequence GEXS participates in sugar binding.…”

Role of Phosphoglucomutase of Stenotrophomonas maltophilia in Lipopolysaccharide Biosynthesis, Virulence, and Antibiotic Resistance

“…PGM activity was assessed by using whole-cell lysates according to a previously published protocol (57). Briefly, 7.5 ml of an overnight culture grown in LB broth (with appropriate antibiotic selection when necessary) was inoculated into 750 ml of LB broth, and the cells were grown for 6 h at 37°C with shaking.…”