Studies on recombinant Acetobacter xylinum α-phosphoglucomutase

Abstract

The phosphoglucomutase (PGM) from Acetobacter xylinum, which had been cloned and expressed in Escherichia coli, has been studied. After expression, the enzyme was purified from the E. coli in a three-step process consisting of (NH4)2SO4 precipitation, gel filtration and anion-exchange chromatography. The purified enzyme gave one band on gel electrophoresis and was judged essentially free of impurities, although it was unstable when diluted without the addition of 15 microM BSA. The isoelectric point for A. xylinum PGM was 4.8 and the molar absorbance was 3.9 x 10(4) M-1.cm-1. The enzyme was reasonably heat-stable below 50 degrees C and was stable throughout the pH 5.5-7.4 range, but was 70% inactivated at pH 10.0 and completely inactivated after standing for 10 min at pH 3.0 or at pH 12.4. When isolated, the recombinant enzyme was fully active without the addition of extra Mg2+. The Km for glucose 1-phosphate was much higher than that of other PGM species reported, which accords with the production of extracellular cellulose in A. xylinum. Glucose 1,6-diphosphate is not considered to be a substrate or coenzyme but an activating cofactor like Mg2+. The following kinetic constants were determined: Vmax 81.1 units/mg; kcat and the turnover rate 135 s-1; Km (glucose 1,6-diphosphate) 0.2 microM; Km (glucose 1-phosphate) 2.6 mM; kcat/Km (glucose 1-phosphate) 5.2 x 10(4) M-1.s-1. The recombinant enzyme is considered to follow a characteristic substituted enzyme or Ping Pong reaction mechanism.

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

“…These values are 8 M (G1P) for PgmU of rabbit muscle (39), 17 M (M1P) and 22 M (G1P) for AlgC of P. aeruginosa (49), and 20 M (G1P) for the PGM of maize leaves (37). However, these K m values are below the value calculated for the specific PGM of A. xylinum (2,600 M) [G1P] (25). Interestingly, this protein was included in the subclass of enzymes specific for G1P that is distinct from the subclass of phosphohexosemutases formed by S. paucimobilis PgmG and other bifunctional enzymes, such as AlgC from P. aeruginosa (51), XanA from X. campestris (24), N. gonorrhoeae PGM (44,50), and PmmA from P. hollandica (13) (Fig.…”

“…695163) and R. prowazekii (ExoC) (3). The PGM enzymes of E. coli and Acetobacter xylinum (CelB) (7,25,27) were very similar to the PGM enzymes of Saccharomyces cerevisiae (Pgm1) (5) and rabbit muscle (PgmU) (39) and are considered to be highly specific for phosphoglucose. Although these two subclasses share activesite and metal-ion-binding domains (box I and II) in box III [GE(M or E)S] that may control substrate specificity (10), the subclass of specific PGMs exhibits the E residue, while in the subclass of bifunctional enzymes, including PgmG, this residue is substituted by M (Fig.…”

“…1B). Although the back reaction has not been studied for A. xylinum PGM, the high K m (G1P) value calculated was considered consistent with the involvement of this enzyme in the production of extracellular cellulose, since a high K m (G1P) favors metabolic flux toward polymer synthesis rather than catabolic pathways (25).…”

Identification and Characterization of an Archaeal Kojibiose Catabolic Pathway in the Hyperthermophilic Pyrococcus sp. Strain ST04

“…The decrease of activity in the absence of G1,6biP has also been observed with Acetobacter xylinus ␣-phosphoglucomutase. The activity without G1,6biP was measured at 70% of the activity with G1,6biP (51).…”

Investigation of ion/molecule reactions as a quantification method for phosphorylated positional isomers: An FT-ICR approach

“…In particular, the phosphate linkage position of carbohydrates is important to regulate biological function. For example, the conversion between the 6-phosphate and the 1-phosphate isomers of several monosaccharides, which is regulated by the corresponding phosphate mutases, plays an important role in the glycosylation of glycoproteins [5,6]. Specifically, the deficiency of the enzyme phosphomannomutase, which catalyzes the conversion of mannose-6-phosphate to mannose-1-phosphate in the early step of biosynthesis of lipid-linked oligosaccharides, is responsible for a disease called CDG-1a [1], in which glycoproteins with truncated N-linked carbohydrates are formed.…”