Purification and characterization of two phosphoglucomutases from Lactococcus lactis subsp. lactis and their regulation in maltose- and glucose-utilizing cells

Abstract

Two distinct forms of phosphoglucomutase were found in Lactococcus lactis subsp. lactis, strains 19435 and 65.1, growing on maltose: beta-phosphoglucomutase (beta-PGM), which catalyzes the reversible conversion of beta-glucose 1-phosphate to glucose 6-phosphate in the maltose catabolism, and alpha-phosphoglucomutase (alpha-PGM). beta-PGM was purified to more than 90% homogeneity in crude cell extract from maltose-grown lactococci, and polyclonal antisera to the enzyme were prepared. The molecular mass of beta-PGM was estimated by gel filtration to be 28 kDa; its isoelectric point was 4.8. The corresponding values for alpha-PGM were 65 kDa and 4.4, respectively. The expression of both PGM enzymes was investigated under different growth conditions. The specific activity and amount of beta-PGM per milliliter of cell extract increased with time in lactococci grown on maltose, but the enzyme was absent in lactococci grown on glucose, indicating enzyme synthesis to be induced by maltose in the growth medium. When glucose was added to maltose-grown lactococci, both the specific activity and amount of beta-PGM per milliliter of cell extract decreased rapidly. This suggests that synthesis of beta-PGM is repressed by glucose in the medium. Although the specific activity of alpha-PGM did not change during growth on maltose or glucose, lactococcal strain 19435 showed a much higher specific activity of both alpha- and beta-PGM than strain 65.1 when grown on maltose.

A Trojan horse transition state analogue generated by MgF 3 − formation in an enzyme active site

“…that kinetic assays of ␣-and ␤-PGM are usually run in the presence of either ␣-or ␤-G16BP, which accelerate the reaction by saturating the enzyme at the intermediate stage or maintaining its activated, phosphorylated state (10,15,19). Only ␤-G16BP can contribute by maintaining a high concentration of the ␤-G16BP͞␤-PGM complex, but other cofactors can stimulate the enzyme by acting as phosphorylating agents, and commercially available ␣-G16BP is used commonly (10,(23)(24)(25). Interestingly ␣-G16BP is not turned over to G6P by ␤-PGM, but is converted to ␣-G1P to generate ␤-PGM* (10).…”

Exopolysaccharides produced by Lactococcus lactis: from genetic engineering to improved rheological properties?

“…β-PGM) and the other apparently specific for α-glucose-1P (i.e. α-PGM) (Qian et al 1994). Since the phosphorolysis of glucose, lactose and other carbon sources usually yields α-glucose-1P, α-PGM could be a keyenzyme in sugar-nucleotide biosynthesis.…”

Evolutionary Genomics of the HAD Superfamily: Understanding the Structural Adaptations and Catalytic Diversity in a Superfamily of Phosphoesterases and Allied Enzymes

“…As a result an acyl phosphate intermediate is formed with the carboxyl group of this aspartate. [27][28][29][30][31] Subsequently, the enzyme enters the open configuration again and allows the leaving group to escape (Figure 1(a)). In the open state bulk solvent enters the active site and a water is deprotonated by the second aspartate of strand one; hydrolyzing the acyl phosphate intermediate and returning the enzyme to the native state.…”

“…Additionally, the first Asp in motif I acts as a nucleophile that forms an aspartyl-intermediate during catalysis. [27][28][29][30][31] In phosphatase and phosphomutase members of the superfamily the second acidic residue acts as a general acid-base. It binds and, in many cases, protonates the substrate leaving group in the first step and deprotonates the nucleophile of the second step.…”