Characterization of methylglyoxal synthase from Clostridium acetobutylicum ATCC 824 and its use in the formation of 1, 2-propanediol

Abstract

A gene encoding a putative 150-amino-acid methylglyoxal synthase was identified in Clostridium acetobutylicum ATCC 824. The enzyme was overexpressed in Escherichia coli and purified. Methylglyoxal synthase has a native molecular mass of 60 kDa and an optimum pH of 7.5. The Km and Vmax values for the substrate dihydroxyacetone phosphate were 0.53 mM and 1.56 mmol min(-1) microgram(-1), respectively. When E. coli glycerol dehydrogenase was coexpressed with methylglyoxal synthase in E. coli BL21(DE3), 3.9 mM 1,2-propanediol was produced.

FIG. 1

Metabolic pathways to 1,2-propanediol from DHAP.

FIG. 2

Alignment of the amino acid sequences of C. acetobutylicum ATCC 824, E. coli, Bacillus subtilis, Haemophilus influenzae, Synechocystis, and Bacillus abortus methylglyoxal synthases. Positions at which more than 50% of the sequences have identical amino acids are shaded.

FIG. 3

SDS-PAGE analysis of fractions from each purification step and total protein. Samples were electrophoresed on a 12% acrylamide gel. The resulting gel was subjected to protein staining. (A) Lane M, molecular weight standards, lane 1, total protein from induced E. coli BL21(DE3)/pMGS1 (12 μg after 4 h of induction with 0.4 mM IPTG); lane 2, soluble protein from E. coli BL21(DE3)/pMGS1 (14 μg); lanes 3 and 4, purified methylglyoxal synthase (1 and 2 μg, respectively). (B) Lane M, molecular weight standards; lane 1, total protein from E. coli BL21(DE3)/pMGS1; lane 2, total protein from E. coli BL21(DE3)/pMGS2; lane 3, total protein from E. coli BL21(DE3)/pGLDH.

FIG. 4

Determination of the apparent Michaelis constant for DHAP. The assay mixtures (total volume, 100 μl) contained 50 mM imidazole buffer (pH 7.5), different amounts of purified enzyme, and different amounts of DHAP. After 5 min of incubation at 30°C, the amount of methylglyoxal formed was measured colorimetrically (8).