TREHALOSE-P SYNTHASE OF MYCOBACTERIA - ITS SUBSTRATE-SPECIFICITY IS AFFECTED BY POLYANIONS

The trehalose-P synthase was purified to near homogeneity from the cyt oplasmic fraction of Mycobacterium sinegmatis. At the final stage of p urification, the enzyme preparation showed one major band of 59 kDa on SDS gels, The 59 kDa band became labeled with N-3-UDP[P-32]-glucose, and this labeling was inhibited in a concentration-dependent manner by either unlabeled UDP-glucose or GDP-glucose. The native enzyme also h ad a molecular weight of about 60 kDa by gel filtration, indicating th at the active enzyme is a monomer. The 59 kDa protein was subjected to endoproteinase Lys-C digestion, and three peptides isolated by HPLC w ere sequenced, The sequences of 56 amino acids in these three peptides showed 60% identity to the trehalose-P synthases of Saccharomyces cer evesiae and Schizosaccharomyces pombe. The purified mycobacterial enzy me catalyzed the synthesis of trehalose-P from glucose-6-P and a varie ty of nucleoside diphosphate glucose derivatives, depending on whether a polyanion was absent or present, Thus, UDP-glucose and GDP-glucose were the best glucosyl donors, but maximum activity with UDP-glucose r equired the presence of a polyanion such as heparin, whereas activity with GDP-glucose was relatively independent of polyanion. The presence of heparin in the incubation mixture increased the affinity of the en zyme for UDP-glucose by a factor of 100, or more, However, the affinit y for GDP-glucose was only twofold better in the presence of heparin. The purified synthase also utilized ADP-glucose and CDP-glucose, but t he K-m for these glucosyl donors was quite high even in the presence o f polyanion, The effect of heparin on UDP-glucose activity was dose-de pendent and maximum at about 1-2 mu g of heparin/incubation. However, the size of the heparin molecule (i.e., the number of monosaccharide r esidues) was critical for activation, and only those heparins with 18 or more monosaccharide units were effective in stimulating activity.