Trehalose-P synthase of mycobacteria: its substrate specificity is affected by polyanions

The trehalose-P synthase was purified to near homogeneity fromthe cytoplasmic fraction of Mycobacterium smegmatis. At thefinal stage of purification, the enzyme preparation showed onemajor band of 59 kDa on SDS gels. The 59 kDa band became labeledwith N₃-UDP³²P]-glucose, and this labeling was inhibited ina concentration-dependent manner by either unlabeled UDP-glucoseor GDP-glucose. The native enzyme also had a molecular weightof about 60 kDa by gel filtration, indicating that the activeenzyme is a monomer. The 59 kDa protein was subjected to endoproteinaseLys-C digestion, and three peptides isolated by HPLC were sequenced.The sequences of 56 amino acids in these three peptides showed60% identity to the trehalose-P synthases of Saccharomyces cerevesiaeand Schizosaccharomyces pombe. The purified mycobacterial enzymecatalyzed the synthesis of trehalose-P from glucose-6-P anda variety of nucleoside diphosphate glucose derivatives, dependingon whether a polyanion was absent or present. Thus, UDP-glucoseand GDP-glucose were the best glucosyl donors, but maximum activitywith UDP-glucose required the presence of a polyanion such asheparin, whereas activity with GDP-glucose was relatively independentof polyanion. The presence of heparin in the incubation mixtureincreased the affinity of the enzyme for UDP-glucose by a factorof 100, or more. However, the affinity for GDP-glucose was onlytwofold better in the presence of heparin. The purified synthasealso utilized ADP-glucose and CDP-glucose, but the K_m for theseglucosyl donors was quite high even in the presence of polyanion.The effect of heparin on UDP-glucose activity was dose-dependentand maximum at about 1–2 µ;g of heparin/incubation.However, the size of the heparin molecule (i.e., the numberof monosaccharide residues) was critical for activation, andonly those heparins with 18 or more monosaccharide units wereeffective in stimulating activity. trehalose polyanions mycobacteria GDP-glucose heparin