Aims: Discovery and utilization of highly active and thermostable phosphoglucomutase (PGM) would be vital for biocatalysis mediated by multiple enzymes, for example, high-yield production of enzymatic hydrogen.
Methods and Results: The thermophilic cellulolytic bacterium Clostridium thermocellum was hypothesized to have a very active PGM because of its key role in microbial cellulose utilization. The Cl. thermocellum ORF Cthe1265 encoding a putative PGM was cloned and expressed in Escherichia coli. The purified enzyme appeared to be a monomer with an estimated molecular weight of 64·9 kDa. This enzyme was found to be a dual-specificity enzyme – PGM/phosphomannomutase (PMM). Mg2+ and Mn2+ were activators. Ser144 was identified as an essential catalytic residue through site-directed mutagenesis. The kcat and Km of PGM were 190 s−1 and 0·41 mmol l−1 on glucose-1-phosphate and 59 s−1 and 0·44 mmol l−1 on mannose-1-phosphate, respectively, at 60°C. Thermostability of PGM at a low concentration (2 nmol l−1, 100 U l−1) was enhanced by 12-fold (i.e. t1/2 = 72 h) at 60°C with addition of bovine serum albumin, Triton X-100, Mg2+and Mn2+.
Conclusions: The ORF Cthe1265 was confirmed to encode a PGM with PMM activity. This enzyme was the most active PGM reported.
Significance and Impact of the Study: This highly active PGM with enhanced thermostability would be an important building block for in vitro synthetic biology projects (complicated biotransformation mediated by multiple enzymes in one pot).