A highly active phosphoglucomutase from Clostridium thermocellum: cloning, purification, characterization and enhanced thermostability.

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). Mg(2+) and Mn(2+) were activators. Ser144 was identified as an essential catalytic residue through site-directed mutagenesis. The k(cat) and K(m) 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 degrees C. Thermostability of PGM at a low concentration (2 nmol l(-1), 100 U l(-1)) was enhanced by 12-fold (i.e. t(1/2) = 72 h) at 60 degrees C with addition of bovine serum albumin, Triton X-100, Mg(2+)and Mn(2+).
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).