Cloning, Expression, and Characterization of a Cold-Adapted Shikimate Kinase from the Psychrophilic Bacterium Colwellia psychrerythraea 34H

Abstract

Most cold-adapted enzymes possess higher K and k values than those of their mesophilic counterparts to maximize the reaction rate. This characteristic is often ascribed to a high structural flexibility and improved dynamics in the active site. However, this may be less convincing to cold-adapted metabolic enzymes, which work at substrate concentrations near K. In this respect, cold adaptation of a shikimate kinase (SK) in the shikimate pathway from psychrophilic (CpSK) was characterized by comparing it with a mesophilic homolog (EcSK). The optimum temperatures for CpSK and EcSK activity were approximately 30°C and 40°C, respectively. The melting points were 33°C and 45°C for CpSK and EcSK, respectively. The ΔG (denaturation in the absence of denaturing agent) values were 3.94 and 5.74 kcal/mol for CpSK and EcSK, respectively. These results indicated that CpSK was a cold-adapted enzyme. However, contrary to typical kinetic data, CpSK had a lower K for its substrate shikimate than most mesophilic SKs, and the k was not increased. This observation suggested that CpSK may have evolved to exhibit increased substrate affinity at low intracellular concentrations of shikimate in the cold environment. Sequence analysis and homology modeling also showed that some important salt bridges were lost in CpSK, and higher Arg residues around critical Arg 140 seemed to increase flexibility for catalysis. Taken together, these data demonstrate that CpSK exhibits characteristics of cold adaptation with unusual kinetic parameters, which may provide important insights into the cold adaptation of metabolic enzymes.

Characterization of 5-Enolpyruvylshikimate-3-Phosphate Synthase from Colwellia psychrerythraea

“…To investigate the thermal liability, we performed thermal unfolding of the CpsEPSPS. The thermal unfolding was determined by monitoring the changes of molar ellipticity at 220 nm over the temperature range of 5−80℃ using a Chirascan Circular Dichroism Spectrometer (Applied photophysics Co., UK) [16]. The melting point (T m ) of CpsEPSPS was 54℃ (Fig.…”