Cloning, Expression, and Characterization of a Cold-Adapted Shikimate Kinase from the Psychrophilic BacteriumColwellia psychrerythraea34H.
Most cold-adapted enzymes possess higher Kmand kcatvalues 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 Km. In this respect, cold adaptation of a shikimate kinase (SK) in the shikimate pathway from psychrophilicColwellia psychrerythraea(CpSK) was characterized by comparing it with a mesophilicEscherichia colihomolog (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 ΔGH2O(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 Kmfor its substrate shikimate than most mesophilic SKs, and the kcatwas 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.
Department of Chemistry, Pukyong National University, Busan 48547, Republic of Korea.
Wahyu Sri Kunto
WS
Nugroho
Department of Chemistry, Pukyong National University, Busan 48547, Republic of Korea.
Dong-Woo
DW
Kim
Southeast Sea Fisheries Research Institute, National Fisheries Research and Development Institute, Tongyeong 53085, Republic of Korea.
Jong-Cheol
JC
Han
Southeast Sea Fisheries Research Institute, National Fisheries Research and Development Institute, Tongyeong 53085, Republic of Korea.
Young Baek
YB
Hur
Department of Biotechnology and Bioengineering, Dong-Eui University, Busan 47340, Republic of Korea.
Soo-Wan
SW
Nam
Department of Chemistry, Pukyong National University, Busan 48547, Republic of Korea.
Hak Jun
HJ
Kim
D034241:Alteromonadaceae / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics; D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D003001:Cloning, Molecular; D003080:Cold Temperature; D004795:Enzyme Stability; D015870:Gene Expression; D007700:Kinetics; D008969:Molecular Sequence Data; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D016415:Sequence Alignment; D012765:Shikimic Acid / Q000737:chemistry / Q000378:metabolism
D001426:Bacterial Proteins; D012765:Shikimic Acid; D017853:Phosphotransferases (Alcohol Group Acceptor); C022642:shikimate kinase
Cold adaptation; Michaelis-Menten constant; psychrophile; shikimate kinase; turnover number