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

Abstract

Psychrophiles can thrive in cold environments [1−3].To grow and proliferate in these habitats, psychrophiles need adaptive strategies to maintain metabolic rates comparable to those of mesophiles [1,2,4]. To compensate for the reduction of biochemical reaction rate caused by low temperatures, they have evolved the enzymes which have higher catalytic activity (k cat ) than those from mesophilic homologs by increasing structural flexibility. However, the increase of flexibility was quite frequently accompanied by the decrease of the substrate affinity (K m ) [3,5]. These kinetic features are usually achieved by the substitutions of amino acids in the primary sequences [6−10]. As a consequence, these substitutions make the cold-adapted enzymes labile to thermal inactivation prior to their thermal unfolding [3,11]. The common characteristics of cold-adapted enzymes are higher catalytic activity (k cat ), lower substrate affinity (K m ), and lower thermal stability.Recently, we reported the crystal structure of 5enolpyruvylshikimate-3-phosphate synthase (EPSPS)Psychrophiles have evolved to produce cold-adapted enzymes to enable survival in low-temperature environments. In this study, the cold adaptation of 5-enolpyruvylshikimate-3-phosphate synthase (CpsEPSPS) from Colwellia psychrerythraea, a model psychrophile, was analyzed. The optimum temperature for the activity of CpsEPSPS was found to be 25℃, with 35% activity remaining at 5℃. However, the unfolding temperature of CpsEPSPS was 54℃. This phenomenon is frequently observed in cold-active enzymes. As is the cases for most cold-active enzymes, the K m values of CpsEPSPS for its substrates were higher than those of Escherichia coli EPSPS. These results indicate that CpsEPSPS is cold-adapted, but not perfectly.