Flexibility and Stability Trade-Off in Active Site of Cold-Adapted Pseudomonas mandelii Esterase EstK

Abstract

Cold-adapted enzymes exhibit enhanced conformational flexibility, especially in their active sites, as compared with their warmer-temperature counterparts. However, the mechanism by which cold-adapted enzymes maintain their active site stability is largely unknown. In this study, we investigated the role of conserved D308-Y309 residues located in the same loop as the catalytic H307 residue in the cold-adapted esterase EstK from Pseudomonas mandelii. Mutation of D308 and/or Y309 to Ala or deletion resulted in increased conformational flexibility. Particularly, the D308A or Y309A mutant showed enhanced substrate affinity and catalytic rate, as compared with wild-type EstK, via enlargement of the active site. However, all mutant EstK enzymes exhibited reduced thermal stability. The effect of mutation was greater for D308 than Y309. These results indicate that D308 is not preferable for substrate selection and catalytic activity, whereas hydrogen bond formation involving D308 is critical for active site stabilization. Taken together, conformation of the EstK active site is constrained via flexibility-stability trade-off for enzyme catalysis and thermal stability. Our study provides further insights into active site stabilization of cold-adapted enzymes.

Discovery, Molecular Mechanisms, and Industrial Applications of Cold-Active Enzymes

“…Typically, the fluorescence quenching constants (as reported by the Stern-Volmer constant) of psychrophilic enzymes are higher than for mesophilic proteins at both low and warm temperatures, thus indicating a more permeable structure (Huston et al, 2008 ; Tang et al, 2012 ), and the variation of fluorescence quenching (i.e., the change in the Stern-Volmer constant) within a temperature range where the native state prevails decreases in the order psychrophilic > mesophilic > thermophilic (D'Amico et al, 2003a ; Georlette et al, 2003 , 2004 ; Cipolla et al, 2012 ), thus indicating that cold-adapted enzymes possess higher flexibility. These experiments can also be combined with mutational analysis to explore the interplay between sequence variation, protein flexibility, and catalytic activity (Cipolla et al, 2011 ; Sigtryggsdóttir et al, 2014 ; Truongvan et al, 2016 ).…”

The Role of Solvent-Accessible Leu-208 of Cold-Active Pseudomonas fluorescens Strain AMS8 Lipase in Interfacial Activation, Substrate Accessibility and Low-Molecular Weight Esterification in the Presence of Toluene

“…The idea of having a rigid active site capable of supporting higher enzyme activity in a cold-active lipase is quite contrary. Supposedly mutations that leave more space lead to a better improvement of enzyme activity [ 12 ]. As for L208A mutation, the active sites and lid 2 regions had been reorganized or reshaped to a level of extreme rigidity which has been addressed in both molecular dynamic and docking studies.…”

“…Recently, cold-adapted esterases (EstK) which harbored the conserved dipeptide sequence Asp-Tyr located at catalytic His-307 loop was also reported to be responsible in maintaining active site conformation as a result to the establishment of hydrogen bond between D308 and W208. Meanwhile, EstK mutant W208Y situated at the wall of active site exhibited high catalytic efficiency and catalytic site thermal stability as compared to wild-type (WT) [ 12 ]. By far, the substitution, insertions and deletions of bulky residues could lead to a more accessible or flexible active site in cold-adapted enzymes.…”

Engineering Lipases: walking the fine line between activity and stability

“…On the other hand, the psychrophilic enzymes have not evolved under thermal selection pressure and generally have lower thermostability but higher flexibility especially in the active site region. [51][52][53][54][55][56] The increased flexibility contributes to the lower thermal stability of psychrophiles. Consequently, flexibility has become a key parameter to bridge the mutations (therefore, effect of a residue) to the observed changes in activity and stability when comparing enzymes.…”