Study of the structure and dynamics of a thermophilic acylphosphatase from Pyrococcus horikoshii by NMR spectroscopy

Abstract

Thermophilic enzymes adapted to perform catalysis at elevated temperatures are often sluggish enzymes at lower temperatures, when comparing to mesophilic homologues. Reduced flexibility is often regarded as the culprit behind the reduced catalytic efficiency of thermophilic enzymes. We have cloned the coding sequence of acylphosphatase from Pyrococcus horikoshii (PhAcP) and expressed the protein in E. coli. We propose to use an acylphosphatase from Pyrococcus horikoshii as a model system to study the stability, activity, and flexibility relationships of enzymes. Acylphosphatase (AcP, ~90-100 residues) is one of the smallest enzymes known. We have recently solved the structure of PhAcP, and measured its stability and kinetics parameters. High-resolution structure of the thermophilic AcP is compared to the structures of mesophilic AcPs to understand the structural adaptation of P. horikoshii AcP in coping with high temperatures. We have found that while PhAcP is extremely stable, with a melting temperature of ~112oC, and a free energy of unfolding of ~54 kJ/mol. Kinetics studies showed that PhAcP is a less efficient enzyme than other mesophilic AcP, for its kcat value of ~95 s-1 is much lower than the value of ~1500 s-1 reported for mesophilic AcP. As there is no structural difference between the active site of PhAcP and mesophilic AcP, we hypothesize that the reduced activity of PhAcP is due to reduced flexibility of the active site. To investigate the structural and dynamical change upon binding of phosphate to the active site, we are going to determine the structure of P. horikoshii AcP-phosphate complex and study the dynamics of free and bound forms of AcP by NMR spectroscopy. The protein dynamics of PhAcP and a mesophilic AcP characterized by NMR relaxation experiments will also be compared.