Enzymatic hydrolysis of PET: functional roles of three Ca2+ ions bound to a cutinase-like enzyme, Cut190*, and its engineering for improved activity

Abstract

Cut190 from Saccharomonospora viridis AHK190 (Cut190) is the only cutinase that exhibits inactive (Ca²⁺-free) and active (Ca²⁺-bound) states, although other homologous cutinases always maintain the active states (Ca²⁺-free and bound). The X-ray crystallography of the S176A mutant of Cut190* (Cut190_S226P/R228S) showed that three Ca²⁺ ions were bound at sites 1-3 of the mutant. We analyzed the roles of three Ca²⁺ ions by mutation and concluded that they play different roles in Cut190* for activation (sites 1 and 3) and structural and thermal stabilization (sites 2 and 3). Based on these analyses, we elucidated the mechanism for the conformational change from the Ca²⁺-free inactive state to the Ca²⁺-bound active state, proposing the novel Ca²⁺ effect on structural dynamics of protein. The introduction of a disulfide bond at Asp250 and Glu296 in site 2 remarkably increased the melting temperatures of the mutant enzymes by more than 20-30 °C (while Ca²⁺-bound) and 4-14 °C (while Ca²⁺-free), indicating that a disulfide bond mimics the Ca²⁺ effect. Replacement of surface asparagine and glutamine with aspartic acid, glutamic acid, or histidine increased the melting temperatures. Engineered mutant enzymes were evaluated by an increase in melting temperatures and kinetic values, based on the hydrolysis of poly(butylene succinate-co-adipate) and microfiber polyethylene terephthalate (PET). A combined mutation, Q138A/D250C-E296C/Q123H/N202H, resulted in the highest thermostability, leading to the maximum degradation of PET film (more than 30%; approximately threefold at 70 °C, compared with that of Cut190* at 63 °C).

Keywords: Ca2+-bound activation; Ca2+-bound thermostabilization; Cutinase-like enzyme; Genetic engineering; PET hydrolase.