Characterization and mechanism insight of accelerated catalytic promiscuity of Sulfolobus tokodaii (ST0779) peptidase for aldol addition reaction

Abstract

A novel peptidase from thermophilic archaea Sulfolobus tokodaii (ST0779) is examined for its catalytic promiscuity of aldol addition, which shows comparable activity as porcine pancreatic lipase (PPL, one of the best enzymes identified for biocatalytic aldol addition) at 30 °C but much accelerated activity at elevated temperature. The molecular catalytic efficiency kcat/Km (M(-1) s(-1)) of this thermostable enzyme at 55 °C adds up to 140 times higher than that of PPL at its optimum temperature 37 °C. The fluorescence quenching analysis depicts that the binding constants of PPL are significantly higher than those of ST0779, and their numbers of binding sites show opposite temperature dependency. Thermodynamic parameters estimated by fluorescence quenching analysis unveil distinctly different substrate-binding modes between PPL and ST0779: the governing binding interaction between PPL and substrates is hydrophobic force, while the dominating substrate-binding forces for ST0779 are van der Waals and H-bonds interactions. A reasonable mechanism for ST0779-catalyzed aldol reaction is proposed based on kinetic study, spectroscopic analysis, and molecular stereostructure simulation. This work represents a successful example to identify a new enzyme for catalytic promiscuity, which demonstrates a huge potential to discover and exploit novel biocatalyst from thermophile microorganism sources.

Bio-fabrication of thermozyme-based nano-biosensors: their components and present scenario

“…The aldol reaction, a vital carbon–carbon bond-forming reaction, forms the core of several organic synthesis for industrial applications, such as flavors, detergents, drugs, plasticizers, etc. [ 47 ]. Currently, acidic or alkaline catalysts are applied in aldol reactions.…”

A transaminase-mediated aldol reaction and applications in cascades to styryl pyridines

“…1 Examples include hydrolytic enzymes such as porcine pancreatic lipase and a peptidase from Sulfolobus tokodaii that have been reported to catalyse aldol reactions. [1][2][3][4] Enzyme mutagenesis has been reported to modify enzyme functionality and an early example was a single mutation in alanine racemase which converted it into an aldolase. 5,6 More recently, a range of new to nature enzyme functionalities have been reported such as photoinduced enzyme catalysis with ene reductases and variants via radical mechanisms for asymmetric reduction, hydroalkylation and conjugate addition.…”

Recent advances in enzyme promiscuity

“…The catalytic efficiency of this enzyme at 55 °C adds up to 140 times higher than that of PPL at its optimum physiological temperature. This study is an example, which signifies the importance to identify a new enzyme with catalytic promiscuity and demonstrates the application of novel biocatalyst from thermophile microorganisms [50].…”

“…The composition of the monolignol monomers determines the degree of lignin condensation and thus the degradability of plant cell walls. Guaiacyl lignin is considered as the condensed structural unit [50]. The active site of a monolignol 4-O-methyltransferase (MOMT5) was precisely remodeled to create an enzyme that specifically methylates the condensed guaiacyl lignin precursor coniferyl alcohol.…”