Enzyme engineering of a haloacid dehalogenase-like phosphatase from Thermotoga neopolitana for optimization of substrate specificity

Sammanfattning: Haloacid dehalogenase (HAD)-like hydrolases represent a vast superfamily characterised by their ability to form covalent enzyme-substrate intermediates facilitating the cleavage of C-Cl, C-P, or CO-P bonds. Within this work one member of this superfamily, HAD from Thermotoga neopolitana (HADTn) exhibiting a phosphatase (P-O bond cleavage) like activity was investigated in the context of altering the substrate specificity. The enzyme shows different affinities for sugar phosphates, e.g. fructose-6-phosphate (F6P) and glucose-6-phosphate (G6P) at high temperatures, which is of great interest for industrial applications. In this study, HADTn was engineered using semi rational site saturation mutagenesis to increase its specificity toward F6P for use in enzymatic cascade reactions. At first, several mutants were generated to deduce the role of different amino acids. Results suggested that serine at position 166 (S166) played an important role in catalysis, presumably in the positioning of Mg (II). Additionally, glutamic acid at position 47 (E47) was identified to influence substrate recognition. Based on these findings, degenerate primers using an NNK motif were designed to generate libraries for six different amino acid positions (Y19, M8, P46, E47, P110, R117) around the catalytic centre. High-throughput screening of the libraries with two different substrates (F6P and G6P) resulted in an improved variant that had a mutation at position 46 from proline to threonine (P46T). Further characterisation of HADTnP46T was performed for F6P and G6P with respect to the wild type and the results showed almost a two-fold increase in the ratio of the catalytic efficiency (kcat/Km). Moreover, thermal stability studies revealed that HADTnP46T had a half-life of 392 min at 94oC; similar to that of the WT. Addition of Mg (II) doubled the half-life for both the enzymes. Fructose and glucose inhibited both enzymes via a noncompetitive mechanism. These findings support the already existing proposal of the presence of a probable fifth loop in the HAD family that plays a pivotal role in substrate specificity.