Alteration of the substrate specificity of Thermus caldophilus ADP-glucose pyrophosphorylase by random mutagenesis through error-prone polymerase chain reaction

Abstract

Expanding the scope of stereoselectivity is of current interest in enzyme catalysis. In this study, using error-prone polymerase chain reaction (PCR), a thermostable adenosine diphosphate (ADP)-glucose pyrophosphorylase (AGPase) from Thermus caldophilus GK-24 has been altered to improve its catalytic activity toward enatiomeric substrates including [glucose-1-phosphate (G-1-P) + uridine triphosphate (UTP)] and [N-acetylglucosamine-1-phosphate (GlcNAc) + UTP] to produce uridine diphosphate (UDP)-glucose and UDP-N-acetylglucosamine, respectively. To elucidate the amino acids responsible for catalytic activity, screening for UDP-glucose pyrophosphorylase (UGPase) and UDP-N-acetylglucosamine pyrophosphorylase (UNGPase) activities was carried out. Among 656 colonies, two colonies showed UGPase activities and three colonies for UNGPase activities. DNA sequence analyses and enzyme assays showed that two mutant clones (H145G) specifically have an UGPase activity, indicating that the changed glycine residue from histidine has the base specificity for UTP. Also, three double mutants (H145G/A325V) showed a UNGPase, and A325 was associated with sugar binding, conferring the specificity for the sugar substrates and V325 of the mutant appears to be indirectly involved in the binding of the N-acetylamine group of N-acetylglucosmine-1-phosphate.

Substrate Specificity and Inhibitor Sensitivity of Plant UDP-Sugar Producing Pyrophosphorylases

“…A common architecture at or nearby substrate-binding domains appears also to be a feature for bacterial nucleotide-sugar producing pyrophosphorylases, which frequently share only less than 10% identity (based on aa sequences) with their plant counterparts ( Geisler et al, 2004 ; Kleczkowski et al, 2004 ). Mild random mutagenesis of a bacterial AGPase resulted in cDNA clones coding for proteins which had their substrate specificity changed to that of UGPase and UAGPase ( Sohn et al, 2006 ). Thus, a change of few aa could bring about a fundamental change in substrate specificity for this protein.…”

UDP-Sugar Producing Pyrophosphorylases: Distinct and Essential Enzymes With Overlapping Substrate Specificities, Providing de novo Precursors for Glycosylation Reactions

“…Structural similarities at or nearby substrate-binding domains are presumably also the case for bacterial nucleotide-sugar producing pyrophosphorylases, even though those proteins frequently share less than 10% identity with their plant counterparts (Kleczkowski et al., 2004). In one study, a mild random mutagenesis of a bacterial AGPase resulted in the production of novel proteins which had their substrate specificity changed to that of UGPase and UAGPase (Sohn et al., 2006). Thus, the nucleotide-sugar producing enzymes do have the capacity for changing their substrate specificity after few mutations.…”

Cloning and Mutagenesis of Catechol 2,3-Dioxygenase Gene from the Gram-Positive <b><i>Planococcus</i></b> sp. Strain S5

“…Error-prone PCR is the widely used method that has been successfully applied to the identification of critical residues for protein activity [Katayama et al, 2000;van Vugt-Lussenburg et al, 2007], improvement of thermal stability [Ahmad et al, 2008;Bordes et al, 2011] and alteration of substrate specificity [Sohn et al, 2006]. In this work, random mutagenesis of C23O was accomplished by the incorporation of the wrong bases during PCR by increasing the Mg 2+ concentration.…”