Effect of the inserted active-site-covering lid loop on the catalytic activity of a mutant B. subtilis γ-glutamyltransferase (GGT)

Abstract

A mutant γ-glutamyltransferase with improve transpeptidase activity was obtained by inserting the active site-covering lid loop on an enzyme naturally lacking it.

Simulating Multiple Substrate-Binding Events by γ-Glutamyltransferase Using Accelerated Molecular Dynamics

“…Although the role of the lid-loop in substrate selection and catalysis , has been experimentally demonstrated, GGTs from a number of microorganisms are devoid of the lid-loop, still sharing sequence, structural, and functional similarities with other GGTs (Figure S14). The lower transpeptidase activity of these enzymes and their ability of accepting polymeric substrates, differently from other GGTs, have been related to the lack of the lid-loop .…”

From Batch to Continuous Flow Bioprocessing: Use of an Immobilized γ-Glutamyl Transferase from B. subtilis for the Synthesis of Biologically Active Peptide Derivatives

“…The reactions were initiated by adding 0.5 UI/mL of BsGGT-GLX-AG (17 UI/g) and incubated at 25 °C under magnetic stirring for 24 h. Samples were periodically withdrawn and derivatized with Sanger's reagent before analysis by following a standard protocol. 15 HPLC analyses were carried out by using a linear gradient of eluent A (water + 0.1% TFA) and eluent B (acetonitrile + eluent A 80: Batch Synthesis of γ-Glutamyl-Derivatives: Semipreparative Scale and Purification. Once the endpoint for each reaction was determined (analytical scale), the reactions were scaled up (15 mL).…”

“…14 Later on, the transpeptidase activity of a mutant GGT, obtained by inserting the lid-loop from Escherichia coli GGT (EcGGT) in the structure of Bacillus subtilis GGT (BsGGT), naturally lacking it, was investigated. 15 The mutant GGT showed enhanced transpeptidase activity with respect to the wild-type enzyme. However, the mutant enzyme maintained the ability of the wild-type parent GGT to catalyze the formation of polyglutamylated derivatives, thus hampering its application in preparative syntheses.…”

“…However, the mutant enzyme maintained the ability of the wild-type parent GGT to catalyze the formation of polyglutamylated derivatives, thus hampering its application in preparative syntheses. 15 The transpeptidase activity of GGTs can be also selectively enhanced by a fine tuning of the reaction conditions (donor/acceptor ratio, pH, time, temperature, and enzyme amount), even assisted by advanced reaction and enzyme engineering tools based on flow biocatalysis and enzyme immobilization, respectively. On the one hand, flow biotransformations combine flow chemistry's throughput to the catalytic power of enzymes, thus providing new solutions to achieve high productivity, better process control, and less waste.…”

“…In order to maximize the formation of transpeptidation products, mutagenesis experiments were carried out to improve the catalytic efficiency of GGTs as biocatalysts according to path a (Scheme ). To this purpose, a few variants of GGTs were reported. − Bacillus licheniformis ER15 GGT ( Bl GGT) was engineered in an attempt of minimizing the autotranspeptidation reaction. The Arg109Lys mutation increased transpeptidation activity and catalytic efficiency in the synthesis of l -theanine (80% vs 60% conversion with the wild-type enzyme, respectively, under the same reaction conditions) .…”