Abstract
γ-Glutamylpeptides are compounds derived from the acylation of an amino acid or a short peptide by the γ-carboxyl carbon of the side chain of glutamic acid. Due to their altered chemico-physical and organoleptic properties, they may be interesting substitutes or precursors of parent compounds used in pharmaceutical, dietetic and cosmetic formulations. Some of them are naturally occurring flavor enhancers or are endowed with biological activities. Enzymatic approaches to the synthesis of γ-glutamyl derivatives based on the use of γ-glutamyltransferases (GGTs, EC 2.3.2.2) have been proposed, which should be able to alleviate the problems connected with the troublesome and low-yielding extraction from natural sources or the non-economical chemical synthesis, which requires protection/deprotection steps. With the aim of overcoming the current limitations in the use of GGTs as biocatalysts, a mutant GGT was investigated. The mutant GGT was obtained by inserting the active-site-covering lid loop of the E. coli GGT onto the structure of B. subtilis GGT. With respect to the wild-type enzyme, the mutant showed a more demanding substrate specificity and a low hydrolase activity. These results represent an attempt to correlate the structural features of a GGT to its different activities. However, the ability of the mutant enzyme to catalyze the subsequent addition of several γ-glutamyl units, inherited by the parent B. subtilis GGT, still represents a limitation to its full application as a biocatalyst for preparative purposes.
Highlights
GeneralL-Glutamine, L-glutamic acid, L-serine, glycilglycine (GlyGly), Lglutamic acid 5-(p-nitroanilide) (GPNA) and 1- uoro-2,4dinitrobenzene (Sanger's reagent) were from Sigma Aldrich (Darmstadt, Germany); L-alanylglycine, L-2-aminobutrylglycine (Abu-Gly) and L-leucylglycyne were from Bachem (Bubendorf, Switzerland)
Introduction gGlutamyl compounds (Fig. 1) are de ned as being derived by the acylation of an amino acid or a short peptide through the gcarboxyl carbon of a glutamic acid moiety
Our results suggest that the lid-loop is able to shield the gglutamyl enzyme intermediate from bulk solvent, preventing hydrolysis and favoring the transpeptidation reaction
Summary
L-Glutamine, L-glutamic acid, L-serine, glycilglycine (GlyGly), Lglutamic acid 5-(p-nitroanilide) (GPNA) and 1- uoro-2,4dinitrobenzene (Sanger's reagent) were from Sigma Aldrich (Darmstadt, Germany); L-alanylglycine, L-2-aminobutrylglycine (Abu-Gly) and L-leucylglycyne were from Bachem (Bubendorf, Switzerland). HPLC analyses were carried out with a Jasco instrument equipped with UV/Vis detector, using a 250 Â 4.6 mm Gemini RP C18 column (Phenomenex, Torrance, CA, USA). The compounds, derivatized with Sanger's reagent, were analyzed with the following gradient: 0– 10 min, eluent A : eluent B 80 : 20 isocratic elution; 10–15 min, linear gradient to eluent A : eluent B 70 : 30; 15–25 min, linear gradient to eluent A : eluent B 40 : 40; 25–35 min, linear. Ion exchange chromatography was performed with Dowex 1 Â 8 resin 200–400 mesh (Aldrich, Darmstadt, Germany) in the acetate form. HPLC-MS was performed with a Thermo Finnigan Surveyor LC pump equipped with a Thermo Finnigan Surveyor photodiode array detector and interfaced with the ESI Thermo Finnigan LCQ Advantage spectrometer using column and elution parameters reported previously.
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