Application of a Dual Internally Quenched Fluorogenic Substrate in Screening for D-Arginine Specific Proteases

Abstract

The application of D -stereospecific proteases ( D SPs) in resolution of racemic amino acids and in the semisynthesis of proteins has been a successful strategy. The main limitation for a broader application is, however, the accessibility of suitable D SPs covering multiple substrate specificities. To identify D SPs with novel primary substrate preferences, a fast specificity screening method using the easily accessible internally quenched fluorogenic substrate aminobenzoyl- D -arginyl- D -alanyl- p -nitroanilide was developed. By monitoring both UV/ vis -absorbance and fluorescence signals at the same time it allows to detect two distinct D -amino acid substrate specificities simultaneously and separately with respect to the individual specificities. In order to identify novel D SP specificities for synthesis applications, D SPs specific for D -arginine were of special interest due to their potential ability as catalysts for substrate mimetics-mediated peptide and protein ligations. D -alanine in the substrate served as positive control and reference based on its known acceptance by numerous D SPs. In silico analysis suggested that D SPs are predominantly present in gram-positive microorganisms, therefore this study focused on the bacilli strains Bacillus thuringiensis and Bacillus subtilis as potential hosts of D -Arg-specific D SPs. While protease activities toward D -alanine were found in both organisms, a novel and so far unknown D -arginine specific D SP was detected within the culture supernatant of B. thuringiensis . Enrichment of this activity via cation exchange and size exclusion chromatography allowed isolation and further characterization of this novel enzyme consisting of a molecular mass of 37.7 kDa and an enzymatic activity of 8.3 U mg -1 for cleaving the D -Arg| D -Ala bond in the detecting substrate. Independent experiments also showed that the identified enzyme shows similarities to the class of penicillin binding proteins. In future applications this enzyme will be a promising starting point for the development of novel strategies for the semisynthesis of all - L -proteins.

Kinetic Resolution of Epimeric Proteins Enables Stereoselective Chemical Mutagenesis

“…We expect that further optimization of reaction conditions, as well as targeted engineering efforts of AD-P activity and specificity, will allow expansion of this technology to enable the stereoselective installation of a multitude of PTMs and their analogues into diverse protein targets. Indeed, the discovery of d -peptidases with inherent specificities for various sequences and residues (including d -Asp and d -Arg) bodes well for the expansion of a powerful toolkit toward the stereoselective chemoenzymatic mutagenesis of proteins.…”