Engineering of protease variants exhibiting high catalytic activity and exquisite substrate selectivity

Abstract

The exquisite selectivity and catalytic activity of enzymes have been shaped by the effects of positive and negative selection pressure during the course of evolution. In contrast, enzyme variants engineered by using in vitro screening techniques to accept novel substrates typically display a higher degree of catalytic promiscuity and lower total turnover in comparison with their natural counterparts. Using bacterial display and multiparameter flow cytometry, we have developed a novel methodology for emulating positive and negative selective pressure in vitro for the isolation of enzyme variants with reactivity for desired novel substrates, while simultaneously excluding those with reactivity toward undesired substrates. Screening of a large library of random mutants of the Escherichia coli endopeptidase OmpT led to the isolation of an enzyme variant, 1.3.19, that cleaved an Ala-Arg peptide bond instead of the Arg-Arg bond preferred by the WT enzyme. Variant 1.3.19 exhibited greater than three million-fold selectivity (-Ala-Arg-/-Arg-Arg-) and a catalytic efficiency for Ala-Arg cleavage that is the same as that displayed by the parent for the preferred substrate, Arg-Arg. A single amino acid Ser223Arg substitution was shown to recapitulate completely the unique catalytic properties of the 1.3.19 variant. These results can be explained by proposing that this mutation acts to "swap" the P(1) Arg side chain normally found in WT substrate peptides with the 223Arg side chain in the S(1) subsite of OmpT.

Fig. 1.

Flow-cytometric schemes and assays described in this study. (A) Two-color discrimination and selection by using the FRET and electrostatic capture substrates. (B) Library sort gate R3, used to isolate positive clones displaying high FL-1 (from the hydrolysis of 1) and low FL-2 (lack of hydrolysis of 2) fluorescence. (C) Flow-cytometric discrimination of E. coli UT5600 (ΔompT) transformed with pML19 (expressing WT OmpT), pML1.2.19, and pML1.3.19 by using FRET substrate 1. Briefly, the cells were washed and resuspended in 1% sucrose and labeled with 50 nM (final concentration) of the AR-FRET substrate 1. A 20-μl aliquot of the labeling reaction was transferred to 0.5 ml of 1% sucrose and analyzed on the flow cytometer.

Fig. 2.

Substrates used for the detection of catalytic activity and for the kinetic analysis.

Fig. 3.

The crystal structure of OmpT. (A) The electrostatic potential surface of OmpT (31) generated by using weblab viewerlite (Accelrys, San Diego). (B) The side chains corresponding to Asp-208 and Ser-223 are shown in addition to the proposed catalytic residues, Asp-83, Asp-85, and Asp-210 and His-212.

Fig. 4.

Arg side-chain swapping. The Arg occupying the P₁ subsite is accommodated by Ser-223 in S₁. In the 1.3.19 mutant, the Ser223Arg only allows occupancy by Ala in the P₁ site of the substrate.

Fig. 5.

Flow-cytometric analysis of the OmpTSer223 mutants. Briefly, the cells were washed and resuspended in 1% sucrose and labeled with 50 nM (final concentration) of the AR-FRET substrate 1. A 20-μl aliquot of the labeling reaction was transferred to 0.5 ml of 1% sucrose and analyzed on the flow cytometer. Dark blue, S223W; orange, S223L; light blue, S223F; dark green, S223K; pink, S223G; red, OmpT; light green, S223R.