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Engineering of Protease Variants Exhibiting High Catalytic Activity and Exquisite Substrate Selectivity

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Engineering of Protease Variants Exhibiting High Catalytic Activity and Exquisite Substrate Selectivity Engineering of protease variants exhibiting high catalytic activity and exquisite substrate selectivity Navin Varadarajan*†‡, Jongsik Gam†‡, Mark J. Olsen*, George Georgiou*§¶, and Brent L. Iverson*†ʈ *Institute for Cellular and Molecular Biology and Departments of †Chemistry and Biochemistry and §Chemical Engineering, University of Texas, Austin, TX 78712 Edited by Robert T. Sauer, Massachusetts Institute of Technology, Cambridge, MA, and approved April 1, 2005 (received for review January 4, 2005) The exquisite selectivity and catalytic activity of enzymes have (13–15). Similarly, the evolution of highly active variants of been shaped by the effects of positive and negative selection aspartate aminotransferases capable of accepting branched or pressure during the course of evolution. In contrast, enzyme aromatic amino acid substrates was accompanied by a relaxation variants engineered by using in vitro screening techniques to of the substrate selectivity (16, 17). accept novel substrates typically display a higher degree of cata- In nature, the evolution of enzymes occurs as the result of lytic promiscuity and lower total turnover in comparison with their positive selective pressure for turnover of physiological sub- natural counterparts. Using bacterial display and multiparameter strates, combined with simultaneous negative selective pressure flow cytometry, we have developed a novel methodology for to eliminate completely, or at least drastically suppress, delete- emulating positive and negative selective pressure in vitro for the rious activities. It follows that the engineering of enzymes isolation of enzyme variants with reactivity for desired novel exhibiting high catalytic activity and substrate selectivity for a substrates, while simultaneously excluding those with reactivity particular desired substrate should be similarly accomplished by toward undesired substrates. Screening of a large library of ran- implementing selection and counterselection assay schemes in dom mutants of the Escherichia coli endopeptidase OmpT led to the the laboratory. The recent directed evolution of novel tRNA isolation of an enzyme variant, 1.3.19, that cleaved an Ala–Arg synthetases and recombinases by capitalizing on in vivo selec- peptide bond instead of the Arg–Arg bond preferred by the WT tions support this expectation (18–19). Unfortunately, many enzyme. Variant 1.3.19 exhibited greater than three million-fold desired enzyme activities are not amenable to in vivo selection selectivity (-Ala–Arg-͞-Arg–Arg-) and a catalytic efficiency for Ala– strategies because cellular growth cannot be linked to the Arg cleavage that is the same as that displayed by the parent for enzyme activity being sought. Consequently, laboratory-directed the preferred substrate, Arg–Arg. A single amino acid Ser223Arg evolution approaches for the isolation of highly selective en- substitution was shown to recapitulate completely the unique zymes must rely on in vitro catalytic assays whereby the activity catalytic properties of the 1.3.19 variant. These results can be toward selection and counterselection substrates is determined explained by proposing that this mutation acts to ‘‘swap’’ the P1 sequentially for each library member (9–11, 13, 14). The suc- Arg side chain normally found in WT substrate peptides with the cessful implementation of the latter approach hinges on satis- 223Arg side chain in the S1 subsite of OmpT. fying the following requirements. First, assays that afford the proper dynamic range must be designed because mutant proteins engineering ͉ flow cytometry are likely to exhibit drastically different kcat and Km values for the various selection and counterselection substrates. Second, the he reprogramming of enzyme catalytic activity and selectiv- isolation of rare, high-activity clones requires the screening of Tity is a central issue in protein biochemistry and biotechnol- large libraries by assaying each clone toward multiple substrates, ogy. Numerous structure-guided and directed evolution strate- thus necessitating the availability of suitable high-throughput gies have been used in search of enzyme variants that exhibit high methods. catalytic rates with poor or inactive substrates of the parental With these considerations in mind, we have extended our enzyme (1–19). As impressive as these successes have been, the previous approach (20) and developed a new two-pronged engineering of enzymes that exhibit turnover rates and selectiv- strategy in which catalytic activities over a wide dynamic range ities with new substrates comparable to their natural counter- for both a selection substrate and one or more counterselection parts has proven quite a challenge, especially when considering substrates are quantified simultaneously at the single-cell level, those enzymes for which a genetic selection strategy is not enabling the rapid screening of mutant libraries. This approach possible. relies on fluorescent substrates of different colors that label the In particular, enzymes engineered through laboratory evolu- surface of E. coli cells upon cleavage by a surface-anchored tion involving in vitro catalytic assays have often been found enzyme (20). Enzymes can be displayed on the surface of lacking, either with respect to turnover rates or selectivity, Gram-negative bacteria by established techniques (21, 22), and relative to catalyst–substrate pairs isolated from natural sources. thus, access to the fluorescent substrate is assured. The net result As a typical example, an extensive directed evolution program is that the cell fluorescence profile accurately reflects the led to the isolation of Escherichia coli ␤-glucuronidase variants catalytic activity and selectivity of the surface-displayed enzyme. with significant ␤-galactosidase (10) or xylanosidase (11) activ- Multiparameter flow cytometry is then used to isolate clones ities, but nonetheless even the best clones exhibited kcat͞Km Ͼ values 1,000 times lower than those of naturally occurring BIOCHEMISTRY This paper was submitted directly (Track II) to the PNAS office. enzymes such as the E. coli ␤-galactosidase or the Thermoanaer- ␤ Abbreviation: BODIPY, 4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-propi- obacterium saccharolyticum -xylosidase. onic acid. This trend appears to be general. In a recent comprehensive ‡N.V. and J.G. contributed equally to this work. study, Aaron et al. (12) demonstrated that the evolution of higher ¶To whom correspondence may be addressed at: Department of Chemical Engineering, activity toward poor substrates did not impair the parental University of Texas, Austin, TX 78712. E-mail: [email protected]. catalytic activity, and, therefore, the evolved enzymes exhibited ʈTo whom correspondence may be addressed at: Department of Chemistry and Biochem- greater promiscuity. Enzymes evolved for higher substrate en- istry, University of Texas, Campus Mail Code A5300, Austin, TX 78712. E-mail: antioselectivity often exhibit lower specific activities toward their [email protected]. new substrates relative to their respective parental enzymes © 2005 by The National Academy of Sciences of the USA www.pnas.org͞cgi͞doi͞10.1073͞pnas.0500063102 PNAS ͉ May 10, 2005 ͉ vol. 102 ͉ no. 19 ͉ 6855–6860 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 (⌬ompT) 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-␮l aliquot of the labeling reaction was transferred to 0.5 ml of 1% sucrose and analyzed on the flow cytometer. expressing enzymes having a desired fluorescence profile from University of Texas peptide synthesis facility. Tetramethylrho- large libraries. damine-5-iodoacetamide (TMRIA), 5-carboxytetramethylrho- The E. coli endoprotease OmpT and its Omptin homologues damine, succinimidyl ester (5-TAMRA, SE), and BODIPY- play important roles in pathogenicity, are of significance in FL-SE were purchased from Molecular Probes. The peptides 3, protein manufacturing, and have been exploited for biotechnol- WCARVGKGRGR-NH2, and 4, WEEGGRRIGRGGK-NH2 ogy applications (23, 24). OmpT has a strong preference for (Ͼ95% purity), were purchased from Cell Essentials (Boston). cleavage between two basic residues (Lys and, especially, Arg) in Ј For the synthesis of the FRET substrate 1, a 50-␮l solution of the P1 and P1 positions of the substrate (25–27). We sought to peptide 1a (5.4 mg, 4.9 ␮mol) in water was added to 100 ␮lof isolate OmpT variants that exhibit (i) hydrolysis of a substrate 1MNa2CO3 and 50 ␮l of 0.5M NaHCO3. A solution of 50 ␮lof that is cleaved poorly by the WT enzyme and (ii) a low rate of ␮ cleavage of dibasic sequences (preferred by the WT enzyme), TMRIA (4.0 mg, 4.8 mol) in dimethylformamide was added to thus conferring (iii) a high selectivity for the new cleavage over the reaction mixture and stirred at room temperature for 1h. The the one preferred by the parental enzyme (Fig. 1A), while reaction mixture was quenched with 5 ml of 0.1% trifluoroacetic maintaining (iv) a very high level of catalytic activity. acid in water, and the product was purified
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