Joel Osuna

Combinatorial mutagenesis of three major groove-contacting residues of EcoRI: single and double amino acid replacements retaining methyltransferase-sensitive activities

A library of mutant ecoRIR genes encoding EcoRI restriction endonuclease was generated using trinucleotide blocks and a combination of recombinant DNA procedures, including primer extension and the polymerase chain reaction. Codons corresponding to three amino acids (E144, R145 and R200), previously implicated in the specific recognition of the DNA substrate, were combinatorially mutated so as to generate a library that potentially contains all 20(3) possible single, double and triple aa replacements, in a balanced distribution. Inspection of the phenotypes of Escherichia coli colonies bearing the mutant genes showed that several of them retained activities that were deleterious to the cells but were still protected by the EcoRI methyltransferase. These included new enzyme variants, including non-conservative single (Thr or Val for Glu144) and double (Val for Glu144 and Thr for Arg145) replacements.

Saturation mutagenesis of His114 of EcoRI reveals relaxed-specificity mutants

EcoRI recognizes and cleaves DNA at GAATTC sites and is one of the best characterized sequence-specific restriction endonucleases (ENases). In previous studies, an EcoRI mutant, which exhibited relaxed substrate specificity and cleaved both canonical and EcoRI star sites, was isolated. This mutant enzyme has Tyr instead of His114. Here, we subjected residue 114 of the EcoRI ENase to saturation mutagenesis. The resulting mutant enzymes were characterized both in vivo and in vitro, resulting in the identification of mutants with canonical (H114K, Q, D, I) or relaxed (H114Y, F, S, T) specificity, as well as one mutant with severely impaired activity (H114P). In the X-ray structure of an EcoRI-substrate complex, His114 is located between the catalytic and recognition regions of EcoRI and may directly contact the DNA phosphate backbone. Based on our genetic and biochemical findings and the X-ray structure, we propose that His114 participates in substrate recognition and catalysis, either directly, via protein-DNA interactions, or indirectly, by mediating conformational changes that trigger DNA cleavage in response to substrate recognition.

Substitution of Asp for Asn at Position 132 in the Active Site of TEM -Lactamase ACTIVITY TOWARD DIFFERENT SUBSTRATES AND EFFECTS OF NEIGHBORING RESIDUES

Using a random, combinatorial scheme of mutagenesis directed against the conserved SDN region of TEM β-lactamase, and selective screening in ampicillin-plates, we obtained the N132D mutant enzyme. The kinetic characterization of this mutant indicated relatively small effects compared to the wild-type. Both pK1 and pK2 for catalysis were decreased about 1 unit relative to the pK′s for the wild type. This effect was predominantly due to changes in K. In contrast to the wild-type, the pH-rate profiles of the mutant showed that K for several side chain-containing penicillin substrates increases when the pH is above 5.5. 6-Aminopenicillanic acid, which lacks a side chain, did not show this effect. With benzylpenicillin, ampicillin, and carbenicillin, k for the mutant showed a similar pH dependence as the wild type. With 6-aminopenicillanic acid, k for the mutant was greater than that for the wild type. The nature of the 104 side chain may affect the environment of Asp; double mutants N132D/E104X (where X can be Q or N) are unable to confer antibiotic resistance to bacterial cells. The computed contact interactions from modeling substrate complexes between benzylpenicillin or 6-aminopenicillanic acid with the N132D mutant confirmed the importance of the protonation state of residue Asp for the complex stability with side chain-containing substrates. The data indicate that the contact between the side chain of residue 132 and the substrate is relevant for the ground state recognition, but because of close contact with several important groups in its neighborhood, residue 132 is also indirectly involved in the catalytic step of the wild-type enzyme.

Homology Modeling and Site-directed Mutagenesis of Pyroglutamyl Peptidase II INSIGHTS INTO OMEGA-VERSUS AMINOPEPTIDASE SPECIFICITY IN THE M1 FAMILY

Pyroglutamyl peptidase II (PPII), a highly specific membrane-bound omegapeptidase, removes N-terminal pyroglutamyl from thyrotropin-releasing hormone (<Glu-His-Pro-NH2), inactivating the peptide in the extracellular space. PPII and enzymes with distinct specificities such as neutral aminopeptidase (APN), belong to the M1 metallopeptidase family. M1 aminopeptidases recognize the N-terminal amino group of substrates or inhibitors through hydrogen-bonding to two conserved residues (Gln-213 and exopeptidase motif Glu-355 in human APN), whereas interactions involved in recognition of pyroglutamyl residue by PPII are unknown. In rat PPII, the conserved exopeptidase residue is Glu-408, whereas the other one is Ser-269. Given that variations in M1 peptidase specificity are likely due to changes in the catalytic region, we constructed three-dimensional models for the catalytic domains of PPII and APN. The models showed a salt bridge interaction between PPII-Glu-408 and PPII-Lys-463, whereas the equivalent APN-Glu-355 did not participate in a salt bridge. Docking of thyrotropin-releasing hormone in PPII model suggested that the pyroglutamyl residue interacted with PPII-Ser-269. According to our models, PPII-S269Q and -K463N mutations should leave Glu-408 in a physicochemical context similar to that found in M1 aminopeptidases; alternatively, PPII-S269E replacement might be sufficient to transform PPII into an aminopeptidase. These hypotheses were supported by site-directed mutagenesis; the mutants lost omegapeptidase but displayed alanyl-aminopeptidase activity. In conclusion, recognition of a substrate without an N-terminal charge requires neutralization of the aminopeptidase anionic binding site; furthermore, shortening of side chain at PPII-269 position is required for adjustment to the pyroglutamyl residue.

Production of a fully functional, permuted single‐chain penicillin G acylase

Penicillin G acylase (PGA) is a heterodimeric enzyme synthesized as a single‐polypeptide precursor that undergoes an autocatalytic processing to remove an internal spacer peptide to produce the active enzyme. We constructed a single‐chain PGA not dependent on autoproteolytic processing. The mature sequence of the β‐domain was expressed as the N terminus of a new polypeptide, connected by a random tetra‐peptide to the α‐domain, to afford a permuted protein. We found several active enzymes among variants differing in their linker peptides. Protein expression analysis showed that the functional single‐chain variants were produced when using a Sec‐dependent leader peptide, or when expressed inside the bacterial cytoplasm. Active‐site titration experiments showed that the single‐chain proteins displayed similar kcat values to the ones obtained with the wild‐type enzyme. Interestingly, the single‐chain proteins also displayed close to 100% of functional active sites compared to 40% to 70% functional yield usually obtained with the heterodimeric protein.

Microbial systems and directed evolution of protein activities.

Recent advances in recombinant DNA methodology have had an important impact on the capacity to manipulate protein-coding sequences. The appearance of new, powerful screening systems completes a scenario for conducting directed evolution experiments. We review here some of the latest developments in experimental approaches to directed evolution, utilizing microbial systems. These include phage display, surface display, operator-repressor systems, and novel mutagenesis approaches. We also highlight the achievements and limitations of current methodologies. We present strategies used by our own group that permitted isolation of specificity mutants of beta-lactamase. Possible improvements for the future of the variation-selection approach to the study and manipulation of proteins are presented.

Combinatorial Multicomponent Access to Natural-Products-Inspired Peptidomimetics: Discovery of Selective Inhibitors of Microbial Metallo-aminopeptidases

The development of selective inhibitors of microbial metallo‐aminopeptidases is an important goal in the pursuit of antimicrobials for therapeutic applications. Herein, we disclose a combinatorial approach relying on two Ugi reactions for the generation of peptidomimetics inspired by natural metallo‐aminopeptidase inhibitors. The library was screened for inhibitory activity against the neutral metallo‐aminopeptidase of Escherichia coli (ePepN) and the porcine kidney cortex metallo‐aminopeptidase (pAPN), which was used as a model of the M1‐aminopeptidases of mammals. Six compounds showed typical dose–response inhibition profiles toward recombinant ePepN, with two of them being very potent and highly selective for ePepN over pAPN. Another compound showed moderate ePepN inhibition but total selectivity for this bacterial enzyme over its mammalian orthologue at concentrations of physiological relevance. This strategy proved to be useful for the identification of lead compounds for further optimization and development.

Microbial sensor for new-generation cephalosporins based in a protein-engineered β- lactamase

A protein-engineered β-lactamase, constructed by site-directed mutagenesis inEscherichia coli (E104M/G238S), and having broadened specificity, was able to degrade cephalosporins of first, second, and third generations. Manipulations of culture conditions allowed an increase in β-lactamase specific activity by up to twofold. The resultant bacteria were used to construct an immersable whole-cell biosensor for the detection of new-generation cephalosporins. Cells were immobilized on agar membranes, which in turn were attached to the surface of a flat pH electrode, thus constituting a biosensor based on the detection of pH changes. The sensor was able to detect second- and third-generation cephalosporins: cefamandole (0.4-4 mM), cefotaxime (0.4-3.5 mM), and cefoperazone (0.3-1.85 mM). Response times were between 3.5 and 11 min, depending on the kind of cephalosporin tested. The biosensor was stable for at least 7 d, time during which up to 100 tests were performed.

A microbial biosensor for 6-aminopenicillanic acid

Abstract A protein-engineered β-lactamase (N132 D) with enhanced selectivity to 6-APA was used to construct a whole-cell immersible biosensor for 6-APA based on the immobilization of β-lactamase-rich cells of Escherichia coli on a flat pH electrode. The response time was between 3.5–7 min. The sensitivity for penicillin was two to five fold lower to that for 6-APA. Selectivity of 6-APA was maximum at pH 7.6 using a 0.05 m phosphate buffer. 6-APA in concentrations up to 1 mg ml −1 could be detected. At least 70 analyses could be performed over 11 days with no loss of sensitivity.

Improvement of an Unusual Twin-Arginine Transporter Leader Peptide by a Codon-Based Randomization Approach

ABSTRACT Secretion of Escherichia coli penicillin acylase was improved by codon-based random mutagenesis of its signal peptide. The mutagenesis technology was applied to the gene region coding for positions Lys2 to Thr13 (N half) and Ala14 to Leu25 (C half) of the signal peptide. Protein secretion was higher in several signal peptide variants (up to fourfold with respect to the wild-type value).