Jonathan M. Blackburn

Directed evolution of new catalytic activity using the α/β-barrelscaffold

In biological systems, enzymes catalyse the efficient synthesis of complex molecules under benign conditions, but widespread industrial use of these biocatalysts depends crucially on the development of new enzymes with useful catalytic functions. The evolution of enzymes in biological systems often involves the acquisition of new catalytic or binding properties by an existing protein scaffold. Here we mimic this strategy using the most common fold in enzymes, the α/β-barrel, as the scaffold. By combining an existing binding site for structural elements of phosphoribosylanthranilate with a catalytic template required for isomerase activity, we are able to evolve phosphoribosylanthranilate isomerase activity from the scaffold of indole-3-glycerol-phosphate synthase. We find that targeting the catalytic template for in vitro mutagenesis and recombination, followed by in vivo selection, results in a new phosphoribosylanthranilate isomerase that has catalytic properties similar to those of the natural enzyme, with an even higher specificity constant. Our demonstration of divergent evolution and the widespread occurrence of the α/β-barrel suggest that this scaffold may be a fold of choice for the directed evolution of new biocatalysts.

Directed evolution of new catalytic activity using the |[alpha]|/|[beta]|-barrelscaffold

In biological systems, enzymes catalyse the efficient synthesis of complex molecules under benign conditions, but widespread industrial use of these biocatalysts depends crucially on the development of new enzymes with useful catalytic functions. The evolution of enzymes in biological systems often involves the acquisition of new catalytic or binding properties by an existing protein scaffold. Here we mimic this strategy using the most common fold in enzymes, the α/β-barrel, as the scaffold. By combining an existing binding site for structural elements of phosphoribosylanthranilate with a catalytic template required for isomerase activity, we are able to evolve phosphoribosylanthranilate isomerase activity from the scaffold of indole-3-glycerol-phosphate synthase. We find that targeting the catalytic template for in vitro mutagenesis and recombination, followed by in vivo selection, results in a new phosphoribosylanthranilate isomerase that has catalytic properties similar to those of the natural enzyme, with an even higher specificity constant. Our demonstration of divergent evolution and the widespread occurrence of the α/β-barrel suggest that this scaffold may be a fold of choice for the directed evolution of new biocatalysts.

A new plasmid display technology for the in vitro selection of functional phenotype-genotype linked proteins.

BACKGROUND Display technologies which allow peptides or proteins to be physically associated with the encoding DNA are central to procedures which involve screening of protein libraries in vitro for new or altered function. Here we describe a new system designed specifically for the display of libraries of diverse, functional proteins which utilises the DNA binding protein nuclear factor kappa B (NF-kappa B) p50 to establish a phenotype-genotype link between the displayed protein and the encoding gene. RESULTS A range of model fusion proteins to either the amino- or carboxy-terminus of NF-kappa B p50 have been constructed and shown to retain the picomolar affinity and DNA specificity of wild-type NF-kappa B p50. Through use of an optimal combination of binding buffer and DNA target sequence, the half-life of p50-DNA complexes could be increased to over 47 h, enabling the competitive selection of a variety of protein-plasmid complexes with enrichment factors of up to 6000-fold per round. The p50-based plasmid display system was used to enrich a maltose binding protein complex to homogeneity in only three rounds from a binary mixture with a starting ratio of 1:10(8) and to enrich to near homogeneity a single functional protein from a phenotype-genotype linked Escherichia coli genomic library using in vitro functional selections. CONCLUSIONS A new display technology is described which addresses the challenge of functional protein display. The results demonstrate that plasmid display is sufficiently sensitive to select a functional protein from large libraries and that it therefore represents a useful addition to the repertoire of display technologies.

Serine and alanine racemase activities of VanT: a protein necessary for vancomycin resistance in Enterococcus gallinarum BM4174.

Vancomycin resistance in Enterococcus gallinarum results from the production of UDP-MurNAc-pentapeptide[D-Ser]. VanT, a membrane-bound serine racemase, is one of three proteins essential for this resistance. To investigate the selectivity of racemization of L-Ser or L-Ala by VanT, a strain of Escherichia coli TKL-10 that requires D-Ala for growth at 42 degrees C was used as host for transformation experiments using plasmids containing the full-length vanT from Ent. gallinarum or the alanine racemase gene (alr) of Bacillus stearothermophilus: both plasmids were able to complement E. coli TKL-10 at 42 degrees C. No alanine or serine racemase activities were detected in the host strain E. coli TKL-10 grown at 30, 34 or 37 degrees C. Serine and alanine racemase activities were found almost exclusively (96%) in the membrane fraction of E. coli TKL-10/pCA4(vanT): the alanine racemase activity of VanT was 14% of the serine racemase activity in both E. coli TKL-10/pCA4(vanT) and E. coli XL-1 Blue/pCA4(vanT). Alanine racemase activity was present mainly (95%) in the cytoplasmic fraction of E. coli TKL-10/pJW40(alr), with a trace (1.6%) of serine racemase activity. Additionally, DNA encoding the soluble domain of VanT was cloned and expressed in E. coli M15 as a His-tagged polypeptide and purified: this polypeptide also exhibited both serine and alanine racemase activities; the latter was approximately 18% of the serine racemase activity, similar to that of the full-length, membrane-bound enzyme. N-terminal sequencing of the purified His-tagged polypeptide revealed a single amino acid sequence, indicating that the formation of heterodimers between subunits of His-tagged C-VanT and endogenous alanine racemases from E. coli was unlikely. The authors conclude that the membrane-bound serine racemase VanT also has alanine racemase activity but is able to racemize serine more efficiently than alanine, and that the cytoplasmic domain is responsible for the racemase activity.

Site‐Directed Mutagenesis of Tyr354 in Geobacillus stearothermophilus Alanine Racemase Identifies a Role in Controlling Substrate Specificity and a Possible Role in the Evolution of Antibiotic Resistance

The pyridoxal 5 -phosphate (PLP) dependent enzyme alanine racemase (EC 5.1.1.1) catalyses the interconversion of the L and D isomers of alanine. In both Gram-negative and Gram-positive bacteria, the D-Ala produced by this enzyme is incorporated into the cell wall as an essential component of the peptidoglycan layer. The structure of the Geobacillus stearothermophilus alanine racemase (Alr) has been solved at 1.9 ä resolution, revealing a homodimer in which each subunit consists of two domains: a ( )8 barrel and a C-terminal domain essentially composed of three sheets (11 strands in total). On dimerisation the ( )8 barrel of one monomer interacts with the C-terminal domain of the other; the active site is a cleft between these two domains. Mutagenic, structural and modelling analyses 4] confirm a catalytic mechanism involving two bases, Lys39 and Tyr265 . The cofactor PLP forms a Schiff base with one of these, Lys39, in the resting enzyme, and in addition to this covalent attachment, a large number of hydrogen bonds fixes PLP rather rigidly in the active site. In particular, the three oxygen atoms of the cofactor phosphate group are involved in an extensive network of hydrogen bonds involving Tyr43, Ser204, Gly221, Ile222 and Tyr354 (Figure 1A). Interestingly however, the structure of an external aldimine form of Alr in which PLP forms a Schiff base with the inhibitor (R)-1-aminoethylphosphonic acid (L-Ala-P) suggests that there is ample space in the active site for larger side chains than that of alanine and provides no simple structural explanation for the observed substrate specificity. In the present study we have demonstrated that, rather than merely contributing to the immobilisation of the cofactor phosphate, Tyr354 plays a crucial role in defining the strict specificity of Alr for alanine by preventing turnover of other potential substrates. cis trans isomerization was achieved by irradiation with visible light from a Xenon lamp (UV Spot Light Source: HAMAMATSU PHOTONICS) for 1 min through a L-42 filter (from Asahi Technoglass). For the experiments represented in Figure 1 and Figure 2, the solution was UV-irradiated immediately after incubation at 37 C started. In the experiment represented by Figure 3, UV and visible-light irradiation was carried out when 40 and 80 min, respectively, had passed after incubation started.

In vitro selection and characterization of a stable subdomain of phosphoribosylanthranilate isomerase.

The (βα)8‐barrel is the most common enzyme fold and it is capable of catalyzing an enormous diversity of reactions. It follows that this scaffold should be an ideal starting point for engineering novel enzymes by directed evolution. However, experiments to date have utilized in vivo screens or selections and the compatibility of (βα)8‐barrels with in vitro selection methods remains largely untested. We have investigated plasmid display as a suitable in vitro format by engineering a variant of phosphoribosylanthranilate isomerase (PRAI) that carried the FLAG epitope in active‐site‐forming loop 6. Trial enrichments for binding of mAb M2 (a mAb to FLAG) demonstrated that FLAG‐PRAI could be identified from a 106‐fold excess of a FLAG‐negative competitor in three rounds of in vitro selection. These results suggest PRAI as a useful scaffold for epitope and peptide grafting experiments. Further, we constructed a FLAG‐PRAI loop library of ≈ 107 clones, in which the epitope residues most critical for binding mAb M2 were randomized. Four rounds of selection for antibody binding identified and enriched for a variant in which a single nucleotide insertion produced a truncated (βα)8‐barrel consisting of (βα)1−5β6. Biophysical characterization of this clone, trPRAI, demonstrated that it was selected because of a 21‐fold increase in mAb M2 affinity compared with full‐length FLAG‐PRAI. Remarkably, this truncated barrel was found to be soluble, structured, thermostable and monomeric, implying that it represents a genuine subdomain of PRAI and providing further evidence that such subdomains have played an important role in the evolution of the (βα)8‐barrel fold.

retraction: Directed evolution of new catalytic activity using the α/β-barrel scaffold

This corrects the article DOI: 35001001

High-level soluble expression and purification of deacetoxycephalosporin C/deacetylcephalosporin C synthase.

Abstract The deacetoxycephalosporin C/deacetylcephalosporin C synthase gene from Cephalosporium acremonium has been expressed at high levels in E. coli under control of the trc promoter in a vector modified by PCR. The enzyme is produced in soluble, highly active form in contrast to previously reported expression in insoluble form under control of the λP l promoter. A three step purification of this unstable enzyme is reported.

Mutational Analysis of the Kinetics and Thermodynamics of Transcription Factor NF-κB Homodimerisation

Dimeric transcription factors of the NF‐κB/Rel family are sequence‐specific DNA‐binding proteins that mediate the inducible expression of immunologically important eukaryotic genes by competing for κB sites. The kinetic and thermodynamic components of these interactions were probed by mutation of the subunit interface of the p50 homodimer, a paradigm for other family members. Guided by the crystal structure, we selected the side chains of five key residues (R255, Y270, L272, A311 and V313) for individual and combinatorial truncation, with the aim of generating a mutant panel. Homodimerisation was assessed indirectly by measurement of DNA binding with an optical biosensor in order to unmask the relative contributions of each residue. Surface plasmon resonance revealed that a unanimous bias for a palindromic κB site over an asymmetric one was mainly the result of a slower dissociation rate for the DNA/homodimer complex in the case of the palindromic κB site. Y270 and L272 were individually the most critical residues in homodimerisation. DNA binding was abolished when all five residues were substituted, which reinforces the notion that only a subset of residues contributes crucial dimer‐forming contacts. The role of Y270 was unique, since its mutation to glycine dramatically slowed both the association and dissociation rates for DNA binding. Surprisingly, R255 was shown to be of little importance in the stability of the p50 homodimer, despite its apparent participation in a salt bridge at the dimer interface. Our results suggest that binding modes inferred from structural data should be treated cautiously.

Erratum: Retraction Note to: Directed evolution of new catalytic activity using the α/β-barrel scaffold

In biological systems, enzymes catalyse the efficient synthesis of complex molecules under benign conditions, but widespread industrial use of these biocatalysts depends crucially on the development of new enzymes with useful catalytic functions. The evolution of enzymes in biological systems often involves the acquisition of new catalytic or binding properties by an existing protein scaffold. Here we mimic this strategy using the most common fold in enzymes, the α/β-barrel, as the scaffold. By combining an existing binding site for structural elements of phosphoribosylanthranilate with a catalytic template required for isomerase activity, we are able to evolve phosphoribosylanthranilate isomerase activity from the scaffold of indole-3-glycerol-phosphate synthase. We find that targeting the catalytic template for in vitro mutagenesis and recombination, followed by in vivo selection, results in a new phosphoribosylanthranilate isomerase that has catalytic properties similar to those of the natural enzyme, with an even higher specificity constant. Our demonstration of divergent evolution and the widespread occurrence of the α/β-barrel suggest that this scaffold may be a fold of choice for the directed evolution of new biocatalysts.