Humberto Flores

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.

Sensitive genome-wide screen for low secondary enzymatic activities: the YjbQ family shows thiamin phosphate synthase activity.

Contemporary enzymes are highly efficient and selective catalysts. However, due to the intrinsically very reactive nature of active sites, gratuitous secondary reactions are practically unavoidable. Consequently, even the smallest cell, with its limited enzymatic repertoire, has the potential to carry out numerous additional, very likely inefficient, secondary reactions. If selectively advantageous, secondary reactions could be the basis for the evolution of new fully functional enzymes. Here, we investigated if Escherichia coli has cryptic enzymatic activities related to thiamin biosynthesis. We selected this pathway because this vitamin is essential, but the cells requirements are very small. Therefore, enzymes with very low activity could complement the auxotrophy of strains deleted of some bona fide thiamin biosynthetic genes. By overexpressing the E. colis protein repertoire, we selected yjbQ, a gene that complemented a strain deleted of the thiamin phosphate synthase (TPS)-coding gene thiE. In vitro studies confirmed TPS activity, and by directed evolution experiments, this activity was enhanced. Structurally oriented mutagenesis allowed us to identify the putative active site. Remote orthologs of YjbQ from Thermotoga, Sulfolobus, and Pyrococcus were cloned and also showed thiamin auxotrophy complementation, indicating that the cryptic TPS activity is a property of this protein family. Interestingly, the thiE- and yjbQ-coded TPSs are analog enzymes with no structural similarity, reflecting distinct evolutionary origin. These results support the hypothesis that the enzymatic repertoire of a cell such as E. coli has the potential to perform vast amounts of alternative reactions, which could be exploited to evolve novel or more efficient catalysts.

Isolated domain II and III from the Bacillus thuringiensis CrylAb δ-endotoxin binds to lepidopteran midgut membranes

The DNA fragment encoding Cry1Ab domain II–III (45.3 kDa) was cloned and expressed. Domain II–III is expressed in low yields. In vitro binding analysis to Manduca sexta and Trichoplusia ni larval midgut tissue sections demonstrated that domain II–III fragment bound along the microvilli of the midgut epithelium, indicating that this fragment retains binding functionality in the absence of domain I. Binding of domain II–III to the midgut brush border membrane proteins from T. ni larvae indicated that Cry1Ab toxin and domain II–III bind to the same 150 kDa protein. In contrast, in M. sexta membranes, Cry1Ab toxin binds to 200 and 120 kDa proteins, and domain II–III only binds to the 200 kDa protein. Finally, binding assays with isolated brush border membrane vesicles showed that the interaction of domain II–III with the membrane vesicles is highly reversible, supporting the proposition that the integration of domain I into the membrane could participate in the irreversible binding of the toxin. These studies confirm that this part of the toxin is involved in binding interactions and could be separated as a discrete fragment that conserves at least part of its functionality.

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.

Sceletium (Aizoaceae) Alkaloids: Total Synthesis of Racemic Mesembranone, Mesembrenone, O-Methylsceletenone and O-Methyl Dihydrosceletenone

Abstract As a continuation of our synthetic studies2 regarding the Sceletium (Aizoaceae) alkaloids3 we decided to test the usefulness of the recently reported4 Arylacetonitrile Route when combined with the well-known5 endocyclic enamine pathway first enunciated by Curphey6, Stevens7 and Tahk8 in 1968.

Reciprocal domain evolution within a transactivator in a restricted sequence space

offhough the concept of domain merging and shuffling as a major force in protein evolution is well established, it has been difficult to demonstrate how domains coadapt. Here we show evidence of coevolution of the Sinorhizobium meliloti NifA (SmNifA) domains. We found that, because of the lack of a conserved glycine in its DNA-binding domain, this transactivator protein interacts weakly with the enhancers. This defect, however, was compensated by evolving a highly efficient activation domain that, contrasting to Bradyrhizobium japonicum NifA (BjNifA), can activate in trans. To explore paths that lead to this enhanced activity, we mutagenized BjNifA. After three cycles of mutagenesis and selection, a highly active derivative was obtained. Strikingly, all mutations changed to amino acids already present in SmNifA. Our artificial process thus recreated the natural evolution followed by this protein and suggests that NifA is trapped in a restricted sequence space with very limited solutions for higher activity by point mutation.

Evolution of a new function in an esterase: simple amino acid substitutions enable the activity present in the larger paralog, BioH.

Gene duplication and divergence are essential processes for the evolution of new activities. Divergence may be gradual, involving simple amino acid residue substitutions, or drastic, such that larger structural elements are inserted, deleted or rearranged. Vast protein sequence comparisons, supported by some experimental evidence, argue that large structural modifications have been necessary for certain catalytic activities to evolve. However, it is not clear whether these activities could not have been attained by gradual changes. Interestingly, catalytic promiscuity could play a fundamental evolutionary role: a preexistent secondary activity could be increased by simple amino acid residue substitutions that do not affect the enzymes primary activity. The promiscuous profile of the enzyme may be modified gradually by genetic drift, making a pool of potentially useful activities that can be selected before duplication. In this work, we used random mutagenesis and in vivo selection to evolve the Pseudomonas aeruginosa PAO1 carboxylesterase PA3859, a small protein, to attain the function of BioH, a much larger paralog involved in biotin biosynthesis. BioH was chosen as a target activity because it provides a highly sensitive selection for evolved enzymatic activities by auxotrophy complementation. After only two cycles of directed evolution, mutants with the ability to efficiently complement biotin auxotrophy were selected. The in vivo and in vitro characterization showed that the activity of one of our mutant proteins was similar to that of the wild-type BioH enzyme. Our results demonstrate that it is possible to evolve enzymatic activities present in larger proteins by discrete amino acid substitutions.

Facile Synthesis of 4-Carbethoxycyclohexanone

Abstract A facile decarboalkoxylative route to 4-carbethoxycyclohexanone (1) from the tricarbethoxy derivative 3 is described.

Sceletium (aizoaceae) alkaloids: total synthesis of racemic mesembranone, joubertinamine and epijoubertinamine

Abstract The total synthesis of the Sceletium alkaloids mesembranone, joubertinamine and epijoubertinamine via the intramolecular cyclization of an enone to a benzensulfo n amide grouping under the conditions of a dissolving metal reduction is described.