Biomedical subjects

Joel Osuna

Publications and source records attributed to Joel Osuna.

8 recordsLinked to original sources

PubMed2006

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-NH(2)), 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.

Amino Acid Sequence↗

PubMed2006

Improvement of an unusual twin-arginine transporter leader peptide by a codon-based randomization approach.

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).

Amino Acid Substitution↗

PubMed2004

Combinatorial codon-based amino acid substitutions.

Twenty Fmoc-protected trinucleotide phosphoramidites representing a complete set of codons for the natural amino acids were chemically synthesized for the first time. A pool of these reagents was incorporated into oligonucleotides at substoichiometric levels to generate two libraries of variants that randomly carry either few or many codon replacements on a region encoding nine amino acids of the bacterial enzyme TEM-1 beta-lactamase. Assembly of the libraries was performed in a completely automated mode through a simple modification of ordinary protocols. This technology eliminates codon redundancy, stop codons and enables complete exploration of sequence space for single, double and triple mutations throughout a protein region spanning several residues. Sequence analysis of many non-selected clones revealed a good incorporation of the trinucleotides, producing combinations of mutations quite different from those obtained using conventional degenerate oligonucleotides. Ceftazidime-selection experiments yielded several never before reported variants containing novel amino acid combinations in the beta-lactamase omega loop region.

Amino Acid Substitution↗

PubMed2004

Protein evolution by codon-based random deletions.

A method to delete in-phase codons throughout a defined target region of a gene has been developed. This approach, named the codon-based random deletion (COBARDE) method, is able to delete complete codons in a random and combinatorial mode. Robustness, automation and fine-tuning of the mutagenesis rate are essential characteristics of the method, which is based on the assembly of oligonucleotides and on the use of two transient orthogonal protecting groups during the chemical synthesis. The performance of the method for protein function evolution was demonstrated by changing the substrate specificity of TEM-1 beta-lactamase. Functional ceftazidime-resistant beta-lactamase variants containing several deleted residues inside the catalytically important omega-loop region were found. The results show that the COBARDE method is a useful new molecular tool to access previously unexplorable sequence space.

Amino Acids↗

PubMed2004

Metabolic engineering and protein directed evolution increase the yield of L-phenylalanine synthesized from glucose in Escherichia coli.

L-phenylalanine (L-Phe) is an aromatic amino acid with diverse commercial applications. Technologies for industrial microbial synthesis of L-Phe using glucose as a starting raw material currently achieve a relatively low conversion yield (Y(Phe/Glc)). The purpose of this work was to study the effect of PTS (phosphotransferase transport system) inactivation and overexpression of different versions of feedback inhibition resistant chorismate mutase-prephenate dehydratase (CM-PDT) on the yield (Y(Phe/Glc)) and productivity of L-Phe synthesized from glucose. The E. coli JM101 strain and its mutant derivative PB12 (PTS(-)Glc(+) phenotype) were used as hosts. PB12 has an inactive PTS, but is capable of transporting and phosphorylating glucose by using an alternative system constituted by galactose permease (GalP) and glucokinase activities (Glk). JM101 and PB12 were transformed with three plasmids, harboring genes that encode for a feedback inhibition resistant DAHP synthase (aroG(fbr)), transketolase (tktA) and either a truncated CM-PDT (pheA(fbr)) or its derived evolved genes (pheA(ev1) or pheA(ev2)). Resting-cells experiments with these engineered strains showed that JM101 and PB12 strains expressing either pheA(ev1) or pheA(ev2) genes produced l-Phe from glucose with Y(Phe/Glc) of 0.21 and 0.33 g/g, corresponding to 38 and 60% of the maximum theoretical yield (0.55 g/g), respectively. In addition, in both engineered strains the reached q(Phe) high levels of 40 mg/g-dcw.h. The metabolic engineering strategy followed in this work, including a strain with an inactive PTS, resulted in a positive impact over the Y(Phe/Glc), enhancing it nearly 57% compared with its PTS(+) counterpart. This is the first report wherein PTS inactivation was a successful strategy to improve the Y(Phe/Glc).

Directed Molecular Evolution↗

PubMed2004

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 beta-domain was expressed as the N terminus of a new polypeptide, connected by a random tetra-peptide to the alpha-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 k(cat) 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.

Amino Acid Sequence↗

PubMed2002

Novel ceftazidime-resistance beta-lactamases generated by a codon-based mutagenesis method and selection.

Four known and nine new ceftazidime-resistance beta-lactamases were generated by a novel, contaminating codon-based mutagenesis approach. In this method, wild-type codons are spiked with a set of mutant codons during oligonucleotide synthesis, generating random combinatorial libraries of primers that contain few codon replacements per variant. Mutant codons are assembled by tandem addition of a diluted mixture of five Fmoc-dimer amidites to the growing oligo and a mixture of four DMTr-monomer amidites to generate 20 trinucleotides that encode a set of 18 amino acids. Wild-type codons are assembled with conventional chemistry and the whole process takes place in only one synthesis column, making its automation feasible. The random and binomial behavior of this approach was tested in the polylinker region of plasmid pUC19 by the synthesis of three oligonucleotide libraries mutagenized at different rates and cloned as mutagenic cassettes. Additionally, the method was biologically assessed by mutating six contiguous codons that encode amino acids 237-243 (ABL numbering) of the TEM(pUC19) beta-lactamase, which is functionally equivalent to the clinically important TEM-1 beta-lactamase. The best ceftazidime-recognizing variant was a triple mutant, R164H:E240K: R241A, displaying a 333-fold higher resistance than the wild-type enzyme.

Amino Acid Substitution↗

PubMed2002

Improving a circularly permuted TEM-1 beta-lactamase by directed evolution.

Circular permutation of proteins is a powerful technique to explore the importance of the polypeptide secondary structure order for attaining the final three-dimensional structure. Here, we designed a circular permutation of the TEM beta-lactamase in order to produce a new domain-forming amino acid arrangement in the polypeptide sequence. Closing the normal N- and C-termini with the connecting peptide GGS and creating new N- and C-termini at position 216, produces a severely impaired permuted protein. Introduction of a connector with random components allows the isolation of enzymes with better activities and indicates a selection for a potential helix-stop signal at the new super-secondary motif. We applied several directed-evolution cycles, starting from permuted enzymes with each of the two different connecting peptides, and selecting for antibiotic resistance and isolated several mutants with resistance levels close to those of the wild-type enzyme. We also analyze some of the data collected on the outcomes and paths of these evolutionary experiments. A purified sixth cycle variant with connector peptide GGS showed catalytic efficiency values approximately 8% of the natural enzyme.

Directed Molecular Evolution↗