Dataset Information

A comprehensive in silico characterization of bacterial signal peptides for the excretory production of Anabaena variabilis phenylalanine ammonia lyase in Escherichia coli.

ABSTRACT: Anabaena variabilis double mutant (C503S/C565S) phenylalanine ammonia-lyase (PAL) is an appealing enzyme in the enzyme-replacement therapy of phenylketonuria. Yet abundant production of this enzyme has been of concern for industrial production. In this study, we have characterized 1175 bacterial signal peptides (SPs) and identified the most efficient ones for the excretory production of mutant AvPAL. Analysis by SignalP 4.1 revealed that more than 61% of SPs had a D-score greater than 0.7, denoting to be highly efficient. The optimum length of a bacterial SP was 25-30. The preferable net positive charge and the second residue of N-region were +?2 and Lys/Arg, respectively. Highly efficient SPs possessed 3-5 Leus in their H-region and A/L/VXA-FF cleavage site. Highly efficient SPs were from Escherichia coli, corroborating the necessity of an agreement between SPs and the host. Physiochemical characterization of mutant AvPAL conjugates via ProtParam and PROSOII, revealed that ~?99.5% of proteins would not be entraped in inclusion bodies. Secretory pathways were identified by EffectiveDB and more than 98% of SPs were cleavable. Chimeras were modeled using the I-TASSER program, being evaluated by the Ramachandran plots. The mRNA secondary structure of mutant AvPAL upon linkage to SPs was assessed using the mfold program. It was shown that the linkage of a SP does not affect mutant AvPAL's stability at the protein or mRNA level. AllergenFP tool demonstrated that chimeras were not allergen. SPs, including FMF4_ECOLX, E2BB_ECOLX, and LPTA_ECOLI exhibited the highest propensity for secretion and appropriate features in all analyses.

SUBMITTER: Owji H

PROVIDER: S-EPMC6240017 | biostudies-literature | 2018 Dec

REPOSITORIES: biostudies-literature

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A comprehensive in silico characterization of bacterial signal peptides for the excretory production of Anabaena variabilis phenylalanine ammonia lyase in Escherichia coli.

Owji Hajar H Hemmati Shiva S

3 Biotech 20181116 12

Anabaena variabilis double mutant (C503S/C565S) phenylalanine ammonia-lyase (PAL) is an appealing enzyme in the enzyme-replacement therapy of phenylketonuria. Yet abundant production of this enzyme has been of concern for industrial production. In this study, we have characterized 1175 bacterial signal peptides (SPs) and identified the most efficient ones for the excretory production of mutant AvPAL. Analysis by SignalP 4.1 revealed that more than 61% of SPs had a D-score greater t ...[more]

PMID: 30498661

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Project description:We have recently observed promising success in a mouse model for treating the metabolic disorder phenylketonuria with phenylalanine ammonia lyase (PAL) from Rhodosporidium toruloides and Anabaena variabilis. Both molecules, however, required further optimization in order to overcome problems with protease susceptibility, thermal stability, and aggregation. Previously, we optimized PAL from R. toruloides, and in this case we reduced aggregation of the A. variabilis PAL by mutating two surface cysteine residues (C503 and C565) to serines. Additionally, we report the structural and biochemical characterization of the A. variabilis PAL C503S/C565S double mutant and carefully compare this molecule with the R. toruloides engineered PAL molecule. Unlike previously published PAL structures, significant electron density is observed for the two active-site loops in the A. variabilis C503S/C565S double mutant, yielding a complete view of the active site. Docking studies and N-hydroxysuccinimide-biotin binding studies support a proposed mechanism in which the amino group of the phenylalanine substrate is attacked directly by the 4-methylidene-imidazole-5-one prosthetic group. We propose a helix-to-loop conformational switch in the helices flanking the inner active-site loop that regulates accessibility of the active site. Differences in loop stability among PAL homologs may explain the observed variation in enzyme efficiency, despite the highly conserved structure of the active site. A. variabilis C503S/C565S PAL is shown to be both more thermally stable and more resistant to proteolytic cleavage than R. toruloides PAL. Additional increases in thermal stability and protease resistance upon ligand binding may be due to enhanced interactions among the residues of the active site, possibly locking the active-site structure in place and stabilizing the tetramer. Examination of the A. variabilis C503S/C565S PAL structure, combined with analysis of its physical properties, provides a structural basis for further engineering of residues that could result in a better therapeutic molecule.

Project description:BACKGROUND: Phenylalanine ammonia lyase (PAL) is a key enzyme of the phenylpropanoid pathway that catalyzes the deamination of phenylalanine to trans-cinnamic acid, a precursor for the lignin and flavonoid biosynthetic pathways. To date, PAL genes have been less extensively studied in gymnosperms than in angiosperms. Our interest in PAL genes stems from their potential role in the defense responses of Pinus taeda, especially with respect to lignification and production of low molecular weight phenolic compounds under various biotic and abiotic stimuli. In contrast to all angiosperms for which reference genome sequences are available, P. taeda has previously been characterized as having only a single PAL gene. Our objective was to re-evaluate this finding, assess the evolutionary history of PAL genes across major angiosperm and gymnosperm lineages, and characterize PAL gene expression patterns in Pinus taeda. METHODS: We compiled a large set of PAL genes from the largest transcript dataset available for P. taeda and other conifers. The transcript assemblies for P. taeda were validated through sequencing of PCR products amplified using gene-specific primers based on the putative PAL gene assemblies. Verified PAL gene sequences were aligned and a gene tree was estimated. The resulting gene tree was reconciled with a known species tree and the time points for gene duplication events were inferred relative to the divergence of major plant lineages. RESULTS: In contrast to angiosperms, gymnosperms have retained a diverse set of PAL genes distributed among three major clades that arose from gene duplication events predating the divergence of these two seed plant lineages. Whereas multiple PAL genes have been identified in sequenced angiosperm genomes, all characterized angiosperm PAL genes form a single clade in the gene PAL tree, suggesting they are derived from a single gene in an ancestral angiosperm genome. The five distinct PAL genes detected and verified in P. taeda were derived from a combination of duplication events predating and postdating the divergence of angiosperms and gymnosperms. CONCLUSIONS: Gymnosperms have a more phylogenetically diverse set of PAL genes than angiosperms. This inference has contrasting implications for the evolution of PAL gene function in gymnosperms and angiosperms.

Dataset Information

A comprehensive in silico characterization of bacterial signal peptides for the excretory production of Anabaena variabilis phenylalanine ammonia lyase in Escherichia coli.

Publications

A comprehensive in silico characterization of bacterial signal peptides for the excretory production of <i>Anabaena variabilis</i> phenylalanine ammonia lyase in <i>Escherichia coli</i>.

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