Protein engineering of the aldoxime dehydratase from Bacillus sp. OxB-1 based on a rational sequence alignment approach.
Ontology highlight
ABSTRACT:
SUBMITTER: Oike K
PROVIDER: S-EPMC8275659 | biostudies-literature |
REPOSITORIES: biostudies-literature
Similar Datasets
Project description:Bacillus sp. OxB-1 has been characterized as a strain that produces a new enzyme, aldoxime dehydratase, which catalyzes the dehydration of aldoxime to form nitrile. Here, its complete genome sequence (3,594,618 bp, with a GC content of 47.85%), comprising a circular chromosome, is announced.
| S-EPMC4342420 | biostudies-literature
Project description:Aldoxime dehydratase (Oxd) catalyzes the dehydration of aldoximes (R-CH=N-OH) to their corresponding nitrile (R-C triple bond N). Oxd is a heme-containing enzyme that catalyzes the dehydration reaction as its physiological function. We have determined the first two structures of Oxd: the substrate-free OxdRE at 1.8 A resolution and the n-butyraldoxime- and propionaldoxime-bound OxdREs at 1.8 and 1.6 A resolutions, respectively. Unlike other heme enzymes, the organic substrate is directly bound to the heme iron in OxdRE. We determined the structure of the Michaelis complex of OxdRE by using the unique substrate binding and activity regulation properties of Oxd. The Michaelis complex was prepared by x-ray cryoradiolytic reduction of the ferric dead-end complex in which Oxd contains a Fe(3+) heme form. The crystal structures reveal the mechanism of substrate recognition and the catalysis of OxdRE.
| S-EPMC2797279 | biostudies-literature
Project description:Rational protein engineering requires a holistic understanding of protein function. Here, we apply deep learning to unlabeled amino-acid sequences to distill the fundamental features of a protein into a statistical representation that is semantically rich and structurally, evolutionarily and biophysically grounded. We show that the simplest models built on top of this unified representation (UniRep) are broadly applicable and generalize to unseen regions of sequence space. Our data-driven approach predicts the stability of natural and de novo designed proteins, and the quantitative function of molecularly diverse mutants, competitively with the state-of-the-art methods. UniRep further enables two orders of magnitude efficiency improvement in a protein engineering task. UniRep is a versatile summary of fundamental protein features that can be applied across protein engineering informatics.
| S-EPMC7067682 | biostudies-literature
Project description:In general, hemoproteins are capable of catalyzing redox reactions. Aldoxime dehydratase (OxdA), which is a unique heme-containing enzyme, catalyzes the dehydration of aldoximes to the corresponding nitriles. Its reaction is a rare example of heme directly activating an organic substrate, unlike the utilization of H2O2 or O2 as a mediator of catalysis by other heme-containing enzymes. While it is unknown whether OxdA catalyzes redox reactions or not, we here for the first time detected catalase activity (which is one of the redox activities) of wild-type OxdA, OxdA(WT). Furthermore, we constructed a His320 → Asp mutant of OxdA [OxdA(H320D)], and found it exhibits catalase activity. Determination of the kinetic parameters of OxdA(WT) and OxdA(H320D) revealed that their Km values for H2O2 were similar to each other, but the kcat value of OxdA(H320D) was 30 times higher than that of OxdA(WT). Next, we examined another redox activity and found it was the peroxidase activity of OxdAs. While both OxdA(WT) and OxdA(H320D) showed the activity, the activity of OxdA(H320D) was dozens of times higher than that of OxdA(WT). These findings demonstrated that the H320D mutation enhances the peroxidase activity of OxdA. OxdAs (WT and H320D) were found to catalyze another redox reaction, a peroxygenase reaction. During this reaction of OxdA(H320D) with 1-methoxynaphthalene as a substrate, surprisingly, the reaction mixture changed to a color different from that with OxdA(WT), which was due to the known product, Russig's blue. We purified and identified the new product as 1-methoxy-2-naphthalenol, which has never been reported as a product of the peroxygenase reaction, to the best of our knowledge. These findings indicated that the H320D mutation not only enhanced redox activities, but also significantly altered the hydroxylation site of the substrate.
| S-EPMC5391958 | biostudies-literature
Project description:R.MwoI is a Type II restriction endonucleases enzyme (REase), which specifically recognizes a palindromic interrupted DNA sequence 5'-GCNNNNNNNGC-3' (where N indicates any nucleotide), and hydrolyzes the phosphodiester bond in the DNA between the 7th and 8th base in both strands. R.MwoI exhibits remote sequence similarity to R.BglI, a REase with known structure, which recognizes an interrupted palindromic target 5'-GCCNNNNNGGC-3'. A homology model of R.MwoI in complex with DNA was constructed and used to predict functionally important amino acid residues that were subsequently targeted by mutagenesis. The model, together with the supporting experimental data, revealed regions important for recognition of the common bases in DNA sequences recognized by R.BglI and R.MwoI. Based on the bioinformatics analysis, we designed substitutions of the S310 residue in R.MwoI to arginine or glutamic acid, which led to enzyme variants with altered sequence selectivity compared with the wild-type enzyme. The S310R variant of R.MwoI preferred the 5'-GCCNNNNNGGC-3' sequence as a target, similarly to R.BglI, whereas the S310E variant preferentially cleaved a subset of the MwoI sites, depending on the identity of the 3rd and 9th nucleotide residues. Our results represent a case study of a REase sequence specificity alteration by a single amino acid substitution, based on a theoretical model in the absence of a crystal structure.
| S-EPMC3458533 | biostudies-literature
Project description:Aldoxime dehydratase (OxdA), which is a unique heme protein, catalyzes the dehydration of an aldoxime to a nitrile even in the presence of water in the reaction mixture. Unlike the utilization of H(2)O(2) or O(2) as a mediator of catalysis by other heme-containing enzymes (e.g., P450), OxdA is notable for the direct binding of a substrate to the heme iron. Here, we determined the crystal structure of OxdA. We then constructed OxdA mutants in which each of the polar amino acids lying within ?6 Å of the iron atom of the heme was converted to alanine. Among the purified mutant OxdAs, S219A had completely lost and R178A exhibited a reduction in the activity. Together with this finding, the crystal structural analysis of OxdA and spectroscopic and electrostatic potential analyses of the wild-type and mutant OxdAs suggest that S219 plays a key role in the catalysis, forming a hydrogen bond with the substrate. Based on the spatial arrangement of the OxdA active site and the results of a series of mutagenesis experiments, we propose the detailed catalytic mechanism of general aldoxime dehydratases: (i) S219 stabilizes the hydroxy group of the substrate to increase its basicity; (ii) H320 acts as an acid-base catalyst; and (iii) R178 stabilizes the heme, and would donate a proton to and accept one from H320.
| S-EPMC3581911 | biostudies-literature
Project description:Chitosanases can catalyze the release of chitooligosaccharides which have a number of medical applications. Therefore, Chitosanases are good candidates for large-scale enzymatic synthesis due to their favorable thermostability properties and high catalytic efficiency. To further improve the thermostability of a chitosanase from Bacillus sp. TS, which has a half-life of 5.32 min, we mutated specific serine residues that we identified as potentially relevant through structure comparison with thermophilic CelA from Clostridium thermocellum. Out of a total of 15 mutants, three, namely S265G, S276A, and S347G, show higher thermostability. Their half-lives at 60 °C were calculated as 34.57 min, 36.79 min and 7.2 min. The Km values of S265G, S276A and S347G mutants show substrate binding ability comparable to that of the wild-type enzyme, while the S265G mutant displays a significant decrease of enzymatic activities. Additionally, we studied the synergistic effects of combined mutations, observing that all double mutants and the triple mutant are more stable than the wild-type enzyme and single mutants. Finally, we investigated the mechanisms which might give a reasonable explanation for the improved thermostability via comparative analysis of the resulting 3D structures.
| S-EPMC8339078 | biostudies-literature
Project description:Antibody immunodominance refers to the preferential and asymmetric elicitation of antibodies against specific epitopes on a complex protein antigen. Traditional vaccination approaches for rapidly evolving pathogens have had limited success in part because of this phenomenon, as elicited antibodies preferentially target highly variable regions of antigens, and thus do not confer long lasting protection. While antibodies targeting functionally conserved epitopes have the potential to be broadly protective, they often make up a minority of the overall repertoire. Here, we discuss recent protein engineering strategies used to favorably alter patterns of immunodominance, and selectively focus antibody responses toward broadly protective epitopes in the pursuit of next-generation vaccines for rapidly evolving pathogens.
| S-EPMC8683408 | biostudies-literature
Project description:Control of enzyme allosteric regulation is required to drive metabolic flux toward desired levels. Although the three-dimensional (3D) structures of many enzyme-ligand complexes are available, it is still difficult to rationally engineer an allosterically regulatable enzyme without decreasing its catalytic activity. Here, we describe an effective strategy to deregulate the allosteric inhibition of enzymes based on the molecular evolution and physicochemical characteristics of allosteric ligand-binding sites. We found that allosteric sites are evolutionarily variable and comprised of more hydrophobic residues than catalytic sites. We applied our findings to design mutations in selected target residues that deregulate the allosteric activity of fructose-1,6-bisphosphatase (FBPase). Specifically, charged amino acids at less conserved positions were substituted with hydrophobic or neutral amino acids with similar sizes. The engineered proteins successfully diminished the allosteric inhibition of E. coli FBPase without affecting its catalytic efficiency. We expect that our method will aid the rational design of enzyme allosteric regulation strategies and facilitate the control of metabolic flux.
| S-EPMC3395594 | biostudies-literature
Project description:The generation of enzymes to catalyze specific reactions is one of the more challenging problems facing protein engineers. Structural similarities between the enzyme scytalone dehydratase with nuclear transport factor 2 (NTF2) suggested the potential for NTF2 to be re-engineered into a scytalone dehydratase-like enzyme. We introduced four key catalytic residues into NTF2 to create a scytalone dehydratase-like active site. A C-terminal helix found in scytalone dehydratase but absent in NTF2 also was added. Mutant NTF2 proteins were tested for catalytic activity by using a spectroscopic assay. One of the engineered enzymes exhibited catalytic activity with minimal kcat and Km values of 0.125 min-1 and 800 microM, respectively. This level of catalytic activity represents minimally a 150-fold improvement in activity over the background rate for substrate dehydration and a dramatic step forward from the catalytically inert parent NTF2. This work represents one of the few examples of converting a protein scaffold into an enzyme, outside those arising from the induction of catalytic activity into antibodies.
| S-EPMC22334 | biostudies-literature