Ether Oxidation by an Evolved Fungal Heme-Peroxygenase: Insights into Substrate Recognition and Reactivity.
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ABSTRACT:
SUBMITTER: Mireles R
PROVIDER: S-EPMC8396878 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Fungal unspecific peroxygenases (UPOs) are hybrid biocatalysts with peroxygenative activity that insert oxygen into non-activated compounds, while also possessing convergent peroxidative activity for one electron oxidation reactions. In several ligninolytic peroxidases, the site of peroxidative activity is associated with an oxidizable aromatic residue at the protein surface that connects to the buried heme domain through a long-range electron transfer (LRET) pathway. However, the peroxidative activity of these enzymes may also be initiated at the heme access channel. In this study, we examined the origin of the peroxidative activity of UPOs using an evolved secretion variant (PaDa-I mutant) from Agrocybe aegerita as our point of departure. After analyzing potential radical-forming aromatic residues at the PaDa-I surface by QM/MM, independent saturation mutagenesis libraries of Trp24, Tyr47, Tyr79, Tyr151, Tyr265, Tyr281, Tyr293 and Tyr325 were constructed and screened with both peroxidative and peroxygenative substrates. These mutant libraries were mostly inactive, with only a few functional clones detected, none of these showing marked differences in the peroxygenative and peroxidative activities. By contrast, when the flexible Gly314-Gly318 loop that is found at the outer entrance to the heme channel was subjected to combinatorial saturation mutagenesis and computational analysis, mutants with improved kinetics and a shift in the pH activity profile for peroxidative substrates were found, while they retained their kinetic values for peroxygenative substrates. This striking change was accompanied by a 4.5°C enhancement in kinetic thermostability despite the variants carried up to four consecutive mutations. Taken together, our study proves that the origin of the peroxidative activity in UPOs, unlike other ligninolytic peroxidases described to date, is not dependent on a LRET route from oxidizable residues at the protein surface, but rather it seems to be exclusively located at the heme access channel.
| S-EPMC8476742 | biostudies-literature
Project description:Unspecific peroxygenases (UPOs) are highly promiscuous biocatalyst with self-sufficient mono(per)oxygenase activity. A laboratory-evolved UPO secreted by yeast was covalently immobilized in activated carriers through one-point attachment. In order to maintain the desired orientation without compromising the enzyme's activity, the S221C mutation was introduced at the surface of the enzyme, enabling a single disulfide bridge to be established between the support and the protein. Fluorescence confocal microscopy demonstrated the homogeneous distribution of the enzyme, regardless of the chemical nature of the carrier. This immobilized biocatalyst was characterized biochemically opening an exciting avenue for research into applied synthetic chemistry.
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Project description:We employ error-corrected density functional theory methods to map out the dependence of reactivity on substrate position for SyrB2, a member of a family of non-heme iron halogenases and hydroxylases that are only reactive toward amino acid substrates delivered via prosthetic phosphopantetheine arms. For the initial hydrogen abstraction step, the inherent flexibility of the phosphopantetheine molecule weakens the position dependence for both the native substrate (threonine for SyrB2) and alternative substrates. Over a 5 Å window of substrate positions, the tethered hydrogen abstraction step proceeds with nearly identical activation energies and donor-acceptor distances in the transition state. The propensity of a particular substrate toward halogenation or hydroxylation is found to depend strongly on the substrate placement following hydrogen abstraction, with deeper substrate delivery into the active (for native substrates) site favoring halogenation and shallower substrate delivery favoring hydroxylation.
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Project description:BackgroundIsoprenoid precursor synthesis via the mevalonate route in humans and pathogenic trypanosomatids is an important metabolic pathway. There is however, only limited information available on the structure and reactivity of the component enzymes in trypanosomatids. Since isoprenoid biosynthesis is essential for trypanosomatid viability and may provide new targets for therapeutic intervention it is important to characterize the pathway components.ResultsPutative mevalonate kinase encoding genes from Leishmania major (LmMK) and Trypanosoma brucei (TbMK) have been cloned, over-expressed in and proteins isolated from procyclic-form T. brucei. A highly sensitive radioactive assay was developed and shows ATP-dependent phosphorylation of mevalonate. Apo and (R)-mevalonate bound crystal structures of LmMK, from a bacterial expression system, have been determined to high resolution providing, for the first time, information concerning binding of mevalonate to an MK. The mevalonate binds in a deep cavity lined by highly conserved residues. His25 is key for binding and for discrimination of (R)- over (S)-mevalonate, with the main chain amide interacting with the C3 hydroxyl group of (R)-mevalonate, and the side chain contributing, together with Val202 and Thr283, to the construction of a hydrophobic binding site for the C3 methyl substituent. The C5 hydroxyl, where phosphorylation occurs, points towards catalytic residues, Lys18 and Asp155. The activity of LmMK was significantly reduced compared to MK from other species and we were unable to obtain ATP-binding data. Comparisons with the rat MK:ATP complex were used to investigate how this substrate might bind. In LmMK, helix alpha2 and the preceding polypeptide adopt a conformation, not seen in related kinase structures, impeding access to the nucleotide triphosphate binding site suggesting that a conformational rearrangement is required to allow ATP binding.ConclusionOur new structural information, consistent with data on homologous enzymes allows a detailed description of how mevalonate is recognized and positioned for catalysis in MK. The mevalonate-binding site is highly conserved yet the ATP-binding site is structurally distinct in LmMK. We are unable to provide a definitive explanation for the low activity of recombinant protein isolated from a bacterial expression system compared to material isolated from procyclic-form Trypanosoma brucei.
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Project description:A recently discovered peroxygenase from the fungus Marasmius rotula (MroUPO) is able to catalyze the progressive one-carbon shortening of medium and long-chain mono- and dicarboxylic acids by itself alone, in the presence of H2 O2 . The mechanism, analyzed using H218 O2 , starts with an α-oxidation catalyzed by MroUPO generating an α-hydroxy acid, which is further oxidized by the enzyme to a reactive α-keto intermediate whose decarboxylation yields the one-carbon shorter fatty acid. Compared with the previously characterized peroxygenase of Agrocybe aegerita, a wider heme access channel, enabling fatty acid positioning with the carboxylic end near the heme cofactor (as seen in one of the crystal structures available) could be at the origin of the unique ability of MroUPO shortening carboxylic acid chains.
| S-EPMC5725704 | biostudies-literature
Project description:A kinetic and spectroscopic characterization of the ferryl intermediate (APO-II) from APO, the heme-thiolate peroxygenase from Agrocybe aegerita, is described. APO-II was generated by reaction of the ferric enzyme with metachloroperoxybenzoic acid in the presence of nitroxyl radicals and detected with the use of rapid-mixing stopped-flow UV-visible (UV-vis) spectroscopy. The nitroxyl radicals served as selective reductants of APO-I, reacting only slowly with APO-II. APO-II displayed a split Soret UV-vis spectrum (370 nm and 428 nm) characteristic of thiolate ligation. Rapid-mixing, pH-jump spectrophotometry revealed a basic pKa of 10.0 for the Fe(IV)-O-H of APO-II, indicating that APO-II is protonated under typical turnover conditions. Kinetic characterization showed that APO-II is unusually reactive toward a panel of benzylic C-H and phenolic substrates, with second-order rate constants for C-H and O-H bond scission in the range of 10-10(7) M(-1)⋅s(-1). Our results demonstrate the important role of the axial cysteine ligand in increasing the proton affinity of the ferryl oxygen of APO intermediates, thus providing additional driving force for C-H and O-H bond scission.
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Project description:Mixed acetals and organotrifluoroborates undergo BF(3)·OEt(2)-promoted cross-couplings to give dialkyl ethers under simple, mild conditions. A survey of reaction partners identified a hydroxamate leaving group that improves the regioselectivity and product yield in the BF(3)·OEt(2)-promoted coupling reaction of mixed acetals and potassium alkynyl-, alkenyl-, aryl- and heteroaryltrifluoroborates to access substituted dialkyl ethers. This leaving group enables the reaction to proceed rapidly under mild conditions (0 °C, 5-60 min) and permits reactions with electron-deficient potassium aryltrifluoroborates that are less reactive with other acetal substrates. A study of the reaction mechanism and characterization of key intermediates by NMR spectroscopy and X-ray crystallography identified a role for the hydroxamate moiety as a reversible leaving group that serves to stabilize the key oxocarbenium intermediate and the need for a slight excess of organodifluoroborane to serve as a catalyst. A secondary role for the boron nucleophile as an activating ligand was also considered. These studies provide the basis for a general class of reagents that lead to dialkyl ethers by a simple, predictable cross-coupling reaction.
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Project description:In this study, we focus on a detailed bioinformatics analysis of hyBpox genes, mainly within the genomes of Sclerotiniaceae (Ascomycota, Leotiomycetes), which is a specifically evolved fungal family of necrotrophic host generalists and saprophytic or biotrophic host specialists. Members of the genus Sclerotium produce only sclerotia and no fruiting bodies or spores. Thus, their physiological role for peroxidases remains open. A representative species, S. cepivorum, is a dangerous plant pathogen causing white rot in Allium species, particularly in onions, leeks, and garlic. On a worldwide basis, the white rot caused by this soil-borne fungus is apparently the most serious threat to Allium-crop production. We have also found very similar peroxidase sequences in the related fungus S. sclerotiorum, although with minor yet important modifications in the architecture of its active centre. The presence of ScephyBpox1-specific mRNA was confirmed by transcriptomic analysis. The presence of Hybrid B peroxidase at the protein level as the sole extracellular peroxidase of this fungus was confirmed in the secretome of S. cepivorum through detailed proteomic analyses. This prompted us to systematically search for all available genes coding for Hybrid B heme peroxidases in the whole fungal family of Sclerotiniaceae. We present here a reconstruction of their molecular phylogeny and analyse the unique aspects of their conserved-sequence features and structural folds in corresponding ancestral sequences.
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Project description:Aromatic peroxygenase (APO) is an extracellular enzyme produced by the agaric basidiomycete Agrocybe aegerita that catalyzes diverse peroxide-dependent oxyfunctionalization reactions. Here we describe the oxygenation of the unactivated aromatic ring of benzene with hydrogen peroxide as co-substrate. The optimum pH of the reaction was around 7 and it proceeded via an initial epoxide intermediate that re-aromatized in aqueous solution to form phenol. Identity of the epoxide intermediate as benzene oxide was proved by a freshly prepared authentic standard using GC-MS and LC-MS analyses. Second and third [per]oxygenation was also observed and resulted in the formation of further hydroxylation and following [per]oxidation products: hydroquinone and p-benzoquinone, catechol and o-benzoquinone as well as 1,2,4-trihydroxybenzene and hydroxy-p-benzoquinone, respectively. Using H218O2 as co-substrate and ascorbic acid as radical scavenger, inhibiting the formation of peroxidation products (e.g., p-benzoquinone), the origin of the oxygen atom incorporated into benzene or phenol was proved to be the peroxide. Apparent enzyme kinetic constants (kcat, Km) for the peroxygenation of benzene were estimated to be around 8 s-1 and 3.6 mM. These results raise the possibility that peroxygenases may be useful for enzymatic syntheses of hydroxylated benzene derivatives under mild conditions.
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Project description:Nitric oxide (NO) produced by mammalian nitric oxide synthases (mNOSs) is an important mediator in a variety of physiological functions. Crystal structures of mNOSs have shown strong conservation of the active-site residue Val567 (numbering for rat neuronal NOS, nNOS). NOS-like proteins have been identified in several bacterial pathogens, and these display striking sequence identity to the oxygenase domain of mNOS (NOSoxy), with the exception of a Val to Ile mutation at the active site. Preliminary studies have highlighted the importance of this Val residue in NO-binding, substrate recognition, and oxidation in mNOSs. To further elucidate the role of this valine in substrate and substrate analogue recognition, we generated five Val567 mutants of the oxygenase domain of the neuronal NOS (nNOSoxy) and used UV-visible and EPR spectroscopy to investigate the effects of these mutations on the heme distal environment, the stability of the heme-FeII-CO complexes, and the binding of a series of substrate analogues. Our results are consistent with Val567 playing an important role in preserving the integrity of the active site for substrate binding, stability of heme-bound gaseous ligands, and potential NO production.
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