The UbiD enzyme plays an important role in bacterial ubiquinone (coenzyme Q) biosynthesis. It belongs to a family of reversible decarboxylases that interconvert propenoic or aromatic acids with the corresponding alkenes or aromatic compounds using a prenylated flavin (prFMN) cofactor. This cofactor is suggested to support (de)carboxylation through a reversible 1,3-dipolar cycloaddition process. Here we report an atomic-level description of the reaction of the UbiD related ferulic acid decarboxylase with substituted propenoic and propiolic acids (data ranging from 1.01 to 1.39 Å). The enzyme is only able to couple (de)carboxylation of cinnamic acid-type compounds to reversible 1,3-dipolar cycloaddition, while formation of dead-end prFMN cycloadducts occurs with distinct propenoic and propiolic acids. The active site imposes considerable strain on covalent intermediates formed with cinnamic and phenylpropiolic acids. Strain reduction through mutagenesis negatively affects catalytic rates with cinnamic acid, indicating a direct link between enzyme-induced strain and catalysis that is supported by computational studies.Many enzymes make use of covalent catalysis to achieve substantial rate enhancements, often by recruiting cofactors such as PLP 1 , TPP 2 and flavins 3 . To ensure high turnover, these enzymes are inherently required to ensure both rapid and reversible cofactor-ligand adduct formation. In the case of the UbiD enzyme family, reversible decarboxylation has been suggested to occur via a 1,3-dipolar cycloaddition process between the substrate and the UbiD-cofactor, prenylated FMN (prFMN) 4 , enabled by the azomethine ylide character of the Users may view, print, copy, and download text and data-mine the content in such documents, for the purposes of academic research, subject always to the full Conditions of use:
Linalool is a monoterpenoid used as a fragrance ingredient, and is a promising source for alternative fuels. Synthetic biology offers attractive alternative production methods compared to extraction from natural sources and chemical synthesis. Linalool/nerolidol synthase (bLinS) from Streptomyces clavuligerus is a bifunctional enzyme, producing linalool as well as the sesquiterpenoid nerolidol when expressed in engineered Escherichia coli harbouring a precursor terpenoid pathway such as the mevalonate (MVA) pathway. Here we identified two residues important for substrate selection by bLinS, L72 and V214, where the introduction of bulkier residues results in variants with reduced nerolidol formation. Terpenoid production using canonical precursor pathways is usually limited by numerous and highly regulated enzymatic steps. Here we compared the canonical MVA pathway to the non‐canonical isopentenol utilization (IU) pathway to produce linalool using the optimised bLinS variant. The IU pathway uses isoprenol and prenol to produce linalool in only five steps. Adjusting substrate, plasmid system, inducer concentration, and cell strain directs the flux towards monoterpenoids. Our integrated approach, combining enzyme engineering with flux control using the artificial IU pathway, resulted in high purity production of the commercially attractive monoterpenoid linalool, and will guide future efforts towards efficient optimisation of terpenoid production in engineered microbes.
Within human biology, combinations of regioisomeric motifs of α2,6- or α2,3-sialic acids linked to galactose are frequently observed attached to glycoconjugates. These include glycoproteins and glycolipids, with each linkage carrying distinct biological information and function. Microbial linkage-specific sialidases have become important tools for studying the role of these sialosides in complex biological settings, as well as being used as biocatalysts for glycoengineering. However, currently, there is no α2,6-specific sialidase available. This gap has been addressed herein by exploiting the ability of a Photobacterium sp. α2,6-sialyltransferase to catalyze trans-sialidation reversibly and in a highly linkage-specific manner, acting as a pseudosialidase in the presence of cytidine monophosphate. Selective, near quantitative removal of α2,6-linked sialic acids was achieved from a wide range of sialosides including small molecules conjugates, simple glycan, glycopeptide and finally complex glycoprotein including both linkages.
<p>In this manuscript we address an important gap in our current carbohydrate active enzyme toolbox, by developing a highly a2,6-selective (over a2,3-selective) de facto sialidase that is necessary both for glycan analysis and glycoconjugate remodeling. Both glycosidic linkages are commonly found in animal biology and each has been shown to have distinct biological function.</p> <p>Our approach is novel in that it harnesses the high selectivity of known glycosyltransferases ‘in reverse’ for effective hydrolysis, converting transferases to hydrolases by reaction engineering. </p> <p>More specifically, we demonstrate that the a2,6-specific pseudosialidase activity of <i>Photobacterium </i>sp. JT-ISH-224 a2,6-sialyltransferase can be used effectively for highly a2,6 selective hydrolysis on a broad range of analytes: small synthetic probes, isolated complex glycans and complex mixtures of glycoproteins. </p>
<p>In this manuscript we address an important gap in our current carbohydrate active enzyme toolbox, by developing a highly a2,6-selective (over a2,3-selective) de facto sialidase that is necessary both for glycan analysis and glycoconjugate remodeling. Both glycosidic linkages are commonly found in animal biology and each has been shown to have distinct biological function.</p> <p>Our approach is novel in that it harnesses the high selectivity of known glycosyltransferases ‘in reverse’ for effective hydrolysis, converting transferases to hydrolases by reaction engineering. </p> <p>More specifically, we demonstrate that the a2,6-specific pseudosialidase activity of <i>Photobacterium </i>sp. JT-ISH-224 a2,6-sialyltransferase can be used effectively for highly a2,6 selective hydrolysis on a broad range of analytes: small synthetic probes, isolated complex glycans and complex mixtures of glycoproteins. </p>
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