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Item type:Publication, Access status: with files , Structural and biochemical characterisation of the N-carbamoyl-β-alanine amidohydrolase from Rhizobium radiobacter MDC 8606(2023); ; 19 40 - Some of the metrics are blocked by yourconsent settings
Item type:Publication, Access status: without files , Biocatalytic carboxylation of phenol derivatives: kinetics and thermodynamics of the biological Kolbe-Schmitt synthesis(Wiley-Blackwell, 2015-02-05); ; ; ; Microbial decarboxylases, which catalyse the reversible regioselective ortho-carboxylation of phenolic derivatives in anaerobic detoxification pathways, have been studied for their reverse carboxylation activities on electron-rich aromatic substrates. Ortho-hydroxybenzoic acids are important building blocks in the chemical and pharmaceutical industries and are currently produced via the Kolbe-Schmitt process, which requires elevated pressures and temperatures (≥ 5 bar, ≥ 100 °C) and often shows incomplete regioselectivities. In order to resolve bottlenecks in view of preparative-scale applications, we studied the kinetic parameters for 2,6-dihydroxybenzoic acid decarboxylase from Rhizobium sp. in the carboxylation- and decarboxylation-direction using 1,2-dihydroxybenzene (catechol) as starting material. The catalytic properties (Km, Vmax) are correlated with the overall thermodynamic equilibrium via the Haldane equation, according to a reversible random bi-uni mechanism. The model was subsequently verified by comparing experimental results with simulations. This study provides insights into the catalytic behaviour of a nonoxidative aromatic decarboxylase and reveals key limitations (e.g. substrate oxidation, CO2 pressure, enzyme deactivation, low turnover frequency) in view of the employment of this system as a 'green' alternative to the Kolbe-Schmitt processes. Microbial decarboxylases are known to catalyze the reversible regioselective ortho-carboxylation of phenolic derivatives in anaerobic detoxification pathways. In order to get new insights into the catalytic action and to resolve bottlenecks in view applications, we studied the kinetics of 2,6-dihydroxybenzoic acid decarboxylase from Rhizobium sp. in the carboxylation- and decarboxylation-direction, correlating the data according to a reversible random bi-uni mechanism.Publicationtype: Journal Article150 - Some of the metrics are blocked by yourconsent settings
Item type:Publication, Access status: without files , Crystal structures of two tetrameric β-carbonic anhydrases from the filamentous ascomycete Sordaria macrospora(Wiley-Blackwell, 2014-02-07); ; ; ; Carbonic anhydrases (CAs) are metalloenzymes catalyzing the reversible hydration of carbon dioxide to bicarbonate (hydrogen carbonate) and protons. CAs have been identified in archaea, bacteria and eukaryotes and can be classified into five groups (α, β, γ, δ, ζ) that are unrelated in sequence and structure. The fungal β-class has only recently attracted attention. In the present study, we investigated the structure and function of the plant-like β-CA proteins CAS1 and CAS2 from the filamentous ascomycete Sordaria macrospora. We demonstrated that both proteins can substitute for the Saccharomyces cerevisiae β-CA Nce103 and exhibit an in vitro CO2 hydration activity (kcat/Km of CAS1: 1.30 × 10 6 m-1·s-1; CAS2: 1.21 × 10 6m-1·s-1). To further investigate the structural properties of CAS1 and CAS2, we determined their crystal structures to a resolution of 2.7 Å and 1.8 Å, respectively. The oligomeric state of both proteins is tetrameric. With the exception of the active site composition, no further major differences have been found. In both enzymes, the Zn2+ -ion is tetrahedrally coordinated; in CAS1 by Cys45, His101 and Cys104 and a water molecule and in CAS2 by the side chains of four residues (Cys56, His112, Cys115 and Asp58). Both CAs are only weakly inhibited by anions, making them good candidates for industrial applications. Structured digital abstract CAS1 and CAS2 bind by x-ray crystallography (View interaction) Database Structural data have been deposited in the Protein Data Bank database under accession numbers 4O1J for CAS1 and 4O1K for CAS2. In this manuscript we determined the crystal structures and the in vitro CO2 hydration activities of the plant-type β-carbonic anhydrases CAS1 and CAS2 of the fungus Sordaria macrospora. The oligomeric state of both proteins is tetrameric. Both CAs are weakly only inhibited by anions making them good candidates for industrial applications. © 2014 FEBS.Publicationtype: Journal ArticleScopus© Citations 41 33 - Some of the metrics are blocked by yourconsent settings
Item type:Publication, Access status: without files , Do N-terminal nucleophile hydrolases indeed have a single amino acid catalytic center?: Supporting amino acid residues at the active site of penicillin G acylaseA new set of experimental kinetic data on the hydrolysis of a series of phenylacetyl p-substituted anilides catalyzed by penicillin G acylase from Escherichia coli (PGA) is presented in this article. The Hammett plot of log(kcat,R-kcat,H) versus p- has three linear segments, which distinguishes the enzyme from the other N-terminal nucleophile hydrolases for which data are available. Three amino acids in the vicinity of the catalytic SerB1 (AsnB241, AlaB69, and GlnB23) were included in the quantum mechanical model. The stable structures and the transition states for acylation were optimized by molecular mechanical modeling and at the AM1 level of theory for three model substrates (with H, a methoxy group or a nitro group in the para position in the leaving group). Intrinsic interactions of several functional groups at the active site of PGA are discussed in relation to the catalytic efficiency of the enzyme. The energy barrier computed for the first step of acylation (the nucleophilic attack of SerB1) is lower than that for the second step (the collapse of the tetrahedral intermediate). However, the electronic properties of the substituent on the leaving group affect the structure of the second transition state. It is shown that the main chain carbonyl group of GlnB23 forms a hydrogen bond with the leaving group nitrogen, thus influencing the hydrolysis rate. On the basis of our computations, we propose an interpretation of the complex character of the Hammett plot for the reaction catalyzed by PGA. We suggest a modified scheme of the catalytic mechanism in which some of the intramolecular interactions essential for catalysis are included. © 2009 FEBS.Publicationtype: Journal Article42