Die Genfusion ist ein wichtiger natürlicher Prozess zur Erzeugung neuer Enzyme aus einfachen Vorläufern. Gerrit J. Poelarends und Mitarbeiter haben diese Strategie in ihrer Zuschrift (e202113970) ...angewendet, um eine promiskuitive homohexamere 4‐Oxalocrotonat‐Tautomerase (4‐OT) zu einem effizienten Enzym für enantioselektive Michael‐Reaktionen zu entwickeln. Sie entwarfen eine tandemfusionierte 4‐OT mit reduzierter Symmetrie, die eine unabhängige Sequenzdiversifizierung benachbarter Untereinheiten ermöglicht und so den Proteinsequenzraum vergrößert, der durch gerichtete Evolution erforscht werden kann.
Cyclodextrin-glycosyltransferase (CGTase) catalyzes the formation of α-, β-, and γ-cyclodextrins (cyclic α-(1,4)-linked oligosaccharides
of 6, 7, or 8 glucose residues, respectively) from starch. ...Nine substrate binding subsites were observed in an x-ray structure
of the CGTase from Bacillus circulans strain 251 complexed with a maltononaose substrate. Subsite â6 is conserved in CGTases, suggesting its importance for the
reactions catalyzed by the enzyme. To investigate this in detail, we made six mutant CGTases (Y167F, G179L, G180L, N193G,
N193L, and G179L/G180L). All subsite â6 mutants had decreased k cat values for β-cyclodextrin formation, as well as for the disproportionation and coupling reactions, but not for hydrolysis.
Especially G179L, G180L, and G179L/G180L affected the transglycosylation activities, most prominently for the coupling reactions.
The results demonstrate that (i) subsite â6 is important for all three CGTase-catalyzed transglycosylation reactions, (ii)
Gly-180 is conserved because of its importance for the circularization of the linear substrates, (iii) it is possible to independently
change cyclization and coupling activities, and (iv) substrate interactions at subsite â6 activate the enzyme in catalysis
via an induced-fit mechanism. This article provides for the first time definite biochemical evidence for such an induced-fit
mechanism in the α-amylase family.
Hevamine is a chitinase from the rubber tree Hevea brasiliensis. Its active site contains Asp125, Glu127, and Tyr183, which interact with the −1 sugar residue of the substrate. To investigate their ...role in catalysis, we have successfully expressed wild‐type enzyme and mutants of these residues as inclusion bodies in Escherichia coli. After refolding and purification they were characterized by both structural and enzyme kinetic studies. Mutation of Tyr183 to phenylalanine produced an enzyme with a lower kcat and a slightly higher Km than the wild‐type enzyme. Mutating Asp125 and Glu127 to alanine gave mutants with ≈ 2% residual activity. In contrast, the Asp125Asn mutant retained substantial activity, with an approximately twofold lower kcat and an approximately twofold higher Km than the wild‐type enzyme. More interestingly, it showed activity to higher pH values than the other variants. The X‐ray structure of the Asp125Ala/Glu127Ala double mutant soaked with chitotetraose shows that, compared with wild‐type hevamine, the carbonyl oxygen atom of the N‐acetyl group of the −1 sugar residue has rotated away from the C1 atom of that residue. The combined structural and kinetic data show that Asp125␣and Tyr183 contribute to catalysis by positioning the␣carbonyl oxygen of the N‐acetyl group near to the C1 atom. This allows the stabilization of a positively charged transient intermediate, in agreement with a previous proposal that the enzyme makes use of substrate‐assisted catalysis.
Haloalkane dehalogenase (DhlA) catalyzes the hydrolysis of haloalkanes via an alkyl−enzyme intermediate. Trp175 forms a halogen/halide-binding site in the active-site cavity together with Trp125. To ...get more insight in the role of Trp175 in DhlA, we mutated residue 175 and explored the kinetics and X-ray structure of the Trp175Tyr enzyme. The mutagenesis study indicated that an aromatic residue at position 175 is important for the catalytic performance of DhlA. Pre-steady-state kinetic analysis of Trp175Tyr-DhlA showed that the observed 6-fold increase of the K m for 1,2-dibromoethane (DBE) results from reduced rates of both DBE binding and cleavage of the carbon−bromine bond. Furthermore, the enzyme isomerization preceding bromide release became 4-fold faster in the mutant enzyme. As a result, the rate of hydrolysis of the alkyl−enzyme intermediate became the main determinant of the k cat for DBE, which was 2-fold higher than the wild-type k cat. The X-ray structure of the mutant enzyme at pH 6 showed that the backbone structure of the enzyme remains intact and that the tyrosine side chain lies in the same plane as Trp175 in the wild-type enzyme. The Clα-stabilizing aromatic rings of Tyr175 and Trp125 are 0.7 Å further apart and due to the smaller size of the mutated residue, the volume of the cavity has increased by one-fifth. X-ray structures of mutant and wild-type enzyme at pH 5 demonstrated that the Tyr175 side chain rotated away upon binding of an acetic acid molecule, leaving one of its oxygen atoms hydrogen bonded to the indole nitrogen of Trp125 only. These structural changes indicate a weakened interaction between residue 175 and the halogen atom or halide ion in the active site and help to explain the kinetic changes induced by the Trp175Tyr mutation.
Carnein is an 80 kDa subtilisin‐like serine protease from the latex of the plant Ipomoea carnea which displays an exceptional resistance to chemical and thermal denaturation. In order to obtain the ...first crystal structure of a plant subtilisin and to gain insight into the structural determinants underlying its remarkable stability, carnein was isolated from I. carnea latex, purified and crystallized by the hanging‐drop vapour‐diffusion method. A data set was collected to 2.0 Å resolution in‐house from a single crystal at 110 K. The crystals belonged to the trigonal space group P3121 or P3221, with unit‐cell parameters a = b = 126.9, c = 84.6 Å, α = β = 90, γ = 120°. Assuming the presence of one molecule per asymmetric unit, the Matthews coefficient is 2.46 Å3 Da−1, corresponding to a solvent content of 50%. Structure determination of the enzyme is in progress.
Epoxide hydrolases catalyze the cofactor-independent hydrolysis of reactive and toxic epoxides. They play an essential role in the detoxification of various xenobiotics in higher organisms and in the ...bacterial degradation of several environmental pollutants. The first x-ray structure of one of these, from Agrobacterium radiobacter AD1, has been determined by isomorphous replacement at 2.1-A resolution. The enzyme shows a two-domain structure with the core having the alpha/beta hydrolase-fold topology. The catalytic residues, Asp107 and His275, are located in a predominantly hydrophobic environment between the two domains. A tunnel connects the back of the active-site cavity with the surface of the enzyme and provides access to the active site for the catalytic water molecule, which in the crystal structure, has been found at hydrogen bond distance to His275. Because of a crystallographic contact, the active site has become accessible for the Gln134 side chain, which occupies a position mimicking a bound substrate. The structure suggests Tyr152/Tyr215 as the residues involved in substrate binding, stabilization of the transition state, and possibly protonation of the epoxide oxygen.
Haloalkane dehalogenase (DhlA) converts haloalkanes to their corresponding alcohols and halide ions. The rate-limiting step in the reaction of DhlA is the release of the halide ion. The kinetics of ...halide release have been analyzed by measuring halide binding with stopped-flow fluorescence experiments. At high halide concentrations, halide import occurs predominantly via the rapid formation of a weak initial collision complex, followed by transport of the ion to the active site. To obtain more insight in this collision complex, we determined the X-ray structure of DhlA in the presence of bromide and investigated the kinetics of mutants that were constructed on the basis of this structure. The X-ray structure revealed one bromide ion firmly bound in the active site and two bromide ions weakly bound on the surface of the enzyme. One of the weakly bound ions is close to Thr197 and Phe294, near the entrance of the earlier proposed tunnel for substrate import. Kinetic analysis of bromide import by the Thr197Ala and Phe294Ala mutants of DhlA at high halide concentration showed that the rate constants for halide binding no longer displayed a wild-type-like parabolic increase with increasing bromide concentrations. This is in agreement with an elimination or a decrease in affinity of the surface-located halide-binding site. Likewise, chloride binding kinetics of the mutants indicated significant differences with wild-type enzyme. The results indicate that Thr197 and Phe294 are involved in the formation of an initial collision complex for halide import in DhlA and provide experimental evidence for the role of the tunnel in substrate and product transport.
Quinoprotein alcohol dehydrogenases are redox enzymes that participate in distinctive catabolic pathways that enable bacteria to grow on various alcohols as the sole source of carbon and energy. The ...x-ray structure of the quinohemoprotein alcohol dehydrogenase from Comamonas testosteroni has been determined at 1.44 Å resolution. It comprises two domains. The N-terminal domain has a β-propeller fold and binds one pyrroloquinoline quinone cofactor and one calcium ion in the active site. A tetrahydrofuran-2-carboxylic acid molecule is present in the substrate-binding cleft. The position of this oxidation product provides valuable information on the amino acid residues involved in the reaction mechanism and their function. The C-terminal domain is an α-helical type I cytochrome c with His608 and Met647 as heme-iron ligands. This is the first reported structure of an electron transfer system between a quinoprotein alcohol dehydrogenase and cytochrome c. The shortest distance between pyrroloquinoline quinone and heme c is 12.9 Å, one of the longest physiological edge-to-edge distances yet determined between two redox centers. A highly unusual disulfide bond between two adjacent cysteines bridges the redox centers. It appears essential for electron transfer. A water channel delineates a possible pathway for proton transfer from the active site to the solvent.
The l-2-haloacid dehalogenase from the 1,2-dichloroethane degrading bacterium Xanthobacter autotrophicus GJ10 catalyzes the hydrolytic dehalogenation of small l-2-haloalkanoic acids to yield the ...correspondingd-2-hydroxyalkanoic acids. Its crystal structure was solved by the method of multiple isomorphous replacement with incorporation of anomalous scattering information and solvent flattening, and was refined at 1.95-Å resolution to an R factor of 21.3%. The three-dimensional structure is similar to that of the homologousl-2-haloacid dehalogenase from Pseudomonassp. YL (1), but the X. autotrophicus enzyme has an extra dimerization domain, an active site cavity that is completely shielded from the solvent, and a different orientation of several catalytically important amino acid residues. Moreover, under the conditions used, a formate ion is bound in the active site. The position of this substrate-analogue provides valuable information on the reaction mechanism and explains the limited substrate specificity of the Xanthobacterl-2-haloacid dehalogenase.
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