The <i>α</i>/<i>β</i> hydrolase fold

Ollis, David L.; Cheah, Eong; Cygler, Mirosław; Dijkstra, Bauke W.; Frolow, Felix; Franken, Sybille; Harel, Michal; Remington, S.J.; Silman, Israel; Schrag, Joseph D.; Sussman, Joel L.; Verschueren, K.H.G.; Goldman, Adrian

doi:10.1093/protein/5.3.197

Cited by 2,050 publications

(1,703 citation statements)

References 35 publications

Supporting

Mentioning

1,617

Contrasting

Unclassified

Order By: Relevance

“…Therefore, these ORFs were suggested to be the esterase gene of Geobacillus sp. The sequences were identified also due to the sequence motif GXSXG which is typical to a/b-hydrolases such as lipases and esterases (Ollis et al 1992). In addition to the GXSXG motif, the esterases contain the highly conserved GX-motif of the oxyanion hole architecture and thus could be assigned to the GX class (Pleiss et al 2000).…”

Section: Resultsmentioning

confidence: 99%

Molecular cloning, over expression and characterization of thermoalkalophilic esterases isolated from Geobacillus sp.

Tekedar

Şanlı‐Mohamed

2010

Extremophiles

View full text Add to dashboard Cite

Due to potential use for variety of biotechnological applications, genes encoding thermoalkalophilic esterase from three different Geobacillus strains isolated from thermal environmental samples in Balçova (Agamemnon) geothermal site were cloned and respective proteins were expressed in Escherichia coli (E.coli) and characterized in detail. Three esterases (Est1, Est2, Est3) were cloned directly by PCR amplification using consensus degenerate primers from genomic DNA of the strains Est1, Est2 and Est3 which were from mud, reinjection water and uncontrolled thermal leak, respectively. The genes contained an open reading frame (ORF) consisting of 741 bp for Est1 and Est2, which encoded 246 amino acids and ORF of Est3 was 729 bp encoded 242 amino acids. The esterase genes were expressed in E. coli and purified using His-Select HF nickel affinity gel. The molecular mass of the recombinant enzyme for each esterase was approximately 27.5 kDa. The three esterases showed high specific activity toward short chain p-NP esters. Recombinant Est1, Est2, Est3 have exhibited similar activity and the highest esterase activity of 1,100 U/mg with p-nitrophenyl acetate (pNPC 2 ) as substrate was observed with Est1. All three esterase were most active around 65°C and pH 9.5-10.0. The effect of organic solvents, several metal ions, inhibitors and detergents on enzyme activity for purified Est1, Est2, Est3 were determined separately and compared.

show abstract

Section: Resultsmentioning

confidence: 99%

Molecular cloning, over expression and characterization of thermoalkalophilic esterases isolated from Geobacillus sp.

Tekedar

Şanlı‐Mohamed

2010

Extremophiles

View full text Add to dashboard Cite

show abstract

“…Thus, some conformational differences in the lid domains occur among our structures. Movement in the lid domain could be analogous to what occurs in other α/β-hydrolases such as lipases (7,8). In BphD, the lid domains make contacts dependent on the tetramer (i.e., the AB' dimer in Figure 6).…”

Section: The Origin Of the Biphasic Kineticsmentioning

confidence: 86%

“…It is a member of the α/β-hydrolase superfamily (7)(8)(9) and contains conserved Ser-His-Asp residues that might be expected to constitute a catalytic triad enabling the acyl-enzyme mechanism common to many well characterized hydrolytic enzymes. In this mechanism, the hydrogen-bonding network between the triad of conserved residues serves to activate the serine hydroxyl for nucleophilic attack at the carbonyl carbon of the scissile bond.…”

mentioning

confidence: 99%

Kinetic and Structural Insight into the Mechanism of BphD, a C−C Bond Hydrolase from the Biphenyl Degradation Pathway

et al. 2006

View full text Add to dashboard Cite

Kinetic and structural analyses of 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoic acid (HOPDA) hydrolase from Burkholderia xenovorans LB400 (BphD LB400 ) provide insight into the catalytic mechanism of this unusual serine hydrolase. Single turnover stopped-flow analysis at 25 °C showed that the enzyme rapidly (1/τ 1 ∼ 500 s −1 ) transforms HOPDA (λ max = 434 nm) to a species with electronic absorption maxima at 473 and 492 nm. The absorbance of this enzyme-bound species (E:S) decayed in a biphasic manner (1/τ 2 = 54 s −1 , 1/τ 3 = 6 s −1 ∼ k cat ) with simultaneous biphasic appearance (48 and 8 s −1 ) of an absorbance band at 270 nm characteristic of one of the products, 2-hydroxypenta-2,4-dienoic acid (HPD). Increasing solution viscosity with glycerol slowed 1/τ 1 and 1/τ 2 , but affected neither 1/τ 3 nor k cat , suggesting that 1/τ 2 may reflect diffusive HPD dissociation, while 1/τ 3 represents an intramolecular event. Product inhibition studies suggested that the other product, benzoate, is released after HPD. Contrary to studies in a related hydrolase, we found no evidence that ketonized HOPDA is partially released prior to hydrolysis, and therefore postulate that the biphasic kinetics reflect one of two mechanisms, pending assignment of E:S (λ max = 492 nm). Crystal structures of wild type, the S112C variant, and S112C incubated with HOPDA were each determined to 1.6 Å resolution. The latter reveals interactions between conserved active site residues and the dienoate moiety of the substrate. Most notably, the catalytic residue His265 is hydrogenbonded to the 2-hydroxy/oxo substituent of HOPDA, consistent with a role in catalyzing ketonization. The data are more consistent with an acyl-enzyme mechanism than with the formation of a gem-diol intermediate.The microbial degradation of aromatic compounds is crucial to maintaining the global carbon cycle (1). Aerobic degradation typically involves oxygenation of the aromatic ring to produce a catechol followed by a dioxygenase-catalyzed ring-opening reaction. In one type of degradation pathway, ring-opening yields a meta-cleavage product (MCP) 1 . A serine hydrolase † This work was funded by the Natural Sciences and Engineering Research Council of Canada (Discovery Grant) and the National Institutes of Health (GM-52381).*To whom correspondence should be addressed: Lindsay D. Eltis, leltis@interchange.ubc.ca, Phone: (604) Fax: (604)822 −6041. § These authors contributed equally to this work. ¶ Present addresses: JK: University of Kentucky, Lexington, KY 40506, USA; SD: Howard Hughes Medical Institute, National Jewish Medical and Research Center, Denver, CO 80206; SS: Department of Molecular and Cellular Biology, University of Guelph, Guelph, ON, N1G 2W1, Canada 1 Abbreviations: MCP, meta-cleavage product; HPD, 2-hydroxy-penta-2,4-dienoic acid; PCB, polychlorinated biphenyl; HOPDA, 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoic acid; DHB, 2,3-dihydroxybiphenyl; DHBD, dihydroxybiphenyl dioxygenase; S k , ketonized substrate; E:S k , enzyme-bound ketonized substrate; P...

show abstract

“…1b shows the structure of CALB, which belongs to the large α/β hydrolase superfamily. [29] Initial experimental efforts with CALB wild type (wt) and S105A mutant (shown to have enhanced catalytic activity for carbon-carbon bond formation [20,22,23,24]) did not indicate any Diels-Alder activity.…”

Section: Fig 1: (A)mentioning

confidence: 99%

Computational design of a lipase for catalysis of the Diels-Alder reaction

et al. 2010

View full text Add to dashboard Cite

Combined molecular docking, molecular dynamics (MD) and density functional theory (DFT) studies have been employed to study catalysis of the Diels-Alder reaction by a modified lipase. Six variants of the versatile enzyme {\em Candida Antarctica} lipase B (CALB) have been rationally engineered {\em in silico} based on the specific characteristics of the pericyclic addition. A kinetic analysis reveals that hydrogen bond stabilization of the transition state and substrate binding are key components of the catalytic process. In the case of substrate binding, which has the greater potential for optimization, both binding strength and positioning of the substrates are important for catalytic efficiency. The binding strength is determined by hydrophobic interactions and can be tuned by careful selection of solvent and substrates. The MD simulations show that substrate positioning is sensitive to cavity shape and size, and can be controlled by a few rational mutations. The well-documented S105A mutation is essential to enable sufficient space in the vicinity of the oxyanion hole. Moreover, bulky residues on the edge of the active site hinders the formation of a sandwich-like near-attack conformer (NAC), and the I189A mutation is needed to obtain enough space above the face of the $\alpha$,$\beta$-double bond on the dienophile. The double mutant S105A/I189A performs quite well for two of three dienophiles. Based on binding constants and NAC energies obtained from MD simulations combined with activation energies from DFT computations, relative catalytic rates ($v_{cat}/v_{uncat}$) of up to $10^3$ are predicted.Response to Reviewers: We are happy with the favorable comments by the reviewers. We have have corrected the manuscript as suggested by reviewer 1 1. A sentence explaining catalytic promiscuity has been added. 2. A paragraph discussing the relationship between the NAC concept and the Reaction Force has been added.We have shortened the manuscript as suggested by reviewer 2. In particular, we have moved information regarding the structural relaxation of protein structure in MD simulations to the supplementary material. Abstract Combined molecular docking, molecular dynamics (MD) and density functional theory (DFT) studies have been employed to study catalysis of the DielsAlder reaction by a modified lipase. Six variants of the versatile enzyme Candida Antarctica lipase B (CALB) have been rationally engineered in silico based on the specific characteristics of the pericyclic addition. A kinetic analysis reveals that hydrogen bond stabilization of the transition state and substrate binding are key components of the catalytic process. In the case of substrate binding, which has the greater potential for optimization, both binding strength and positioning of the substrates are important for catalytic efficiency. The binding strength is determined by hydrophobic interactions and can be tuned by careful selection of solvent and substrates. The MD simulations show that substrate positioning is sensitive to cavity s...

show abstract

The α/β hydrolase fold

Cited by 2,050 publications

References 35 publications

Molecular cloning, over expression and characterization of thermoalkalophilic esterases isolated from Geobacillus sp.

Molecular cloning, over expression and characterization of thermoalkalophilic esterases isolated from Geobacillus sp.

Kinetic and Structural Insight into the Mechanism of BphD, a C−C Bond Hydrolase from the Biphenyl Degradation Pathway

Computational design of a lipase for catalysis of the Diels-Alder reaction

Contact Info

Product

Resources

About