Improving enzyme properties: when are closer mutations better?

Morley, Krista L.; Kazlauskas, Romas J.

doi:10.1016/j.tibtech.2005.03.005

Cited by 400 publications

(314 citation statements)

References 34 publications

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“…The current results validate the contact mapping strategy for identifying amino acids and positions within these enzymes that dictate the penultimate reaction steps; however, extending the contact mapping strategy to synthases that catalyze reactions diverging at earlier steps represents an important future target. Long-term, identifying the structural features that govern reaction product specificity for various members of the terpene synthase families and classes should provide a unique perspective into the evolution of this family of enzymes (18,19) and the potential for using this information in the rational design and engineering of different catalytic activities into existing terpene synthase enzymes (20,21).…”

Section: Discussionmentioning

confidence: 99%

Identifying and manipulating structural determinates linking catalytic specificities in terpene synthases

Greenhagen

O’Maille

Noel

et al. 2006

Proc. Natl. Acad. Sci. U.S.A.

200

181

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Terpene synthases are a mechanistically intriguing family of enzymes that catalyze complex, multistep reactions that are capable of generating hundreds of structurally diverse hydrocarbon and oxygenated scaffolds of biological and commercial importance. Interestingly, distantly related terpene synthases from fungi to plants all contain an invariant three-dimensional fold, and molecular comparisons of their active sites indicate that they are enriched with relatively inert amino acid residues that do not react directly with the reaction intermediates. Therefore, catalytic specificity appears to rely on the contour and dynamics of the active site created by the positioning of amino acid backbones and side chains on this catalytic surface and by supporting layers of residues surrounding the synthase active site cavity. Despite the high degree of structural relatedness among terpene synthases, previous studies suggest that no clear relationship between phylogenic organization and catalytic specificities is easily deciphered. We now report on the reciprocal interconversion of catalytic specificities between two distinct yet evolutionarily related terpene synthases based on the systematic identification and mutational replacement of variable residues within and surrounding the active site. Furthermore, we uncover previously undocumented biosynthetic activity during the interconversion, activity that could have been present in a common ancestor of these two highly related synthases. These results provide a simplified means for mapping structural features that are responsible for functional attributes and a strategy for identifying residues that differentiate divergent biosynthetic properties in phylogenetically related terpene synthases.phylogenetic relationships ͉ rational design ͉ sesquiterpene ͉ structure-function T erpenes comprise the most diverse collection of natural products known. Terpene hydrocarbon scaffolds are generated by the action of mono-, sesqui-, and diterpene synthases that catalyze multistep reactions with diphosphorylated substrates of 10 (geranyl diphosphate), 15 [farnesyl diphosphate (FPP)] or 20 (geranylgeranyl diphosphate) carbons (1). The reactions catalyzed by terpene synthases are unparalleled relative to other classes of enzymes because they often consist of a series of stereochemically complex steps. These reactions include ionization of the diphosphate substituent creating an acyclic and reactive carbocation intermediate. In some cases, the isomerization of the all-trans substrate configuration to a cis-trans isomer also occurs before subsequent reactions. Additional steps add increasing complexity and include regio-and stereospecific formation of single or multiple rings, proton eliminations to form double bonds, water quenching of carbocations to create terpene alcohols, and stereospecific hydride, methyl, and methylene migrations. Equally intriguing, the three-dimensional structure of terpene synthases from fungi to plants is highly conserved, including a ''terpene fold'' composed lar...

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Section: Discussionmentioning

confidence: 99%

Identifying and manipulating structural determinates linking catalytic specificities in terpene synthases

Greenhagen

O’Maille

Noel

et al. 2006

Proc. Natl. Acad. Sci. U.S.A.

200

181

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“…This preference for the alternative electron acceptors over oxygen is further expressed by the substrate specificity values, i.e., the ratio of the specificity constants k cat /K m for the two substrates [51]. All variants show increased ratios of Dehydrogenase (Dh kcat/ Km ) to Oxidase (Ox kcat/Km ) catalytic efficiency compared to wildtype POx, regardless of the electron acceptor used (Fig.…”

Section: Protein Expression Purification and Kinetic Characterizationmentioning

confidence: 97%

Engineering of pyranose 2-oxidase for modified oxygen reactivity

2014

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“…On the other side, rational design can be used to guide the direct mutations of residues at specific positions of the enzyme. 9 Molecular simulations appear to be nowadays a promising option for computer-based design.…”

Section: Introductionmentioning

confidence: 99%

Enzyme Promiscuity in Enolase Superfamily. Theoretical Study of o-Succinylbenzoate Synthase Using QM/MM Methods

et al. 2015

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The promiscuous activity of the enzyme o-Succinylbenzoate Synthase (OSBS) from the actinobacteria Amycolatopsis is investigated by means of QM/MM methods, using both Density Functional Theory and Semiempirical Hamiltonians. This enzyme catalyzes not only the dehydration of 2-succinyl-6R-hydroxy-2,4-cyclohexadiene-1R-carboxylate but also catalyzes racemization of different acylaminoacids, being N-succinyl-Rphenylglycine the best substrate. We investigated the molecular mechanisms for both reactions exploring the Potential Energy Surface. Then, Molecular Dynamics simulations were performed to obtain the free energy profiles and the averaged interaction energies of enzymatic residues with the reacting system. Our results confirm the plausibility of the reaction mechanisms proposed in the literature, with a good agreement between theoretical and experimentally-derived activation free energies. Our simulations unravel the role played by the different residues in each of the two possible reactions. The presence of flexible loops in the active site and the selection of structural modifications in the substrate seem to be key elements to promote the promiscuity of this enzyme.

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Improving enzyme properties: when are closer mutations better?

Cited by 400 publications

References 34 publications

Identifying and manipulating structural determinates linking catalytic specificities in terpene synthases

Identifying and manipulating structural determinates linking catalytic specificities in terpene synthases

Engineering of pyranose 2-oxidase for modified oxygen reactivity

Enzyme Promiscuity in Enolase Superfamily. Theoretical Study of o-Succinylbenzoate Synthase Using QM/MM Methods

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