Mechanisms of Protein Evolution and their Application to Protein Engineering

Glasner, Margaret E.; Gerlt, J.A.; Babbitt, Patricia C.

doi:10.1002/9780471224464.ch3

Cited by 30 publications

(29 citation statements)

References 115 publications

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“…amino acid biosynthesis | enzyme evolution | mycobacteria | active site substrate adaptability | protein structure symmetry E nzymes that fold into ðβ∕αÞ 8 barrels provide the most widely found prototype scaffold to study the molecular mechanisms underlying enzymatic diversity (1,2). Among those investigated, many catalyze specific reactions, but there are also examples of enzymes with broader substrate profiles (2,3).…”

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confidence: 99%

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Bisubstrate specificity in histidine/tryptophan biosynthesis isomerase from Mycobacterium tuberculosis by active site metamorphosis

Due

Kuper

Geerlof

et al. 2011

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

124

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In histidine and tryptophan biosynthesis, two related isomerization reactions are generally catalyzed by two specific singlesubstrate enzymes (HisA and TrpF), sharing a similar ðβ∕αÞ 8 -barrel scaffold. However, in some actinobacteria, one of the two encoding genes (trpF) is missing and the two reactions are instead catalyzed by one bisubstrate enzyme (PriA). To unravel the unknown mechanism of bisubstrate specificity, we used the Mycobacterium tuberculosis PriA enzyme as a model. Comparative structural analysis of the active site of the enzyme showed that PriA undergoes a reaction-specific and substrate-induced metamorphosis of the active site architecture, demonstrating its unique ability to essentially form two different substrate-specific actives sites. Furthermore, we found that one of the two catalytic residues in PriA, which are identical in both isomerization reactions, is recruited by a substrate-dependent mechanism into the active site to allow its involvement in catalysis. Comparison of the structural data from PriA with one of the two single-substrate enzymes (TrpF) revealed substantial differences in the active site architecture, suggesting independent evolution. To support these observations, we identified six small molecule compounds that inhibited both PriA-catalyzed isomerization reactions but had no effect on TrpF activity. Our data demonstrate an opportunity for organism-specific inhibition of enzymatic catalysis by taking advantage of the distinct ability for bisubstrate catalysis in the M. tuberculosis enzyme.amino acid biosynthesis | enzyme evolution | mycobacteria | active site substrate adaptability | protein structure symmetry

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confidence: 99%

“…Among those investigated, many catalyze specific reactions, but there are also examples of enzymes with broader substrate profiles (2,3). Generally the level of functional promiscuity seems to be correlated with active site conformational plasticity (2, 4), conceptually described as "metamorphic proteins" (5).…”

mentioning

confidence: 99%

Bisubstrate specificity in histidine/tryptophan biosynthesis isomerase from Mycobacterium tuberculosis by active site metamorphosis

Due

Kuper

Geerlof

et al. 2011

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

124

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“…Enzyme function sharply contrasts with this situation since it requires a multidimensional definition more difficult to express. Quantitative descriptors, such as catalytic efficiencies (k cat /K m ), are frequently preferred for functional description due to their easy correspondence to intrinsic thermodynamic parameters in the case of a perfect Michaelian enzyme [56]. But determining catalytic efficiencies on a large set of activities for several orthologous enzymes is a time-consuming task.…”

Section: Data Pre-processing (Normalization)mentioning

confidence: 99%

Differences in Functional Clustering of Endogenous and Exogenous Substrates Between Members of the CYP1A Subfamily

Urban¹,

Truan²,

Pompon³

2009

Open Drug Metab J

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Abstract:The ability of four mammalian cytochromes P450 (CYP) of the CYP1A subfamily, human and mouse CYP1A1s and human and rabbit CYP1A2s, to metabolize a series of steroids and related compounds was investigated using high throughput approaches. Oxidation rates and metabolite patterns for 16 steroid substrates and for 20 polycyclic aromatic hydrocarbon (PAH) substrates were determined in standardized automated conditions. Multivariate statistics of normalized activity data sets was used to sort out significant information and to compare functional signatures of assayed enzymes. Interestingly, for steroid substrates, rabbit CYP1A2 unambiguously aggregates with human and mouse CYP1A1s and appears functionally divergent from human CYP1A2. In contrast, the functional classification was found consistent with the sequence classification when exogenous PAH substrates were tested. The observed features rely on a large set of substrates, all presenting a similar chemical scaffold but decorated with different substituents similar to chemical series used in drug development. Differential functional clusters are thus evidenced for endogenous and exogenous substrates with CYP1A enzymes. A few residues on rabbit CYP1A2 that may account for its unusual 1A1-like specificity toward steroids have been identified both within the active site and at the protein surface. These specific residues thus seem to play a controlling role for global substrate class discrimination, potentially by involving substrate bulkiness and shape sensing.

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“…[9] However, nonclassical TS inhibitors that are structurally dissimilar from the substrate can discriminate specifically between the two bacterial species and are therefore important tools for probing species specificity for bacterial enzymes. [7,8,[10][11][12][13] By combining virtual screening with solid-phase in-parallel chemistry, a small library of O-dansyl-l-tyrosine derivatives was synthesized previously. Among these derivatives, N,O-didansyll-tyrosine (DDT) was the most potent inhibitor (K i A C H T U N G T R E N N U N G (LcTS) = 1.4 mm).…”

Section: Introductionmentioning

confidence: 99%

Constrained Dansyl Derivatives Reveal Bacterial Specificity of Highly Conserved Thymidylate Synthases

et al. 2008

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The elucidation of the structural/functional specificities of highly conserved enzymes remains a challenging area of investigation, and enzymes involved in cellular replication are important targets for functional studies and drug discovery. Thymidylate synthase (TS, ThyA) governs the synthesis of thymidylate for use in DNA synthesis. The present study focused on Lactobacillus casei TS (LcTS) and Escherichia coli TS (EcTS), which exhibit 50 % sequence identity and strong folding similarity. We have successfully designed and validated a chemical model in which linear, but not constrained, dansyl derivatives specifically complement the LcTS active site. Conversely, chemically constrained dansyl derivatives showed up to 1000-fold improved affinity for EcTS relative to the inhibitory activity of linear derivatives. This study demonstrates that the accurate design of small ligands can uncover functional features of highly conserved enzymes.

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Mechanisms of Protein Evolution and their Application to Protein Engineering

Cited by 30 publications

References 115 publications

Bisubstrate specificity in histidine/tryptophan biosynthesis isomerase from Mycobacterium tuberculosis by active site metamorphosis

Bisubstrate specificity in histidine/tryptophan biosynthesis isomerase from Mycobacterium tuberculosis by active site metamorphosis

Differences in Functional Clustering of Endogenous and Exogenous Substrates Between Members of the CYP1A Subfamily

Constrained Dansyl Derivatives Reveal Bacterial Specificity of Highly Conserved Thymidylate Synthases

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