Application of Rigidity Theory to the Thermostabilization of Lipase A from Bacillus subtilis

Rathi, Prakash Chandra; Fulton, Alexander; Jaeger, Karl‐Erich; Gohlke, Holger

doi:10.1371/journal.pcbi.1004754

Cited by 49 publications

(66 citation statements)

References 93 publications

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“…Kinetic analysis. To investigate effects of the mutations on enzyme kinetics, 4-nitrophenyl laurate (pNPL) hydrolysis was selected on the basis of its wide usage in lipase studies (61)(62)(63)(64), including those with LipT6 (47,48). The kinetic constants (Table 5) were calculated on the basis of activity under native conditions without methanol.…”

Section: Resultsmentioning

confidence: 99%

Filling the Void: Introducing Aromatic Interactions into Solvent Tunnels To Enhance Lipase Stability in Methanol

Gihaz

Kanteev

Pazy

et al. 2018

Appl Environ Microbiol

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Enhanced stability in organic solvents is a desirable feature for enzymes implemented under industrial conditions. Lipases potential as biocatalysts is mainly limited by denaturation in polar alcohols. In this study we focused on selected solvent tunnels in lipase from T6 to improve its stability in methanol during biodiesel synthesis. Using rational mutagenesis, bulky aromatic residues were incorporated in order to occupy solvent channels and induce aromatic interactions leading to a better inner core packing. Each solvent tunnel was systematically analyzed with respect to its chemical and structural characteristics. Selected residues were replaced with Phe, Tyr or Trp. Overall, 16 mutants were generated and screened in 60% methanol, from which 3 variants showed elevated stability up to 81-fold compared with wild-type. All stabilizing mutations were found in the longest tunnel detected in the "closed-lid" x-ray structure. Combining the Phe substitutions created double mutant A187F/L360F with an increase in T of +7°C in methanol and a 3-fold increase in biodiesel synthesis yield from waste chicken oil. Kinetic analysis with -nitrophenyl laurate revealed that all mutants displayed lower hydrolysis rates ( ), though mostly their stability properties determined the transesterification capability. Seven crystal structures of different variants were solved disclosing new π-π or CH/π intramolecular interactions emphasizing the significance of aromatic interactions to improved solvent stability. This rational approach could be implemented in other enzymes for stabilization in organic solvents. Enzymatic synthesis in organic solvents holds increasing industrial opportunities in many fields, however, one major obstacle is the limited stability of biocatalysts in such a denaturing environment. Aromatic interactions play a major role in protein folding and stability and we were inspired by this to re-design enzyme voids. Rational protein engineering of solvent tunnels of lipase from is presented here, offering a promising approach to introduce new aromatic interactions within the enzyme core. We discovered that longer tunnels leading from the surface to the enzyme active site were more beneficial targets for mutagenesis for improving lipase stability in methanol during biodiesel biosynthesis. Structural analysis of the variants confirmed the generation of new interactions involving aromatic residues. This work provides insights into stability-driven enzyme design by targeting solvent channels void.

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Section: Resultsmentioning

confidence: 99%

Filling the Void: Introducing Aromatic Interactions into Solvent Tunnels To Enhance Lipase Stability in Methanol

Gihaz

Kanteev

Pazy

et al. 2018

Appl Environ Microbiol

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“…Therefore, strict rigidity theory based methods rely on exceedingly local motions to estimate conformational flexibility, 20,27,28 ligand binding, 31 entropy, 33,34 or thermo-stability 38–40 compared to ENM or NMA. Nonetheless, they show convincing agreement with experimental data, if energy cutoffs to determine constraints are carefully chosen.…”

Section: Discussionmentioning

confidence: 99%

“…Monitoring changes in protein rigidity from diluting the constraint network by gradually raising the energy threshold has informed on thermostability 38–40 and the evolution of folding cores. 29,41 However, the topological and combinatorial nature of this approach denies explicit access to the kinematics of the underlying 3-dimensional molecular structure.…”

Section: Introductionmentioning

confidence: 99%

Kinematic Flexibility Analysis: Hydrogen Bonding Patterns Impart a Spatial Hierarchy of Protein Motion

Budday

Leyendecker

Bedem

2018

J. Chem. Inf. Model.

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Elastic network models (ENMs) and constraint-based, topological rigidity analysis are two distinct, coarse-grained approaches to study conformational flexibility of macromolecules. In the two decades since their introduction, both have contributed significantly to insights into protein molecular mechanisms and function. However, despite a shared purpose of these approaches, the topological nature of rigidity analysis, and thereby the absence of motion modes, has impeded a direct comparison. Here, we present an alternative, kinematic approach to rigidity analysis, which circumvents these drawbacks. We introduce a novel protein hydrogen bond network spectral decomposition, which provides an orthonormal basis for collective motions modulated by noncovalent interactions, analogous to the eigenspectrum of normal modes. The zero modes decompose proteins into rigid clusters identical to those from topological rigidity, while nonzero modes rank protein motions by their hydrogen bond collective energy penalty. Our kinematic flexibility analysis bridges topological rigidity theory and ENM, enabling a detailed analysis of motion modes obtained from both approaches. Analysis of a large, structurally diverse data set revealed that collectivity of protein motions, reported by the Shannon entropy, is significantly reduced for rigidity theory compared to normal mode approaches. Strikingly, kinematic flexibility analysis suggests that the hydrogen bonding network encodes a protein-fold specific, spatial hierarchy of motions, which goes nearly undetected in ENM. This hierarchy reveals distinct motion regimes that rationalize experimental and simulated protein stiffness variations. Kinematic motion modes highly correlate with reported crystallographic B factors and molecular dynamics simulations of adenylate kinase. A formal expression for changes in free energy derived from the spectral decomposition indicates that motions across nearly 40% of modes obey enthalpy–entropy compensation. Taken together, our results suggest that hydrogen bond networks have evolved to modulate protein structure and dynamics, which can be efficiently probed by kinematic flexibility analysis.

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“…Residue X IV+1 has also been mentioned as part of the hydrophobic binding pocket [39] and the helical domain that occurs after strand β6 [20], while in some cases it can affect the enzymatic activity [42][43][44]. To our knowledge, there is only one mutational study that refers to residue X IV+1 , where its substitution has resulted in nearly complete loss of catalytic activity [45]. The tripeptide residue X IV -residue X IV+1 -residue X IV+2 retains its amino-acid conservation in some ABH fold enzyme families, and thus, it is suggested that it can be used as an additional sequence identifier of different ABH fold enzyme families [32,37].…”

Section: The Conserved Structural Elements That Line the Catalytic Stmentioning

confidence: 99%

The acid-base-nucleophile catalytic triad in ABH-fold enzymes is coordinated by a set of structural elements

et al. 2020

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The alpha/beta-Hydrolases (ABH) are a structural class of proteins that are found widespread in nature and includes enzymes that can catalyze various reactions in different substrates. The catalytic versatility of the ABH fold enzymes, which has been a valuable property in protein engineering applications, is based on a similar acid-base-nucleophile catalytic mechanism. In our research, we are concerned with the structure that surrounds the key units of the catalytic machinery, and we have previously found conserved structural organizations that coordinate the catalytic acid, the catalytic nucleophile and the residues of the oxyanion hole. Here, we explore the architecture that surrounds the catalytic histidine at the active sites of enzymes from 40 ABH fold families, where we have identified six conserved interactions that coordinate the catalytic histidine next to the catalytic acid and the catalytic nucleophile. Specifically, the catalytic nucleophile is coordinated next to the catalytic histidine by two weak hydrogen bonds, while the catalytic acid is directly involved in the coordination of the catalytic histidine through by two weak hydrogen bonds. The imidazole ring of the catalytic histidine is coordinated by a CH-π contact and a hydrophobic interaction. Moreover, the catalytic triad residues are connected with a residue that is located at the core of the active site of ABH fold, which is suggested to be the fourth member of a "structural catalytic tetrad". Besides their role in the stability of the catalytic mechanism, the conserved elements of the catalytic site are actively involved in ligand binding and affect other properties of the catalytic activity, such as substrate specificity, enantioselectivity, pH optimum and thermostability of ABH fold enzymes. These properties are regularly targeted in protein engineering applications, and thus, the identified conserved structural elements can serve as potential modification sites in order to develop ABH fold enzymes with altered activities.

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Application of Rigidity Theory to the Thermostabilization of Lipase A from Bacillus subtilis

Cited by 49 publications

References 93 publications

Filling the Void: Introducing Aromatic Interactions into Solvent Tunnels To Enhance Lipase Stability in Methanol

Filling the Void: Introducing Aromatic Interactions into Solvent Tunnels To Enhance Lipase Stability in Methanol

Kinematic Flexibility Analysis: Hydrogen Bonding Patterns Impart a Spatial Hierarchy of Protein Motion

The acid-base-nucleophile catalytic triad in ABH-fold enzymes is coordinated by a set of structural elements

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