Stability, Unfolding, and Structural Changes of Cofactor-Free 1<i>H</i>-3-Hydroxy-4-oxoquinaldine 2,4-Dioxygenase

Beermann, Bernd; Guddorf, Jessica; Boehm, Kristian; Albers, Alexander; Kolkenbrock, Stephan; Fetzner, Susanne; Hinz, Hans‐Jürgen

doi:10.1021/bi0622423

Cited by 15 publications

(21 citation statements)

References 14 publications

(33 reference statements)

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“…The unfolding trajectory (Figs 1b and 2 ) reveals the early segregation of loops from the largest rigid cluster, followed by the segregation of α-helices and, finally, the segregation and disintegration of the β-sheet region. This order of segregation is in agreement with experimental findings on the unfolding of other α/β hydrolase proteins [ 46 , 47 ]. The realistic description of WT Bs LipA thermal unfolding encouraged us to identify weak spots at major phase transitions along the unfolding trajectory ( Fig 1c ).…”

Section: Resultssupporting

confidence: 91%

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

et al. 2016

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Protein thermostability is a crucial factor for biotechnological enzyme applications. Protein engineering studies aimed at improving thermostability have successfully applied both directed evolution and rational design. However, for rational approaches, the major challenge remains the prediction of mutation sites and optimal amino acid substitutions. Recently, we showed that such mutation sites can be identified as structural weak spots by rigidity theory-based thermal unfolding simulations of proteins. Here, we describe and validate a unique, ensemble-based, yet highly efficient strategy to predict optimal amino acid substitutions at structural weak spots for improving a protein’s thermostability. For this, we exploit the fact that in the majority of cases an increased structural rigidity of the folded state has been found as the cause for thermostability. When applied prospectively to lipase A from Bacillus subtilis, we achieved both a high success rate (25% over all experimentally tested mutations, which raises to 60% if small-to-large residue mutations and mutations in the active site are excluded) in predicting significantly thermostabilized lipase variants and a remarkably large increase in those variants’ thermostability (up to 6.6°C) based on single amino acid mutations. When considering negative controls in addition and evaluating the performance of our approach as a binary classifier, the accuracy is 63% and increases to 83% if small-to-large residue mutations and mutations in the active site are excluded. The gain in precision (predictive value for increased thermostability) over random classification is 1.6-fold (2.4-fold). Furthermore, an increase in thermostability predicted by our approach significantly points to increased experimental thermostability (p < 0.05). These results suggest that our strategy is a valuable complement to existing methods for rational protein design aimed at improving thermostability.

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Section: Resultssupporting

confidence: 91%

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

et al. 2016

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“…Thus, a helix will persist as an independent rigid cluster during the thermal unfolding simulation until all backbone hydrogen bonds break almost simultaneously at a high temperature, which most likely represents an overstabilization of a helix [ 74 ]. Considering this behavior, the unfolding pathway identified for the wild type Bs LipA is in good agreement with respect to the early segregation of α-helices with experimental findings on the unfolding of proteins with an α/β hydrolase fold [ 75 , 76 ]. This indicates that side chain-mediated interactions between amino acids are well represented by the applied definitions of non-covalent constraints in the network.…”

Section: Resultssupporting

confidence: 81%

Structural Rigidity and Protein Thermostability in Variants of Lipase A from Bacillus subtilis

2015

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Understanding the origin of thermostability is of fundamental importance in protein biochemistry. Opposing views on increased or decreased structural rigidity of the folded state have been put forward in this context. They have been related to differences in the temporal resolution of experiments and computations that probe atomic mobility. Here, we find a significant (p = 0.004) and fair (R 2 = 0.46) correlation between the structural rigidity of a well-characterized set of 16 mutants of lipase A from Bacillus subtilis (BsLipA) and their thermodynamic thermostability. We apply the rigidity theory-based Constraint Network Analysis (CNA) approach, analyzing directly and in a time-independent manner the statics of the BsLipA mutants. We carefully validate the CNA results on macroscopic and microscopic experimental observables and probe for their sensitivity with respect to input structures. Furthermore, we introduce a robust, local stability measure for predicting thermodynamic thermostability. Our results complement work that showed for pairs of homologous proteins that raising the structural stability is the most common way to obtain a higher thermostability. Furthermore, they demonstrate that related series of mutants with only a small number of mutations can be successfully analyzed by CNA, which suggests that CNA can be applied prospectively in rational protein design aimed at higher thermodynamic thermostability.

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“…HodC-H251A protein, termed iHodC, which is virtually inactive due to its inability to initiate catalysis by deprotonating the organic substrate (Frerichs-Deeken et al, 2004), was used in control experiments. Purification of the recombinant His 6 -tagged HodC proteins from recombinant E. coli strains (Table 1) was performed as described by Beermann et al (2007). For storage at −80°C, 10% glycerol (vol/vol) was added to the protein stock solutions.…”

Section: Methodsmentioning

confidence: 99%

“…1 Unit of enzyme activity is defined as the amount of HodC catalyzing the conversion of 1 μmol substrate per minute under the conditions of the assay. Concentrations of HodC were determined by absorption measurements using an extinction coefficient (ε 280nm ) of 1.937 ml mg −1 cm −1 (Beermann et al, 2007). …”

Section: Methodsmentioning

confidence: 99%

Enzyme-Mediated Quenching of the Pseudomonas Quinolone Signal (PQS) Promotes Biofilm Formation of Pseudomonas aeruginosa by Increasing Iron Availability

et al. 2016

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The 2-alkyl-3-hydroxy-4(1H)-quinolone 2,4-dioxygenase HodC was previously described to cleave the Pseudomonas quinolone signal, PQS, which is exclusively used in the complex quorum sensing (QS) system of Pseudomonas aeruginosa, an opportunistic pathogen employing QS to regulate virulence and biofilm development. Degradation of PQS by exogenous addition of HodC to planktonic cells of P. aeruginosa attenuated production of virulence factors, and reduced virulence in planta. However, proteolytic cleavage reduced the efficacy of HodC. Here, we identified the secreted protease LasB of P. aeruginosa to be responsible for HodC degradation. In static biofilms of the P. aeruginosa PA14 lasB::Tn mutant, the catalytic activity of HodC led to an increase in viable biomass in newly formed but also in established biofilms, and reduced the expression of genes involved in iron metabolism and siderophore production, such as pvdS, pvdL, pvdA, and pvdQ. This is likely due to an increase in the levels of bioavailable iron by degradation of PQS, which is able to sequester iron from the surrounding environment. Thus, HodC, despite its ability to quench the production of virulence factors, is contraindicated for combating P. aeruginosa biofilms.

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Stability, Unfolding, and Structural Changes of Cofactor-Free 1H-3-Hydroxy-4-oxoquinaldine 2,4-Dioxygenase

Cited by 15 publications

References 14 publications

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

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

Structural Rigidity and Protein Thermostability in Variants of Lipase A from Bacillus subtilis

Enzyme-Mediated Quenching of the Pseudomonas Quinolone Signal (PQS) Promotes Biofilm Formation of Pseudomonas aeruginosa by Increasing Iron Availability

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