Flexibility of cold- and heat-adapted subtilisin-like serine proteinases evaluated with fluorescence quenching and molecular dynamics

Sigtryggsdóttir, Ásta Rós; Papaleo, Elena; Thorbjarnardóttir, Sigrídur H.; Kristjánsson, Magnús M.

doi:10.1016/j.bbapap.2014.02.009

Cited by 18 publications

(6 citation statements)

References 49 publications

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“…Typically, the fluorescence quenching constants (as reported by the Stern-Volmer constant) of psychrophilic enzymes are higher than for mesophilic proteins at both low and warm temperatures, thus indicating a more permeable structure (Huston et al, 2008 ; Tang et al, 2012 ), and the variation of fluorescence quenching (i.e., the change in the Stern-Volmer constant) within a temperature range where the native state prevails decreases in the order psychrophilic > mesophilic > thermophilic (D'Amico et al, 2003a ; Georlette et al, 2003 , 2004 ; Cipolla et al, 2012 ), thus indicating that cold-adapted enzymes possess higher flexibility. These experiments can also be combined with mutational analysis to explore the interplay between sequence variation, protein flexibility, and catalytic activity (Cipolla et al, 2011 ; Sigtryggsdóttir et al, 2014 ; Truongvan et al, 2016 ).…”

Section: Evolutionary and Molecular Mechanisms Of The Cold-adaptationmentioning

confidence: 99%

Discovery, Molecular Mechanisms, and Industrial Applications of Cold-Active Enzymes

et al. 2016

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Cold-active enzymes constitute an attractive resource for biotechnological applications. Their high catalytic activity at temperatures below 25 • C makes them excellent biocatalysts that eliminate the need of heating processes hampering the quality, sustainability, and cost-effectiveness of industrial production. Here we provide a review of the isolation and characterization of novel cold-active enzymes from microorganisms inhabiting different environments, including a revision of the latest techniques that have been used for accomplishing these paramount tasks. We address the progress made in the overexpression and purification of cold-adapted enzymes, the evolutionary and molecular basis of their high activity at low temperatures and the experimental and computational techniques used for their identification, along with protein engineering endeavors based on these observations to improve some of the properties of cold-adapted enzymes to better suit specific applications. We finally focus on examples of the evaluation of their potential use as biocatalysts under conditions that reproduce the challenges imposed by the use of solvents and additives in industrial processes and of the successful use of cold-adapted enzymes in biotechnological and industrial applications.

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Section: Evolutionary and Molecular Mechanisms Of The Cold-adaptationmentioning

confidence: 99%

Discovery, Molecular Mechanisms, and Industrial Applications of Cold-Active Enzymes

et al. 2016

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“…11,13,14 Cold-adaptation mechanisms are likely to vary across different enzyme families. 8,15 Serine proteases or esterases contain a catalytic triad consisting of Ser, His, and Asp residues, whereas other residues in the active site serve to support the structure of the active site by providing correct conformation. 16 Amino acid sequence alignment has shown that the aromatic amino acid residue Trp is highly conserved in the active site wall of cold-adapted esterases including EstK (Pseudomonas mandelii) 17,18 and rPPE (Pseudomonas putida), 19 whereas Tyr is preferred in the corresponding position of hyperthermophilic esterases such as EstE1 (thermal environmental sample), 20 Est2 (Alicyclobacillus acidocaldarius), 21 and AFEST (Archaeoglobus f ulgidus) 22 (Figure 1a).…”

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confidence: 99%

Flexibility and Stability Trade-Off in Active Site of Cold-Adapted Pseudomonas mandelii Esterase EstK

2016

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Cold-adapted enzymes exhibit enhanced conformational flexibility, especially in their active sites, as compared with their warmer-temperature counterparts. However, the mechanism by which cold-adapted enzymes maintain their active site stability is largely unknown. In this study, we investigated the role of conserved D308-Y309 residues located in the same loop as the catalytic H307 residue in the cold-adapted esterase EstK from Pseudomonas mandelii. Mutation of D308 and/or Y309 to Ala or deletion resulted in increased conformational flexibility. Particularly, the D308A or Y309A mutant showed enhanced substrate affinity and catalytic rate, as compared with wild-type EstK, via enlargement of the active site. However, all mutant EstK enzymes exhibited reduced thermal stability. The effect of mutation was greater for D308 than Y309. These results indicate that D308 is not preferable for substrate selection and catalytic activity, whereas hydrogen bond formation involving D308 is critical for active site stabilization. Taken together, conformation of the EstK active site is constrained via flexibility-stability trade-off for enzyme catalysis and thermal stability. Our study provides further insights into active site stabilization of cold-adapted enzymes.

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“…Whilst, enzymes from thermophiles seems to be more rigid, conformationally stable protein structures, which are reflected in lower catalytic activity at lower temperatures. However, the relationship between catalytic activity, thermal stability and molecular flexibility is vastly complex and the fundamental principles are still poorly understood [25].…”

Section: Resultsmentioning

confidence: 99%

Combining Docking and Molecular Dynamic of Protease from Bacillus lehensis G1

Sulaiman¹

2018

JESR

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Protease is an enzyme that catalysed the hydrolysis of protein into peptide. Application of protease in industry has been linked with cost effective substrates and complex of enzyme-substrate stability. Molecular docking approach has identified casein as a preference substrates. However, lack of data on casein mode of binding to protease and enzyme stability represents a limitation for its production and structural optimization. In this study, we have used a molecular dynamic (MD) to examine the stability of complex enzyme-substrate of protease from Bacillus lehensis G1. The 3D structure of protease (BleG1_1979) was docked with substrate casein using AutoDock Vina. Structural analysis of the substrate-binding cleft revealed a binding site of casein was predominantly at the hydrophobic region of BleG1_1979. The MD of complex BleG1_1979-casein was tested with two temperatures; 298 K and 310 K using GROMACS v5.1.4. MD simulation showed a stable behaviour of BleG1_1979 over the 20 ns simulation period. The molecular docking and MD simulation suggested that the production of protease from B. lehensis G1 by utilization of casein and the stability of complex protease-casein could be a potential application to generate a cost effective enzyme to be develop for industrial use

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Flexibility of cold- and heat-adapted subtilisin-like serine proteinases evaluated with fluorescence quenching and molecular dynamics

Cited by 18 publications

References 49 publications

Discovery, Molecular Mechanisms, and Industrial Applications of Cold-Active Enzymes

Discovery, Molecular Mechanisms, and Industrial Applications of Cold-Active Enzymes

Flexibility and Stability Trade-Off in Active Site of Cold-Adapted Pseudomonas mandelii Esterase EstK

Combining Docking and Molecular Dynamic of Protease from Bacillus lehensis G1

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