Protein Engineering of Nitrile Hydratase Activity of Papain:  Molecular Dynamics Study of a Mutant and Wild-Type Enzyme

2002 ◽  
Vol 124 (44) ◽  
pp. 12979-12990 ◽  
Author(s):  
Swarnalatha Y. Reddy ◽  
Kalju Kahn ◽  
Ya-Jun Zheng ◽  
Thomas C. Bruice
Keyword(s):  
Molecular Dynamics ◽  
Protein Engineering ◽  
Nitrile Hydratase ◽  
Wild Type ◽  
Wild Type Enzyme ◽  
Hydratase Activity ◽  
Nitrile Hydratase Activity

scholarly journals Hydration change during the aging of phosphorylated human butyrylcholinesterase: importance of residues aspartate-70 and glutamate-197 in the water network as probed by hydrostatic and osmotic pressures

1999 ◽  
Vol 343 (2) ◽  
pp. 361-369 ◽  
Author(s):  
Patrick MASSON ◽  
Cécile CLÉRY ◽  
Patrice GUERRA ◽  
Arnaud REDSLOB ◽  
Christine ALBARET ◽  
...  
Keyword(s):  
Molecular Dynamics ◽  
Hydrostatic Pressure ◽  
Transition State ◽  
Osmotic Pressure ◽  
Active Site ◽  
Aging Process ◽  
Wild Type ◽  
Water Network ◽  
Wild Type Enzyme ◽  
Human Butyrylcholinesterase

Wild-type human butyrylcholinesterase (BuChE) and Glu-197 → Asp and Asp-70 → Gly mutants (E197D and D70G respectively) were inhibited by di-isopropyl phosphorofluoridate under standard conditions of pH, temperature and pressure. The effect of hydrostatic and osmotic pressures on the aging process (dealkylation of an isopropyl chain) of phosphorylated enzymes [di-isopropylated (DIP)-BuChE] was investigated. Hydrostatic pressure markedly increased the rate of aging of wild-type enzyme. The average activation volume (δV≠) for the dealkylation reaction was -170 ml/mol for DIP wild-type BuChE. On the other hand, hydrostatic pressure had little effect on the aging of the DIP mutants (δV≠ = -2.6 ml/mol for E197D and -2 ml/mol for D70G), suggesting that the transition state of the aging process was associated with an extended hydration and conformational change in wild-type BuChE, but not in the mutants. The rate of aging of wild-type and mutant enzymes decreased with osmotic pressure, allowing very large positive osmotic activation volumes (δV≠osm) to be estimated, thus probing the participation of water in the aging process. Molecular dynamics simulations performed on the active-site gorge of the wild-type DIP adduct showed that the isopropyl chain involved in aging was highly solvated, supporting the idea that water is important for stabilizing the transition state of the dealkylation reaction. Wild-type BuChE was inhibited by soman (pinacolyl methylphosphonofluoridate). Electrophoresis performed under high pressure [up to 2.5 kbar (1 bar = 105 Pa)] showed that the soman-aged enzyme did not pass through a pressure-induced, molten-globule transition, unlike the native wild-type enzyme. Likewise, this transition was not seen for the native E197D and D70G mutants, indicating that these mutants are resistant to the penetration of water into their structure. The stability energetics of native and soman-aged wild-type BuChE were determined by differential scanning calorimetry. The pH-dependence of the midpoint transition temperature of endotherms indicated that the high difference in stabilization energy between aged and native BuChE (δδG = 23.7 kJ/mol at pH 8.0) is mainly due to the salt bridge between protonated His-438 and PO-, with pKHis-438 = 8.3. A molecular dynamics simulation on the MIP adduct showed that there is no water molecule around the ion pair. The ‘hydrostatic versus osmotic pressure’ approach probed the importance of water in aging, and also revealed that Asp-70 and Glu-197 are the major residues controlling both the dynamics and the structural organization of the water/hydrogen-bond network in the active-site gorge of BuChE. In wild-type BuChE both residues function like valves, whereas in the mutant enzymes the water network is slack, and residues Gly-70 and Asp-197 function like check valves, i.e. forced penetration of water into the gorge is not easily achieved, thereby facilitating the release of water.

Synthesis of enantiomerically pure (S)-mandelic acid using an oxynitrilase–nitrilase bienzymatic cascade: a nitrilase surprisingly shows nitrile hydratase activity

2006 ◽  
Vol 17 (3) ◽  
pp. 320-323 ◽  
Author(s):  
Cesar Mateo ◽  
Andrzej Chmura ◽  
Sven Rustler ◽  
Fred van Rantwijk ◽  
Andreas Stolz ◽  
...  
Keyword(s):  
Mandelic Acid ◽  
Nitrile Hydratase ◽  
Enantiomerically Pure ◽  
Hydratase Activity ◽  
Nitrile Hydratase Activity

scholarly journals Nitrile hydratase activity of Nocardia corallina B-276

2005 ◽  
Vol 16 (6a) ◽  
Author(s):  
Herminia I. Pérez ◽  
Norberto Manjarrez ◽  
Héctor Luna ◽  
Aída Solís ◽  
Concepción Ramírez
Keyword(s):  
Nitrile Hydratase ◽  
Nocardia Corallina ◽  
Hydratase Activity ◽  
Nitrile Hydratase Activity

scholarly journals Modulation of the Activity and Regioselectivity of a Glycosidase: Development of a Convenient Tool for the Synthesis of Specific Disaccharides

Molecules ◽  
2021 ◽  
Vol 26 (18) ◽  
pp. 5445
Author(s):  
Yari Cabezas-Pérusse ◽  
Franck Daligault ◽  
Vincent Ferrières ◽  
Olivier Tasseau ◽  
Sylvain Tranchimand
Keyword(s):  
Molecular Dynamics ◽  
Molecular Modeling ◽  
Fold Increase ◽  
Building Blocks ◽  
Wild Type ◽  
Wild Type Enzyme ◽  
Modeling Methodology ◽  
Glycosidic Bonds ◽  
Predictive Quality ◽  
Good Agreement

The synthesis of disaccharides, particularly those containing hexofuranoside rings, requires a large number of steps by classical chemical means. The use of glycosidases can be an alternative to limit the number of steps, as they catalyze the formation of controlled glycosidic bonds starting from simple and easy to access building blocks; the main drawbacks are the yields, due to the balance between the hydrolysis and transglycosylation of these enzymes, and the enzyme-dependent regioselectivity. To improve the yield of the synthesis of β-d-galactofuranosyl-(1→X)-d-mannopyranosides catalyzed by an arabinofuranosidase, in this study we developed a strategy to mutate, then screen the catalyst, followed by a tailored molecular modeling methodology to rationalize the effects of the identified mutations. Two mutants with a 2.3 to 3.8-fold increase in transglycosylation yield were obtained, and in addition their accumulated regioisomer kinetic profiles were very different from the wild-type enzyme. Those differences were studied in silico by docking and molecular dynamics, and the methodology revealed a good predictive quality in regards with the regioisomer profiles, which is in good agreement with the experimental transglycosylation kinetics. So, by engineering CtAraf51, new biocatalysts were enabled to obtain the attractive central motif from the Leishmania lipophosphoglycan core with a higher yield and regioselectivity.

scholarly journals Engineering nitrile hydratase activity into a cysteine protease by a single mutation

Biochemistry ◽  
1995 ◽  
Vol 34 (50) ◽  
pp. 16382-16388 ◽  
Author(s):  
Eric Dufour ◽  
Andrew C. Storer ◽  
Robert Menard
Keyword(s):  
Cysteine Protease ◽  
Nitrile Hydratase ◽  
Single Mutation ◽  
Hydratase Activity ◽  
Nitrile Hydratase Activity

Immobilization of Rhodococcus ruber strain gt1, possessing nitrile hydratase activity, on carbon supports

2007 ◽  
Vol 43 (2) ◽  
pp. 173-177 ◽  
Author(s):  
A. Yu. Maksimov ◽  
Yu. G. Maksimova ◽  
M. V. Kuznetsova ◽  
V. F. Olontsev ◽  
V. A. Demakov
Keyword(s):  
Nitrile Hydratase ◽  
Rhodococcus Ruber ◽  
Carbon Supports ◽  
Hydratase Activity ◽  
Nitrile Hydratase Activity

Nitrile Hydratase Activity of a Recombinant Nitrilase

2006 ◽  
Vol 348 (18) ◽  
pp. 2597-2603 ◽  
Author(s):  
Bruno C. M. Fernandes ◽  
Cesar Mateo ◽  
Christoph Kiziak ◽  
Andrzej Chmura ◽  
Jan Wacker ◽  
...  
Keyword(s):  
Nitrile Hydratase ◽  
Hydratase Activity ◽  
Nitrile Hydratase Activity

scholarly journals Protein Engineering of the 4-Methyl-5-Nitrocatechol Monooxygenase from Burkholderia sp. Strain DNT for Enhanced Degradation of Nitroaromatics

2006 ◽  
Vol 72 (6) ◽  
pp. 3933-3939 ◽  
Author(s):  
Thammajun Leungsakul ◽  
Glenn R. Johnson ◽  
Thomas K. Wood
Keyword(s):  
Protein Engineering ◽  
Saturation Mutagenesis ◽  
Second Step ◽  
Amino Acid Residues ◽  
Wild Type ◽  
Wild Type Enzyme ◽  
Nitrite Removal ◽  
The Individual ◽  
Enhanced Degradation

ABSTRACT 4-Methyl-5-nitrocatechol (4M5NC) monooxygenase (DntB) from Burkholderia sp. strain DNT catalyzes the second step of 2,4-dinitrotoluene degradation by converting 4M5NC to 2-hydroxy-5-methylquinone with the concomitant removal of the nitro group. DntB is a flavoprotein that has a very narrow substrate range. Here, error-prone PCR was used to create variant DntB M22L/L380I, which accepts the two new substrates 4-nitrophenol (4NP) and 3-methyl-4-nitrophenol (3M4NP). At 300 μM of 4NP, the initial rate of the variant expressing M22L/L380I enzyme (39 � 6 nmol/min/mg protein) was 10-fold higher than that of the wild-type enzyme (4 � 2 nmol/min/mg protein). The values of k cat/Km of the purified wild-type DntB enzyme and purified variant M22L/L380I were 40 and 450 (s−1 M−1), respectively, which corroborates that the variant M22L/L380I enzyme has 11-fold-higher efficiency than the wild-type enzyme for 4NP degradation. In addition, the variant M22L/L380I enzyme has fourfold-higher activity toward 3M4NP; at 300 μM, the initial nitrite release rate of M22L/L380I enzyme was 17 � 4 nmol/min/mg protein, while that of the wild-type enzyme was 4.4 � 0.7 nmol/min/mg protein. Saturation mutagenesis was also used to further investigate the role of the individual amino acid residues at positions M22, L380, and M22/L380 simultaneously. Mutagenesis at the individual positions M22L and L380I did not show appreciable enhancement in 4NP activity, which suggested that these two sites should be mutated together; simultaneous saturation mutagenesis led to the identification of the variant M22S/L380V, with 20% enhanced degradation of 4NP compared to the variant M22L/L380I. This is the first report of protein engineering for nitrite removal by a flavoprotein.

scholarly journals Bioconversion of acrylonitrile using nitrile hydratase activity of Bacillus sp. APB-6

3 Biotech ◽  
2018 ◽  
Vol 8 (5) ◽  
Author(s):  
Rajendra Singh ◽  
Deepak Pandey ◽  
Shilpa Dhariwal ◽  
Priyanka Sood ◽  
Duni Chand
Keyword(s):  
Nitrile Hydratase ◽  
Bacillus Sp ◽  
Hydratase Activity ◽  
Nitrile Hydratase Activity

scholarly journals Exploration of the cofactor specificity of wild-type phosphite dehydrogenase and its mutant using molecular dynamics simulations

RSC Advances ◽  
2021 ◽  
Vol 11 (24) ◽  
pp. 14527-14533
Author(s):  
Kunlu Liu ◽  
Min Wang ◽  
Yubo Zhou ◽  
Hongxiang Wang ◽  
Yudong Liu ◽  
...  
Keyword(s):  
Molecular Dynamics ◽  
Molecular Dynamics Simulations ◽  
Wild Type ◽  
Cofactor Specificity ◽  
Phosphite Dehydrogenase ◽  
Dynamics Simulations

Phosphite dehydrogenase (Pdh) catalyzes the NAD-dependent oxidation of phosphite to phosphate with the formation of NADH.

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