Search This Blog

Friday 22 April 2016

Active Site investigations on the Fe-centred NHase from Comamonas testosteroni Ni1

Analyzing the catalytic role of active site residues in the Fe-type nitrile hydratase from Comamonas testosteroni Ni1

Salette Martinez, Rui Wu, Karoline Krzywda, Veronika Opalka, Hei Chan, Dali Liu , Richard C. Holz


A strictly conserved active site arginine residue (αR157) and two histidine residues (αH80 and αH81) located near the active site of the Fe-type nitrile hydratase from Comamonas testosteroni Ni1 (CtNHase), were mutated. These mutant enzymes were examined for their ability to bind iron and hydrate acrylonitrile. For the αR157A mutant, the residual activity (k cat = 10 ± 2 s−1) accounts for less than 1 % of the wild-type activity (k cat = 1100 ± 30 s−1) while the K m value is nearly unchanged at 205 ± 10 mM. On the other hand, mutation of the active site pocket αH80 and αH81 residues to alanine resulted in enzymes with k cat values of 220 ± 40 and 77 ± 13 s−1, respectively, and K m values of 187 ± 11 and 179 ± 18 mM. The double mutant (αH80A/αH81A) was also prepared and provided an enzyme with a k cat value of 132 ± 3 s−1 and a K m value of 213 ± 61 mM. These data indicate that all three residues are catalytically important, but not essential. X-ray crystal structures of the αH80A/αH81A, αH80W/αH81W, and αR157A mutant CtNHase enzymes were solved to 2.0, 2.8, and 2.5 Å resolutions, respectively. In each mutant enzyme, hydrogen-bonding interactions crucial for the catalytic function of the αCys104-SOH ligand are disrupted. Disruption of these hydrogen bonding interactions likely alters the nucleophilicity of the sulfenic acid oxygen and the Lewis acidity of the active site Fe(III) ion.


BASF opens new bio-acrylamide plant in Bradford, UK

From


“BRADFORD, UK: BASF said it has opened a new world-scale bio-acrylamide (BioACM) production facility at its site in Bradford, a major investment that will help ensure long-term future of one of the UK’s largest chemical manufacturing facilities, which employs around 600 people.”

“The development of a biocatalytic manufacturing process for acrylamide started in Bradford with collaboration with Huddersfield University. Subsequent work by scientists from Britain, Germany, South Africa and US, made significant improvements to the performance of the biocatalyzed conversion technology.”


Image from BASF.com

Tuesday 19 April 2016

Creating bioactive molecules from online data

From a genome sequence to that protein in your hand is a well –established process


Until I started collaborating closely with biologists ten years ago, my impression of how easy it was to get a sample of a complex biomolecule like a protein was based on popular science stories describing days/months of tedious purification of buckets of biomass (sea sponges, exotic fungi, cow bile, etc) to end up with a single vial containing so little it was invisible to the naked eye.

Follow the link to see the schematic explained.


Monday 18 April 2016

A poster on Production of 2,6-difluorobenzamide using the NHase from Aurantimonas manganoxydans

Production of 2, 6- difluorobenzamide via whole-cell biocatalysis by nitrile hydratase from Aurantimonas manganoxydans

Lirong Yang

Nitrile hydratases (NHases) are enzymes which catalyze the hydration of nitriles, converting them into their corresponding amides. Amides are an important intermediates for pharmaceutical and pesticide industry. For example, 2, 6 – difluorobenzamide is used for the synthesis of fluorinated benzoyl urea pesticide.
Four NHase genes from Aurantimonas manganoxydans ATCC BAA-1229, Klebsiella oxytoca KCTC 1686, Pseudomonas putida NRRL-18668, Comamonas testosteroni 5-MGAM-4D were cloned and functionally expressed in Escherichia coli BL21 (DE3). All of the recombinant NHases can catalyze the hydration of 2, 6-difluorobenzonitrile to produce 2, 6-difluorobenzamide. Among them, the NHase from Aurantimonas manganoxydans ATCC BAA-1229 showed the highest activity.

Assisting soluble expression of recombinant and active Co-centred NHase

Chaperones-assisted soluble expression and maturation of recombinant Co-type nitrile hydratase in Escherichia coli to avoid the need for a low induction temperature
Xiaolin Pei, Qiuyan Wang, Lijun Meng, Jing Li, Zhengfen Yang, Xiaopu Yin, Lirong Yang, Shaoyun Chen and Jianping Wu
Journal of Biotechnology, Volume 203, 10 June 2015, Pages 9–16

Nitrile hydratase (NHase) is an important industrial enzyme that biosynthesizes high-value amides. However, most of NHases expressed in Escherichia coli easily aggregate to inactive inclusion bodies unless the induction temperature is reduced to approximately 20 °C. The NHase from Aurantimonas manganoxydans has been functionally expressed in E. coli, and exhibits considerable potential for the production of nicotinamide in industrial application. In this study, the effects of chaperones including GroEL/ES, Dnak/J-GrpE and trigger factor on the expression of the recombinant Co-type NHase were investigated. The results indicate that three chaperones can significantly promote the active expression of the recombinant NHase at 30 °C. The total NHase activities reached to 263 and 155 U/ml in shake flasks when the NHase was co-expressed with GroEL/ES and DnaK/J-GrpE, which were 52- and 31-fold higher than the observed activities without chaperones, respectively. This increase is possibly due to the soluble expression of the recombinant NHase assisted by molecular chaperones. Furthermore, GroEL/ES and DnaK/J-GrpE were determined to promote the maturation of the Co-type NHase in E. coli under the absence of the parental activator gene. These knowledge regarding the chaperones effect on the NHase expression are useful for understanding the biosynthesis of Co-type NHase.

NHase hydration of alicyclic 𝜶,𝝎-dinitrile 1-cyanocyclohexaneacetonitrile to 1-cyanocyclohexaneacetamide

Highly regioselective and efficient production of 1-cyanocyclohexaneacetamide by Rhodococcus aetherivorans ZJB1208 nitrile hydratase
Ren-Chao Zheng, Xin-Jian Yina and Yu-Guo Zheng
DOI 10.1002/jctb.4724

A newly isolated NHase producing strain, Rhodococcus aetherivorans ZJB1208, was successfully used for hydration of1-CCHAN. Some key parameters of the biocatalytic process, including reaction temperature, pH, catalyst loading and substrate loading, were optimized. The fed-batch biotransformation was performed in non-buffered water system with the continuous precipitation of 1-cyanocyclohexaneacetamide. The substrate loading was increased up to 864 g L−1 (6.0 mol L−1), giving a product concentration of 966.7 g L−1 and biocatalyst yield (g product/g cat) of 204.2.

Clues towards the role of amide carbonyl groups in the active site of a cobalt centred NHase

Role of the Amide Carbonyl Groups in the Nitrile Hydratase Active Site for Nitrile Coordination Using Co(III) Complex with N2S3-type Ligand
Takuma Yano,  Tomohiro Ikeda, Tomonori Shibayama,  Tomohiko Inomata, Yasuhiro Funahashi,2 Tomohiro Ozawa,* and Hideki Masuda*
The role of the amide carbonyl oxygens in the nitrile hydratase (NHase) active site for the nitrile coordination to the metal center was studied using a distorted square-pyramidal N2S3-type Co(III) complex at room temperature in the presence of a folded-sheet mesoporous material (FSM) or sodium cation.

Monday 4 April 2016

A proposed catalytic mechanism for NHase via a cyclic intermediate assessed by QM/MM

Catalytic Mechanism of Nitrile Hydratase Subsequent to Cyclic Intermediate Formation: A QM/MM Study
Megumi Kayanuma, Mitsuo Shoji, Masafumi Yohda, Masafumi Odaka, and Yasuteru Shigeta
J. Phys. Chem. B, Article ASAP
DOI: 10.1021/acs.jpcb.5b11363

The catalytic mechanism of an Fe-containing nitrile hydratase (NHase) subsequent to the formation of a cyclic intermediate was investigated using a hybrid quantum mechanics/molecular mechanics (QM/MM) method. We identified the following mechanism: (i) proton transfer from βTyr72 to the substrate via αSer113, and cleavage of the S–O bond of αCys114–SO and formation of a disulfide bond between αCys109 and αCys114; (ii) direct attack of a water molecule on the sulfur atom of αCys114, which resulted in the generation of both an imidic acid and a renewed sulfenic cysteine; and (iii) isomerization of the imidic acid to the amide. In addition, to clarify the role of βArg56K, which is one of the essential amino residues in the enzyme, we analyzed a βR56K mutant in which βArg56 was replaced by Lys. The results suggest that βArg56 is necessary for the formation of disulfide intermediate by stabilizing the cleavage of the S–O bond via a hydrogen bond with the oxygen atom of αCys114–SO.