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

JBIC Journal of Biological Inorganic Chemistry (2015), 20/5, 885-894

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.

3D content at http://proteopedia.org/w/Journal:JBIC:32

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

AGM Society for Industrial Microbiology & Biotechnology

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.

A switch in a substrate tunnel for directing regioselectivity of nitrile hydratases towards α,ω-dinitriles

A switch in a substrate tunnel for directing regioselectivity of nitrile hydratases towards α,ω-dinitriles

Zhongyi Cheng, Wenjing Cui, Zhongmei Liu, Li Zhou, Min Wang, Michihiko Kobayashi and Zhemin Zhou 

The β37 residue of nitrile hydratase (NHase) from Pseudomonas putida and NHase from Comamonas testosteroni played a critical role in directing enzyme regioselectivity. Amino acid substitution in this site modulated or even inverted enzyme regioselectivity towards aliphatic α,ω-dinitriles.

Cartoon model of the substrate access tunnel of (a) wild-type PpNHase and its (b) L37F and (c) L37Y variants, and (d) wild-type CtNHase and its (e) F37L and (f) F37P variants. The protein structures of PpNHase and CtNHase are shown as the grey cartoon. The β37 residues of NHases are shown as blue sticks. The purple balls and sticks represent the catalytic site of NHase. The bottleneck-forming amino acids are shown as red sticks. The tunnels are shown as green spheres, and the tunnel bottlenecks are coloured in yellow. All the figures share the same size proportion.

Rotation

The quick and simple pdb2MGIF tool is available again at http://www.glycosciences.de/modeling/pdb2mgif/

Here is the nitrile hydratase from Comamonas tetosteroni Ni1 (4FM4) in two different rotating animations. I have cut the PDB file down so it only shows a single A/B dimer.

Overlaying the NHases in 4FM4 and 2QDY

The iron centred NHase which I have most experience of is the Rhodococcus erythropolis AJ270. I have overlayed it onto the structure for the Comomonas testosteroni Ni1, and it is very similar as you might expect both at the secondary structure level (4FM4 in red, 2QDY in blue and yellow) and as an alignment. (The original paper makes in-depth analysis with 1AHJ from Rhodococcus sp. r312.)

Modelling of 4FM4

The structure of the NHase from Comamonas testosteroni is now available as a PDB file. I have been having a quick look at it.

And now a cartoon view with the metal binding motif highlighted

A molecular surface showing the entrance to the active site

3D structure of Wild Type Fe-type Nitrile Hydratase from Comamonas testosteroni Ni1

There is a new NHase PDB file available. With the code 4FM4, it refers to the NHase from the recent Holz paper.

Wild Type Fe-type Nitrile Hydratase from Comamonas testosteroni Ni1

The PDB from Comamonas testosteroni Ni1 is currently waiting (3/7.12) to be processed at the Protein Data Bank but will be called 4FM4.

New paper, new crystal structure

There is a new paper just released online in Biochemical and Biophysical Research Communications which is very interesting:

The Fe-Type Nitrile Hydratase from Comamonas testosteroni Ni1 Does Not Require an Activator Accessory Protein for Expression in Escherichia coli by Misty L. Kuhn, Salette Martinez, Natalie Gumataotao, Uwe Bornscheuer, Dali Liu  and Richard C. Holz.

http://dx.doi.org/10.1016/j.bbrc.2012.06.036

This paper reports something that has been something we have wondered about for a while… how important is that activator protein that is commonly cloned into E.coli clones alongside the DNA for the alpha and beta subunits. They show that one of the reasons for poor expression or activity for iron centred E. coli clones could well be down to codon bias. This isn’t obviously the full story because they find that whilst you don’t need the activator for the NHase from Comamonas (which they call CtNHase), you sure do for the one from Rhodococcus equi TG328-2.

The icing on the cake for this paper is that they have a crystal structure (though as of today 25/6.12 it isn’t on http://www.rcsb.org/) of CtNHase. This shows a slightly different arrangement of side chains in the active site but possibly more interestingly this active site isn’t at the end of a long dark tunnel but is relatively solvent-exposed, allowing easy direct access to the axial position on a bound iron. It only got assayed by the standard acrylonitrile assay but one wonders whether it would be rather less sensitive to steric crowding around the nitrile being hydrated than is usual with NHases.

PS This paper also has my favourite use of the word “recently” in a communication… to reference 12 from 2003.

Du Pont and NHases

One of the interesting things about the paper "Evidence for Participation of Remote Residues...etc" by Ondrechen and Ringe is that the source of the recombinant NHase is Du Pont. Du Pont have quite a history of applying this class of enzyme to chemical problems. A search of the chemical or patent literature for the names "Robert Di Cosimo" (sometimes DiCosimo on Espacenet), "Mark S Payne" and "Robert Fallon" pulls up a range of uses of NHases.

For instance, WO 2006049618 which protects the NHase and amidase from Comamonas testosteroni 5-MGMA-4D from both authors, or the 1997 paper in Applied Microbiology and Biotechnology from Fallon, Stieglitz and Turner called "A Pseudomonas putida capable of stereoselective hydrolysis of nitriles" which describes work on the strain NRRL-18668.