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

Sol-gel encapsulation of Cobalt centred nitrile hydratase

The nitrile hydratase from Pseudonocardia thermophila is one of the most stable NHases currently described. It is cobalt centred, and one strain of it has an entry in the PDB. Holz and co-workers have just published a paper entitled "Acrylamide Production using Encapsulated Nitrile Hydratase from Pseudonocardia thermophila in a Sol-gel matrix" in the Journal of Molecular Catalysis A. They show that their system is able to convert acrylonitrile to acrylamide neatly, demonstrating the advantages of their enzyme support system in terms of increasing the robustness of these still rather sensitive enzymes. Interesting to me is that their orthosilicate sol-gels boost this enzyme's stability in methanol to take as much as a70% v/v mix happily.

Identification of an active site bound Nitrile Hydratase Intermediate through Single Turnover Stopped-Flow Spectroscopy

This is a paper in the Journal of Biological Chemistry by Natalie Gumataotao, Misty L. Kuhn, Natalia Hajnas and Richard C. Holz (DOI:10.1074/jbc.M112.398909). Using stopped-flow methodology on the NHase from Rhodococcus equi TG328-2, the authors observe evidence of the first Fe3+-nitrile intermediate species ever reported, and hence show the direct ligation of nitrile to metal during catalytic turnover.

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.

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.