New paper on eukaryotic nitrile hydratases

One of the things that has refocussed me on NHases is the new paper in PLoS ONE on eukaryotic nitrile hydratases. It looks fascinating on a brief flick through.

Nitrile Hydratase Genes Are Present in Multiple Eukaryotic Supergroups by Alan O. Marron, Michael Akam and Giselle Walker in PLoS ONE here.

They have found more single subunit NHases in small eukaryotes, and discount their presence in anything bigger than a protist (like the ones in Ricinus communis) as prokaryotic contamination.

I'll be having a good perusal of this paper.

It's been a while...

I have been concentrating on my other research interests recently (anti-fouling surfaces, paint analysis and mass spectrometry as it happens) so I haven't updated this blog for a couple of months. I have had one student (Hello Luke!) working away on NHases, and he reckons that two of our NHases (including the one from Monosiga brevicollis) can monohydrate a substituted malonitrile.

S-M bond lengths

I was wondering what a "normal" bond length might be for a thiol to iron or cobalt might be, and then what might happen to that bond length might do if that bond was oxidized once and then twice. The best way to do this is to look at the small molecule crystallographic data so I got my friend Ross to comb through the usual database to find examples of the various bonding motifs.

It turns out that there are no examples of 1b in the CCD, and precious few of 1c (only 4), 2b (only 5) and 2c (a more respectable 15). There are loads of examples of 1d (actually 286) and 2d (102).
Anyway, there isnt much of a difference in average bond lengths between these types: 1c- 2.221 angstroms, 1d- 2.276 angstroms, 2b- 2.225 angstroms, 2c- 2.208 angstroms and 2d- 2.252 angstroms.
Here are a few histograms showing the distributions that lie behind these means.

Just Accepted NHase manuscripts

• Enantioselective biocatalytic hydrolysis of beta-aminonitriles to beta-amino-amides using Rhodococcus rhodochrous ATCC BAA-870, by Varsha Chhiba, Moira L. Bode, Kgama Mathiba, Wendy Kwezi, Dean Brady in Journal of Molecular Catalysis B: Enzymatic [doi:10.1016/j.molcatb.2011.12.005]

“A range of β-aminonitriles (3-amino-3-phenylpropanenitrile and derivatives) were synthesised by reaction of various benzonitriles with acetonitrile and subsequent reduction of the resulting acrylonitrile products. These compounds were hydrolysed to the corresponding amides using the nitrile biocatalytic activity of Rhodococccus rhodocrous ATCC BAA-870. Results showed that the nitrile hydratase enzyme was enantioselective for these compounds, in particular 3-amino-3-p-tolylpropanenitrile and 3-amino-3-(4-methoxyphenyl)propanenitrile and the corresponding amides (up to 85% in one case).” They found interesting levels of steric hindrance (probably) when the amino group was functionalized, and they had most success when the biotransformations were run at pH9 to minimize as much as possible protonation of the amine if it wasn’t derivatized. Obviously it being a Rhodococccus, this is an example of enantioselectivity with an iron-centred NHase.

• Biotransformation of the Neonicotinoid Insecticide Thiacloprid by Bacterium Variovorax boronicumulans Strain J1 and Mediation of the Major Metabolic Pathway by Nitrile Hydratase by Hui-Juan Zhang , Qian-Wen Zhou , Guang-Can Zhou , Yu-Min Cao , Yi-Jun Dai , Wei-Wei Ji , Guang-Dong Shang , and Sheng Yuan in Journal of Agricultural and Food Chemistry [DOI: 10.1021/jf203232u]

“A neonicotinoid insecticide thiacloprid-degrading bacterium strain J1 was isolated from soil and identified as Variovorax boronicumulans by 16S rRNA gene sequence analysis… A 2.6-kb gene cluster from V. boronicumulans J1 that includes the full length of the nitrile hydratase gene was cloned and investigated by degenerate primer polymerase chain reaction (PCR) and inverse PCR. The nitrile hydratase gene has a length of 1304 bp and codes a cobalt-type nitrile hydratase with an alpha-subunit of 213 amino acids and a beta-subunit of 221 amino acids.” This protein was then expressed in an active form in E. coli BL21. This bacterium is a boron-accumulating microbe isolated from soil.

Aligning cobalt centre NHases

I have been using the FATCAT protocol to look at structural alignments of differing cobalt centred NHases. This is available as a tool here.
Here is a picture of the alpha chains of 1V29 and 1 IRE aligned.

And here is a picture of the alignment of the beta chains. A bit more variation there.

A new review: Industrial Biotechnology- the future of green chemistry

The most recent issue of Green Chemistry has a 41 page review (DOI: 10.1039/C1GC15579B) by Udo Kragl and his co-workers Stefanie Wenda, Sabine Illner and Annett Mell, entitled  "Industrial Biotechnology- the future for green chemistry" which is a useful overview of where biocatalysis is now. A nice feature it has that I havent seen before is the use of little boxes which are labelled "critical remarks" to discuss highlight problems sometimes in the perception and sometimes in the reality of using biocatalytic process industrially.
As is necessary in this sort of review, there is a discussion (p. 3011) on how good nitrile hydratase is for bulk synthesis of acrylamide though it is interesting to see the scale of the use is "more than 50,000 tons per year" referring back to a viewpoint paper in ChemCatChem authored by Yuryev and Liese which actually says the Mitsubishi Rayon process "runs on scales up to 50,000 tons per year"- whatever that actually means! So it would appear that my suspicion that no one actually knows how successful (in terms of level of adoption) one of the most successfully implemented biocatalytic processes is, has not been contradicted!
Directly following the discussion of acrylamide manufacture is a discussion comparing the chemical and the Lonza chemoenzymatic routes to nicotinamide, with some excellent leading references.

Polymers and nitrile hydratase activity

There have been a few papers over the years looking at the possibility that nitrile-active enzymes might be able to attack nitrile groups on the surface of nitrile-containing polymers such as polyacrylonitrile.  An early example of this is the paper by Gübitz and co-workers [Nitrile Hydratase and Amidase from Rhodococcus rhodochrous Hydrolyze Acrylic Fibers and Granular Polyacrylonitriles, from Appl Environ Microbiol (2000)] which uses a cell free extract from Rhodococcus rhodochrous NCIMB 11216 to create pendant carboxylate groups on the fibres.

More recently there has been another paper looking at this topic using a different cell-free extract

Vikash Babu, Bijan Choudhury, Competitive adsorptions of nitrile hydratase and amidase on polyacrylonitrile and its effect on surface modification, Colloids and Surfaces B: Biointerfaces, Volume 89, 1 January 2012, Pages 277-282, 10.1016/j.colsurfb.2011.08.025.

This uses an extract from Amycolatopsis, and they do show conversion of the surface to carboxylate, by functionally tracking a NHase activity and an amidase activity. I am not sure how they know there isnt a nitrilase in there helping along too (NCBI records currently an Amycolatopsis species nitrilase).

In the light of the congested active site entrance which is generally found with NHase, it is quite interesting this works.