How widespread are NHases in eukaryotes?

There is a neat diagram summarizing the findings of the paper by Marron, Akram and Walker called "Nitrile Hydratase Genes Are Present in Multiple Eukaryotic Supergroups" in PLoS One to be found here in full. There is quite a lot of legend that goes with it that I recommend you look at the paper for, but crucially asterisk * means evidence of one eukaryotic NHase subunit (and hence ** means evidence of both), and hash # means a dodgy read more suggestive of prokaryotic contamination.

So that's a black mark for any NHase in plants and animals then...

NHase in harmful algal blooms

Last year I put up a blogpost about finding what looked like the alpha chain of a nitrile hydratase in the eukaryote Aurecococcus anophagefferens which is something that makes up brown tide algal blooms. The paper that introduces the genomic data which provided my hit back then is

Christopher Gobler, Dianna Berry, Sonya Dyhrman, and Steven Wilhelm. "Niche of harmful alga Aureococcus anophagefferens revealed through ecogenomics" Proceedings of the National Academy of Sciences of the USA 108.11 (2011): 4352-4357

This little beastie has a whole load of enzymes involved in degrading organic nitrogen containing compounds. The authors of the paper suggest that since it lives in an ecological niche which is typically low in organic nitrogen but high in organic nitrogen so having a suite of enzymes to harvest nitrogen from organics is a strategy to give a competitive advantage over other phytoplankton which don't have this ability. There is a supplemental figure which shows the list of these enzymes and whether competing species have them. A rough cut and paste below shows that it is really out on its own from this data.

This organism also seems to be a Guinness world record holder for the number of selenium containing proteins in its proteome (56). Interesting but not entirely relevant for this blog!

How have the database numbers changed in a year?

Last year I did a quick text search for "nitrile hydratase" as a search term under proteins on the NCBI website. This gave me 2869, of which 1042 were RefSeq data. Today when I checked there are 3573 (+25%), of which 1369 (+31%) were RefSeq.
No new PDB files have been deposited of nitrile hydratases since March 2011 which was the NHase from Pseudomonas putida.

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.