Nitrile hydration is exothermic

I spotted a reference to the fact that the hydration of a nitrile is an exothermic process, and as such at scale needs some form of refrigeration. Obviously this is an issue in biocatalytic processes where your enzyme might be thermal sensitive but is also true of the plants which operate the copper based chemical process (like the one I was shown round in Bradford then run by Ciba- thanks Yvonne!).

I used Hyperchem running a semiempirical PM3 method to model the process of conversion of acrylonitrile, benzonitrile and nicotinonitrile to the relevant amide and they all give an exotherm of about 20kcal per mole. I repeated one using a moderately intensive DFT expt and it agreed with this figure.

Castor oil plant nitrile hydratases

Another of the projects from JCVI is the castor oil plant (Ricinus communis) genome. The auto-annotation has picked up two alpha subunits of nitrile hydratase, and there is at least one thing that looks like a beta in there too.

Nitrile hydratases and the Gordon and Betty Moore Foundation’s Marine Microbiology Initiative

Since the most exciting new source of nitrile hydratase sequences has been marine organisms recently, I thought I would have a quick look at the preponderance of nitrile hydratase sequences in marine bacteria. I decided to look at the Moore Foundation's Marine Microbiology Initiative which was run by JCVI. I used the alpha sequence from the nitrile hydratase from Rhodopseudomonas palustris CGA009 as a probe sequence and BLASTped this sequence against the genomes produced by this initiative. This was a slightly more tedious job than I imagined because the BLAST server at JCVI wasn't working, so I had to work through a NCBI BLASTp input, which involves a fair bit of cutting and pasting, AND the taxonomy of the names listed isn't always the same in both databases. Anyway, of the 177 marine microbes which had their genomes listed, 30 produced sequences which had query coverages of greater than 68%, and all were auto-annotated as nitrile hydratase alpha subunits and contained the appropriate metal binding sequence.

A predicted nitrile hydratase from a psychrophilic bacterium

I have been scanning the sequences, and by chance I came across the fact that there is a predicted nitrile hydratase from a bacterium called Octadecabacter antarcticus strain 307 which is a marine organism found at the poles. From the looks of the primary amino acid sequence of the alpha subunit (gb|EDY76981.1|), it is a cobalt-centred NHase.

Automated Protein Structure prediction... or probably not.

I was browsing through a bioinformatics textbook last week, and read up about the work of Prof  David Baker in the field of protein structure prediction. One of the resources that the Baker Lab offer for free to academic customers is access to their Rosetta methodology via their Robetta structure prediction software hosted on their own computers. It is a really easy to use resource with minimal hassle in terms of registration. Anyway I thought it might be interesting to see what an automated program might be able to make of the single protein NHase of Monosiga brevicollis which is made up of the standard two separate alpha and beta subunit proteins tied together, both of which are well known from 41 (and counting) fairly similar PDB files which have a high degree of similarity. First of all, you submit your FASTA sequence, and it parses it using the Ginzu domain prediction technique.

I was pretty pleased with this start as it spotted the two domains by BLAST, and match them to two PDB files of cobalt containing NHases. I then set it away to do the full structure prediction thinking that knowing those two starting points, most of the heavy lifting in terms of the structure prediction had been done. The prediction said it would take seven days but actually was finished after two (not a busy weekend for their computers it would appear!). Its output is the five best structures...

I have to say I have no confidence in any of those structures... all look a bit long and dangly for me. If you contrast them with a couple of PDB structures of NHases of the usual type below you can see that they tend to be much more compact- look where the two subunits' beta sheets are in the PDBs and in the prediction.

I guess it was asking a lot to get a reasonable guess at the histidine-heavy chain in the middle of the Monosiga NHase, and that is a crucial part of this prediction. No need to welcome our robot overlords just yet, and I need to go and talk to a human homology modeller!

NCBI numbers for July

The usual text search for "nitrile hydratase" under proteins on NCBI's website shows 3003 hits for the phrase (+134 since May), and 1105 RefSeq hits (+63 in the same period).

No change in the number of structures listed on the Protein Data Bank.

Comparing the access of the active site of 3QXE and 3HHT

If you orient the pdb file of the Geobacillus which is coded as 3HHT as I have done for 3QXE and 2QDY in the previous posts, you get the following two images.

The cobalt is no longer visible. However if you twirl the structure around you can find a view of it through a different aperture. I wonder how this impacts on the selectivity of this enzyme.

Looking inside for the active site of 3QXE

This is just a couple of rough pictures of 3QXE (the nitrile hydratase from Pseudomonas putida), once again from PyMol, showing a ribbon view of it with the active site cobalt-binding residues shown as CPK spheres, followed by a surface model with some of the active site just visible inside. I have been careful to keep it oriented similarly to the AJ270 nitrile hydratase in the previous post.

Looking for the iron in AJ270

 Using PyMol I have been looking to see what the tunnel which connects the iron centre to the outside world looks like. Here are some pictures. Iron is represented as a warmpink sphere, and the pdb structure used is 2QDY.

Or close up as...