Nitrile hydratase in March's Advanced Organic Chemistry

As a graduate student, March's Advanced Organic Chemistry was my "go-to" text for leading references for synthetic chemistry questions. It was an incredibly thick book with an extraordinary number of references, and it has only increased in size since my days in TCD, and it is now in its seventh edition.
As my indexing system these days needs more key strokes to navigate than is needed to find things on the Web using Google, I decided to find my first paper on nitrile hydratase online to see which substrates I should have in my "interesting nitriles" cupboard which were ortho-substituted. Sure enough the paper was there but so also was a link to that paper as a reference in March. Yes, it's reference 103 on page 1080 of the seventh edition, as a reference to consult about amide formation though the reason for its inclusion is a bit weird. Anyway, I am very pleased!

Making an approximate 3D model of an enzyme

Sometimes we want to check out how an amino acid sequence from a database might translate into a 3D dimensional structure. This is straightforward enough if there exists an x-ray crystal structure for that exact enzyme in the Protein Data Bank or, like often happens in the nitrile hydratase class, there is very limited variation within sequences so overlaying a short portion of the sequence "by eye" on an existing enzyme is possible.
We are currently working on nitrilases, and despite increasing interest in their use as biocatalysts, the number of nitrilases (i.e. enzymes that convert nitriles to carboxylic acids not the other looser biochemical definition) which exist as structures in the PDB s precisely one. It is from Pyrococcus abyssi and is pretty much restricted in substrate tolerance to small aliphatic nitriles like fumaronitrile. It should not be a surprise that it isnt a great model for many other nitrilases.
If the PDB has come up short we tend to use a link out of the Uniprot database to give a prediction of 3D structure. So if you want to see an estimate of what the nitrilase from Aurantimonas manganoxydans looks like, than the link labelled "ModBase" under the subtitle "3D structure databases" leads you to a page of predictions and further tools. For this specific enzyme it is suggested that a mouse nitrilase superfamily structure contains the best 3D match.

A nitrile hydratase for cyanopyridines... and it's a bit more stable than usual.

There is a paper in Process Biochemistry which describes a new NHase from Aurantimonas manganoxydans which shows improved stability than you can normally expect from a NHase. It is entitled “Efficient cloning and expression of a thermostable nitrile hydratase in Escherichia coli using an auto-induction fed-batch strategy”, and it is by Xiaolin Peia, Hongyu Zhang, Lijun Meng, Gang Xu, Lirong Yang and Jianping Wu. This NHase is four times more rapid at converting 3-cyanopyridine to its corresponding amide as valeronitrile, and the authors emphasize their enzyme's stability though in the world of NHases where nothing is what you might describe as thermophilic, please don't get too expectant! They have a great table of NHase thermostability which I reproduce with their enzyme's data inserted.

 

A nitrile hydratase for cyanopyridines... (but it prefers aliphatic nitriles)

There has been a recent paper in Journal of Molecular Catalysis B on a nitrilase that converted cyanopyridines. This enzyme came from a strain of Pseudomonas putida. There is also a recent paper in the same journal entitled “Discovery of a new Fe-type nitrile hydratase efficiently hydrating aliphatic and aromatic nitriles by genome mining” by Xiaolin Peia, Lirong Yang, Gang Xu, Qiuyan Wang and Jianping Wu.which describes a nitrile hydratase, this time, from a Pseudomonas putida strain (F1) which they have shown to be able to turn over 3-cyanopyridine in a 1L fed batch reactor. Their activity data suggests it actually prefers aliphatic nitriles: acrylonitrile rates as 941 U/mg and valeronitrile as 535 U/mg as compared to 3-cyanopyridine at 26 U/mg. They describe how it was cloned into E. coli, needing the inclusion of an activator protein to get activity. They also include a nice phylogenetic tree showing the spread of known iron type NHases... plenty of examples in the Rhodococcus but spreading outwards into Pseudomonas.

Scaling up a nitrilase based hydrolysis

Efficient Production of (R)-o-Chloromandelic Acid by Recombinant Escherichia coli Cells Harboring Nitrilase from Burkholderia cenocepacia J2315 by Dongzhi Wei and co-workers is published in Organic Process Research and Development at  DOI:10.1021/op400174a.  It being OPRD and from a group working in a Laboratory of Bioreactor Engineering, it’s going to be an interesting read on doing a nitrilase reaction on the larger scale.  We are currently working on scaling up a different nitrilase-based reaction currently from bench scale to the “too heavy to use normal glassware” scale so it’s good to see how others do it.

 

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A nitrilase for cyanopyridines...

Characterization and functional cloning of an aromatic nitrilase from Pseudomonas putida CGMCC3830 with high conversion efficiency toward cyanopyridine by Hong Xu and co-workers in Journal of Molecular Catalysis B: Enzymatic is the first report of cloning of an aromatic nitrilase from Pseudomonas genus. It has a particular penchant for 3-cyanopyridine.

Zaparucha nitrilase activities

One of the joys of the Zaparucha nitrilase paper recently published in Advanced Synthesis and Catalysis is that their search for nitrilase activity is not confined to those enzymes which have already been automated annotated as "nitrilase". Within their enzyme set are proteins annotated as amidases, cyanide hydrolases, ureidopropionases, hydrolases (nice and broad that!) and even glycosyl. Here is my analysis of their protein types done by text searching their supplementary materials.

Text search strings are across the top and obviously "nitrilase" will also find those in "nitrilase/cyanide", and "hydrolase" occurs all over the shop. The second line indicates the totals after my removal of those proteins which didnt show any nitrilase activity for them. (You can take the chemist stance "something wrong with the protein" or the molecular biologists stance "you havent found the right substrate yet" at your own whim!). A very interesting set of annotations!