Selectivity in nitrilases

One of the things we are interested in looking at with our increasingly wide panel of nitrilases is how substrate selectivity works with classic Group 1 nitrilases.
 Our standard panel of substrates we are assaying our nitrilases against has fifty substrates so we span quite a range of different structural types and traits. Here is an excerpt from our posters' table of data.

Colour coding mirrors the response that we see with our assay and carries handy semi-quantitative values as well which we get from UV/vis spectroscopy. What we have here is a table with different substrates along the top and different nitrilases down the side.
The top row here shows a nitrilase which hydrolyzes arylaliphatic substrates with great gusto (showing a 10 against 2-phenylacetonitrile) but has not a lot of interest in alkyl or aryl nitriles generally. The fifth row shows a nitrilase which is an enthusiast for aromatic nitriles peaking at para-substituted aromatics but with some interest in almost everything else!
To some extent the other patterns of activties are also interesting... we see some nitrilases which show activities which are active against only a few compounds which are quite structurally diverse. This may be a low expression level only letting us see the most active members of wider groupings (because this data table comes out of a screening programme of cell-free extracts), and this is something we will be investigating. It would be nice to hope that there was something a bit deeper going on though.
Finally we have some enzymes like one in the fourth line here which appear to be oblivious to everything. These show up a difference of response within our team. I, as the chemist, say "perhaps it just isn't active at all"; our molecular biologists say "nonsense- with that sequence it's just got to be a nitrilase, you haven't found the right substrate yet"!
We'll keep looking... for new nitrilases and new substrates.

Nitrile active enzyme posters

We are at Biotrans 2013 in Manchester this week. Whilst it has to be said that transaminases is the most frequently described enzyme type described here, there are a few posters describing nitrile active enzymes.
Nicola D'Antona has a poster entitled "Biotransformations of nitrile ferrocenes catalyzed by the bienzymatic system of nitrile hydratase/amidase: first examples of molecular recognition and enantioselectivity of chiral planar substrates". This poster describes the use of a couple of Rhodococcus species to hydrate and hydrolyze either one or two cyano groups appended to the double decker ferrocene sandwich. Considering NHases' dislike of bulky substrates that is a neat discovery. They even get some enantioselectivity especially in the acids where the amidase in the whole cell is strongly selective in determining the sterics of the outcome.
Birgit Wilding has a poster entitled "Enantioselective synthesis of a taxol side chain precursor with bacterial and fungal nitrilases". Obviously finding a biocatalytic way to build even a portion of a pharmaceutical is a great demonstration of using this technology to provide a green and inherently stereoselective route for the commercial arena. An interesting finding reported on this poster is the tendency of the bacterial nitrilases (two of which came from our Nzomics panel) in her panel to serve up not just the acid but the primary amide of her side chain precursors too. The ratio of amide to acid is sensitive to substrate concentration... Intriguing- I had one person previously complain about getting amide when they wanted acid with one of these enzymes but I don't know what their substrate was!
Cintia Milagre is displaying a poster called "Synthesis of alkyl and aryl substituted amides by nitrile hydratases" summarizing work on nitrile hydratase discovery from Brazilian soil samples and their utility at hydrating alpha substituted nitriles.
Carine Vergne-Vaxelaire is displaying a poster entitled "Biocatalytic tools for organic chemists: new nitrilases for synthesis of building blocks" which outlines the high throughput screening of 125 nitrilases against 25 structurally diverse nitriles, and is linked to a lecture being given later in the week by Anne Zaparucha.
Sander van Pelt has a poster called "Nitrile Hydratases: from genes to immobilized biocatalyst" highlighting the extra utility that a covalently linked enzyme aggregate preparation from CLEA Technologies has in terms of stability. It is nice to see that the NHases from AJ270 and CGA009 that we have worked on together are still useful examples in this field.
Finally hiding undercover at P387 is our poster on nitrilases. Graeme Turnbull has written up his semi-quantitative colorimetric methodology for assaying the activity of nitrilases as cell-free extracts (which screw up a pH driven colorimetric method) and then showing how he has applied it to a panel of 14 enzymes and 40 diverse substrates.

Poster at Biotrans2013

We'll be at Biotrans 2013 in Manchester next week showing a poster entitled "A novel, high-throughput screening method for group 1 nitrilase activity" which gives an outline of how we screen our nitrilases (as cell free extracts) colorimetrically against substrates on 96 well plates.

 

New mini-review on genome mining for nitrilases

In Applied Microbiology and Biotechnology's August 2013 issue, there is a mini review entitled "Metagenomic technology and genome mining: emerging areas for exploring novel nitrilases" from G-S Gong et al which will be relevant for nitrilase fanatics (DOI 10.1007/s00253-013-4932-8).

Animated GIF of 3IVZ

Using the useful PDB2MultiGIF page, this is a rotating image of the Pyroccocus abyssi GE5 PDB file 3IVZ.

What's in a name?

Despite the fact that the term "nitrilase" seems to have quite an obvious origin using the obvious enzyme naming template that a chemist might use (nitrilase = nitrile + ase... so must be something that acts on nitriles), there is a flaw in this. "Nitrilase" has actually been taken by biologists to mean a class of similar enzymes that attack molecules with C-N bonds. In fact, it is only Group 1 nitrilases actually do the "nitrile to carboxylic acid" conversion which we are currently working on, and the other groups which do other types of C-N bond breaking reactions. Table 1 in Charles Brenner's 2002 "Catalysis in the Nitrilase Superfamily" review in Current Opinions in Structural Opinions shows the breadth of other chemistries that appear in the nitrilase superfamily.This broad use of a name explains why there are so many PDB files listed for a search for "nitrilase" which do not actually do the nitrile-acid conversion as well as why there is great confusion between nitrile hydratase and nitrilase activities in many databases.

If you look through the 29 PDB files which are labelled as nitrilase, there is in fact only one enzyme which is a Group 1 nitrilase- and that is the nitrilase from the hyperthermophile Pyrococcus abyssi GE5 (this is an example of a PDB of this enzyme) which was described in a 2011 Journal of Structural Biology paper (doi: 10.1016/j.jsb.2010.11.017) by Rypniewski. As Martinkova has commented in her 2010 Current Opinions in Chemical Biology review (doi: 10.1016/j.cbpa.2009.11.018), this lack of crystal structures made structure–function relationship studies for nitrilases difficult.

How specific are nitrilases for their substrates?

As part of our ongoing nitrilase discovery project, we are trying a panel of about 50 substrates of various types against an ever-growing panel of nitrilases. The first round panel has 20 enzymes, and using a chromogenic assay we are seeing the rather nice selectivity that the literature indicates these enzymes show. Below, an example of two nitrilases (with each repeated) and their response to 4 simple nitriles- the depth of colour scales nicely with the amount of ammonia released by the nitrile hydrolysis.

There are similar levels of diversity of response for aryl over alkyl nitriles, and short chain over long chain nitriles. After the colour screening we are moving onto GCMS for confirmatory results, as well as doing a bit of NMR for that final structural evidence.