NHase numbers to May 2013, and now nitrilase numbers too

Looking at the bare search term "nitrile hydratase" amongst protein sequences (and remember, most aren’t but it’s a rough measure), today gives me 4782 hits (+ 14% since last November), of which 2440 (+ 50% since August) were RefSeq data. It appears that there has been a lot of RefSeq going on with this enzyme class.

There are 24848 sequences labelled as "nitrilase" (not sure how robust that is currently), of which 12037 are pegged as RefSeq data.

An example of gentle hydration using a nitrile hydratase

Using an extreme of pH to carve up a nitrile bond is going to be problematic when there are other, sensitive parts to a molecule. An example would be a nitrile appended to a ferrocene. D'Antona and coworkers are reporting the use of the nitrile hydratase from two Rhodococcus erythropolis strains in whole cell mode to convert nitrile to amide. [DOI:10.1039/C3RA22737E]. The presence of an amidase in the whole cell then coverts a minority through to the related carboxylic acid.

Slight pivot

I have been doing this blog for 2 years and just over 100 posts, and over that time I have focussed very narrowly on nitrile hydratases. Whilst they are lovely enzymes and I will continue to work on/with them, a growing interest of mine has become their cousins, the nitrilases. I now have more research effort going into nitrilases than nitrile hydratases, so from henceforth I intend to write about NHases and nitrilases. I don't think I am going to stretch to amidases, even if they are on the same operon (or whatever molecular biologists call it!). Just to add a bit of contemporary experimental data to the usual reports, I intend to report some interesting and as yet unreported results here (obviously without compromising my ability to publish them eventually) on an ongoing nitrilase discovery project as they happen.

What might happen after the nitrile hydratase has done its job in a whole cell system?

...You've got to work with the amidase which converts the amide to the corresponding carboxylic acid.
Laura Cantarella, Alberto Gallifuoco, Agata Spera and Maria Cantarella look at the amidase in the NHase/amidase system of Microbacterium imperiale CBS 498-74 in the paper entitled "Nitrile, amide and temperature effects on amidase-kinetics during acrylonitrile bioconversion by nitrile-hydratase/amidase in-situ cascade system" in the journal Bioresource Technology with DOI:10.1016/j.biortech.2013.04.126.

E. coli expression of active nitrile hydratase from Aurantimonas manganoxydans needs cobalt ions

This paper in Biotechnology Letters from Pei and co-workers gives details of a cobalt centred nitrile hydratase from Aurantimonas manganoxydans, and the importance of getting the concentration right for good level of expression and activity. (DOI 10.1007/s10529-013-1215-5)

Sequential Oxidations of Thiolates and the Cobalt Metallocenter in a Synthetic Metallopeptide: Implications for the Biosynthesis of Nitrile Hydratase

This is a paper in Inorganic Chemistry from the group of Anne K Jones looking at the use of a heptapeptide they call SODA (ACDLPCG) to model the oxidation process for the thiols and metal in the active site of cobalt centre NHases (DOI: 10.1021/ic400171z). First to oxidize is one of the thiolates to sulfinate, then the cobalt is oxidized to triply charged cobalt. Further sulphur oxidation is not observed and the complex is catalytically inactive. This complex does not have an axial thiolate ligand which may explain these last two obervations.

Identification of an active site bound Nitrile Hydratase Intermediate through Single Turnover Stopped-Flow Spectroscopy

This is a paper in the Journal of Biological Chemistry by Natalie Gumataotao, Misty L. Kuhn, Natalia Hajnas and Richard C. Holz (DOI:10.1074/jbc.M112.398909). Using stopped-flow methodology on the NHase from Rhodococcus equi TG328-2, the authors observe evidence of the first Fe3+-nitrile intermediate species ever reported, and hence show the direct ligation of nitrile to metal during catalytic turnover.

Improving stability of nitrile hydratase by bridging the salt-bridges in specific thermal-sensitive regions

This is a paper in Journal of Biotechnology by Jie Chen, Huimin Yu, Changchun Liu, Jie Liu and Zhongyao Shen (doi:10.1016/j.jbiotec.2013.01.021) on methodology to stabilize notoriously temperature sensitive nitrile hydratase. It involves the transfer of three regions which are prone to thermal deformation from thermophilic Bacillus SC-105-1 and Pseudonocardia thermophila JCM3095 into mesophilic Rhodococcus ruber TH.
Three types of salt bridges—active-center-adjacent (in A1), internal neighboring-residue-bridged (in A2) and C-terminal-residue-bridged (A3) were tried but only the type with A3 had increased efficacy at higher temperatures.

A Japanese patent for an "improved nitrile hydratase"

See this or this to see a Japanese patent awarded to Dia-Nitrix Co Ltd, F. Watanabe, T. Ambo and A. Hara, and published on 6th December 2012.

(It is in Japanese)

Self-Subunit Swapping Occurs in Another Gene Type of Cobalt Nitrile Hydratase

Liu Y, Cui W, Xia Y, Cui Y, Kobayashi M, et al. (2012) Self-Subunit Swapping Occurs in Another Gene Type of Cobalt Nitrile Hydratase. PLoS ONE 7(11): e50829. doi:10.1371/journal.pone.0050829.