New papers for August

There are two new papers, both on Rhodococcus rhodochrous NHases, out there:

• Cloning, Sequencing, and Expression of Nitrile Hydratase Gene of Mutant 4D Strain of Rhodococcus rhodochrous PA 34 in E. coli, by Amit Pratush, Amit Seth and T. C. Bhalla in Applied Biochemistry and Biotechnology, DOI: 10.1007/s12010-012-9790-9.

A clone carrying a new NHase which is cobalt centred and very similar to Rhodococcus rhodochrous J1 is reported.

• Effect of growth media on cell envelope composition and nitrile hydratase stability in Rhodococcus rhodochrous strain DAP 96253, by Trudy-Ann Tucker, Sidney A. Crow Jr. and George E. Pierce in Journal of Industrial Microbiology & Biotechnology, DOI: 10.1007/s10295-012-1168-z

This looks at how being in a Rhodococcus species might impact on the activity of the NHase enzyme on board.  They show that altering sugars in the growth medium affects the cell envelope components and impacts on NHase activity. I had heard this sort of thing can affect the activity of NHases in other prokaryotes too. In fact, I know someone who improved their enzyme activity by accidentally leaving something going overnight…

No extremes

I am reading a very interesting general microbiology book at the moment called "Discover the World of Microbes" by Gerhard Gottschalk.

In the chapter on extremophiles it mentions two amazing microbes, Picrophilus torridus and Natronomonas pharaonis. The first is perfectly happy to grow at pH 0 and 65 degrees celsius, and the second originates from soda lakes where high salinity and a pH 11 are part of the ambient conditions. A quick fossick through their genomes indicates to me that they do not have anything approaching a nitrile hydratase about their genome, sadly. I suspect that in conditions with such extremes of low pH, nitriles aren't commonly encountered, which would remove the reason for something with a streamlined genome to retain related enzyme activities. In our OBC paper on nitrile hydratases, we did assay one from Nitriliruptor alkaphilus which came from a soda lake, and there are other genomic hints of alkaline-tolerant nitrile hydratase containing organisms. Perhaps that is the only type of extremophilic nitrile hydratase we can expect to find?

Desymmetrizing dinitrile substrates

Desymmetrization of dinitrile substrates using nitrile-active enzymes like NHases to get a chiral compound has been known about for ages, (Turner and Sugai & Ohta published early papers on this in 1993).

A nice later example of this is a biocatalytic synthesis of a (S)-methylDOPA precursor by Sugai and Ohta again:-

Realization of the synthesis of a,a-disubstituted carbamylacetates and cyanoacetates by either enzymatic or chemical functional group transformation, depending upon the substrate specificity of Rhodococcus amidase by Masahiro Yokoyama, Mieko Kashiwagi, Masakazu Iwasaki, Ken-ichi Fuhshuku, Hiromichi Ohta and Takeshi Sugai in Tetrahedron Asymmetry doi:10.1016/j.tetasy.2004.04.047

In the recent book “Practical Methods for Biocatalysis and Biotransformations” edited by Whittall and Sutton, there is a section on desymmetrization using nitrile active enzymes (though two are whole cell methods and the last is use of a Codexis nitrilase) written by Wijdeven, Kielbasinski and Rutjes (Section 5.5, p186-189).

NHase reviews in recent books

I have recently come across two book chapters which would be of interest to those interested in nitrile hydratase (and indeed nitrilases) enzymes.

Biocatalysis for the Pharmaceutical Industry: Discovery, Development, and Manufacturing edited by Junhua (Alex) Tao, Guo-Qiang Lin and Andreas Liese has a chapter on “Applications of Nitrile Hydratases and Nitrilases” written by Grace DeSantis and Robert DiCosimo.

Biology of Rhodococcus edited by Héctor Alvarez, whilst sounding a touch unpromising has a chapter co-written by Ludmila Martınkova entitled “Catabolism of Nitriles in Rhodococcus” which runs through a lot of the synthetic potential that the NHases from this type of prokaryote possess in detail I haven’t seen altogether in one place before.

Wild Type Fe-type Nitrile Hydratase from Comamonas testosteroni Ni1

The PDB from Comamonas testosteroni Ni1 is currently waiting (3/7.12) to be processed at the Protein Data Bank but will be called 4FM4.

Connecting B to A

Looking back on the bioinformatic analysis of eukaryotic nitrile hydratases in Marron, Akam and Walker's PLOS One paper to be found here, I thought it would be interesting to look at the link between the two segments which are separate subunits in prokaryotic NHases.
I mocked up this model of where the link had to go from two prokaryotic subunits which had most similarity to the Monosiga brevicollis NHase. The dangling ends of the right hand side of the structure indicate where the linker has to go.

As the easiest to access, I chose to align the protein sequences suggested by the transcriptions of ESTs from the four organisms held within the Broad Institute's Origins of Multicellularity project, namely M. brevicollis, Thecamonas trahens, Salpingoeca rosetta and Sphaeroforma arctica. (NB order in alignment: MB, SR, TT and last SA)

As Marron's paper comments:
"This histidine-rich region is prominent in T. trahens (12 residues), S. arctica (11 residues) and M. brevicollis (17 residues), but shorter in [...] S. rosetta (2 residues)."
That seems quite a lot of variation with only M. brevicollis having a string of pure histidines. In contrast the sequences are around the metal binding segment are quite similar.

New paper, new crystal structure

There is a new paper just released online in Biochemical and Biophysical Research Communications which is very interesting:

The Fe-Type Nitrile Hydratase from Comamonas testosteroni Ni1 Does Not Require an Activator Accessory Protein for Expression in Escherichia coli by Misty L. Kuhn, Salette Martinez, Natalie Gumataotao, Uwe Bornscheuer, Dali Liu  and Richard C. Holz.

http://dx.doi.org/10.1016/j.bbrc.2012.06.036

This paper reports something that has been something we have wondered about for a while… how important is that activator protein that is commonly cloned into E.coli clones alongside the DNA for the alpha and beta subunits. They show that one of the reasons for poor expression or activity for iron centred E. coli clones could well be down to codon bias. This isn’t obviously the full story because they find that whilst you don’t need the activator for the NHase from Comamonas (which they call CtNHase), you sure do for the one from Rhodococcus equi TG328-2.

The icing on the cake for this paper is that they have a crystal structure (though as of today 25/6.12 it isn’t on http://www.rcsb.org/) of CtNHase. This shows a slightly different arrangement of side chains in the active site but possibly more interestingly this active site isn’t at the end of a long dark tunnel but is relatively solvent-exposed, allowing easy direct access to the axial position on a bound iron. It only got assayed by the standard acrylonitrile assay but one wonders whether it would be rather less sensitive to steric crowding around the nitrile being hydrated than is usual with NHases.

PS This paper also has my favourite use of the word “recently” in a communication… to reference 12 from 2003.