Modelling of 4FM4

The structure of the NHase from Comamonas testosteroni is now available as a PDB file. I have been having a quick look at it.

And now a cartoon view with the metal binding motif highlighted

A molecular surface showing the entrance to the active site

Another 4D mutant of Rhodococcus rhodochrous PA-34 paper

There is a new paper on the 4D mutant of Rhodococcus rhodochrous PA34 from Pratush, Seth and Bhalla.

Purification and characterization of nitrile hydratase of mutant 4D of Rhodococcus rhodochrous PA-34 in 3 Biotech, DOI: 10.1007/s13205-012-0081-5.

They give methodology for how to purify the NHase out of the mutant cells, and then test it against a set of standard compounds under a range of conditions.

As a cobalt-centred NHase, you might expect it to turnover aryl nitriles better than alkyl nitriles if the simplistic “rule of thumb” operated but this seems to be pretty undifferentiated. (see one panel from their Figure 4 below).

This mutant also seems to have a temperature maximum somewhere in the region of 45-60^oC (they go for 55^oC) which as the authors note is sort of high for a NHase (and higher than for wild type Rhodococcus rhodochrous PA34 which is quoted as 40^oC). Another panel of their Figure 4 shows this below.

3D structure of Wild Type Fe-type Nitrile Hydratase from Comamonas testosteroni Ni1

There is a new NHase PDB file available. With the code 4FM4, it refers to the NHase from the recent Holz paper.

Looking for nitrile active enzymes

I was out for a walk at the weekend at Bishop Middleham quarry which is a nature reserve and an example of Magnesian limestone grassland. It is a nice spot and very quiet so there is plenty of wildlife to look at. One thing we spotted fluttering all around the grassland on the quarry floor was a six spotted burnet moth, Zygaena filipendulae. After a bit we got one to settle and we got a photo.

All very ecological but what's this got to do with nitrile hydratase? I know nothing about moths, so I looked this up when we got him to identify it. The intriguing thing about this moth is that it survives in the wild despite not being at all camouflaged (it is a blur of metallic grey, red and orange on the wing). Its defence strategy is based on containing cyanogenic glucosides which liberate HCN given half a chance making them a very poor choice of meal for anything tempted to snack on them. A browse of the literature suggests that it gets its cyanide both from absorption from its food source when a larva (such as bird's foot trefoil) and also biosynthesis. It seemed logical to me that if it has something to make nitriles, it might have some pathway to dispose of them too so that its biochemistry doesnt get swamped.
Rather amazingly a genome search of NCBI shows that Zygaena filipendulae has had its transcriptome sequenced. Apparently there are all sorts of biosynthetic apparatus for making cyanogenic compounds in there but when I did a bit of BLASTing nothing that looked vaguely like a nitrile hydratase. Maybe there are nitrile hydratase enzymes in the intestinal flora of this moth and its larva. That hasnt been examined yet!

NHase in halophilic archaeon DL31

I notice that both an alpha chain and a beta chain of a cobalt centred NHase have been recently annotated (14 June 2012) in the genome of the rather unprepossessingly named "halophilic archaeon DL31". It must be quite an elusive beast because Google Images doesn't deliver a picture of anything suitable.
Halophilicity quite often comes with tolerance to alkaline conditions, which can mean all sorts of utility in the world of synthetic biocatalysis. As I mentioned in the "No extremes" post we published with Roger Sheldon's group on the synthetic potential of one of these halophilic NHases, but this is a good example of a genome-sourced potential halophilic NHase. BLASTing both the alpha and beta chains against prokaryotic genomes shows that the beta chain is most similar to beta chain of Caldalkalibacillus thermarum TA2.A1 (I think we can guess the proclivities of this microbe) with 3 of the next 4 best hits being the sequences from the crystal structures 3HHT, 2DPP and 1V29 all of which are moderately thermophilic.

Interestingly there is a slightly different aspect to the alpha chain BLAST: Pseudonocardia dioxanivorans and Rhodopseudomonas palustris make up the top hits before Caldalkalibacillus thermarum TA2.A1 turns up.

NHase number changes since May 2012

Continuing my regular rough and ready look at the extent of "nitrile hydratase" sequences (most aren’t but it’s a rough measure), I have just used that as a search term under proteins on the NCBI website. This gave me 3935 (+ 10% since May), of which 1493 (+ 9% since May) were RefSeq data.

No new PDB files have been deposited of nitrile hydratases since March 2011 which was the NHase from Pseudomonas putida though one from Comamonas testosteroni Ni1 is in the process of release.

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…