NHase numbers to November 2012

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 4199 hits (+ 7% since August), of which 1605 (+ 8% since August) were RefSeq data.

No new PDB files have been deposited of nitrile hydratases in the last 3 months.

No nitrile hydratases in Acidobacteria

There is another interesting taxonomic post on All Things Considered called "A Whiff of Taxonomy: The Acidobacteria". They are a phylum of difficult to culture soil borne bacteria. I couldn't find one that contained a nitrile hydratase anything like the one in Rhodopseudomonas palustris CGA009. No evidence of the key metal binding sequence for either Co or Fe NHase at all. Some do appear to have nitrilase type proteins.

New thermophilic NHase sequence

I have the NCBI database set up to send me a weekly email digest of newly uploaded nitrile hydratase sequences. One of the ones which appeared this week is from the proteome of Mycobacterium hassiacum DSM 44199. This organism is one of the Mycobacterium which use humanity as its primary habitat, and this particular one was isolated from a urine sample collected in Germany in 1995. The literature reports that this organism is comfortable with temperatures up to 65 degrees C, and can deal with up to 5% salt, all of which might well offer typical "moderately thermophilic" properties to its nitrile hydratase. The alpha chain shows a "CTLCSC" sequence suggesting it is cobalt-centred, and BLASTing this sequence shows that 7 other Mycobacterium species are between 89-85% sequence similar, with non-Mycobacterium sequences being significantly different (starting below 55% similar). Interestingly most of the other Mycobacteria which are sources of these sequences are from environmental samples with much lower temperature ranges. I cant find any reference to a paper where a Mycobacterium has been exploited for its nitrile hydratase activity either in the Prasad and Bhalla review from 2010 or from a Google Scholar search. Perhaps here's one to start with.

NHases in Roseobacter

On the "Small Things Considered" blog (run by Prof Moselio Schaechter for the American Society for Microbiology), they have started an occasional series on interesting taxonomic groups of bacteria. The first that Prof Schaechter is giving is a pen picture of Roseobacter.

This group of bacteria are marine in origin, “make up 25% of the bacterial biomass in some coastal marine waters from the tropics to the poles” and have quite a lot of diversity in their microbiology so it seemed natural to see how much diversity there is in the recorded genomes for Roseobacter.

I did a search for nitrile hydratase alpha chains in organisms explicitly labelled as Roseobacter, and there are 7 which have RefSeq levels of quality in the NCBI database. There are two from Roseobacter litoralis Och 149, one from Roseobacter denitrificans OCh 114 and four from species various labelled sp. AZwk-3b, sp. CCS2, sp. SK209-2-6 and sp. MED193.  The two from Och 149 are very different but it can be seen from the COBALT alignment shown below that they are all cobalt containing NHases. According to a Clustal2.1 alignment, no two sequences are more than 88% similar (the second and third sequences are most similar, with the sixth/seventh pair next) with the average similarity being approximately 55%.

Diversity in Rhodococcus Sequences on NCBI

Downloading all the alpha chain amino acid sequences of nitrile hydratases of Rhodococcus origin from NCBI, you get over 100 sequences. By eliminating all the ones which are from PDB entries, you get 98 sequences. By using the usual amino acid sequence tag to identify which metal centre present, you get 68 iron-centred NHases and 24 cobalt-centred NHases, and six bits of rubbish. Of the iron centred ones, 61 have the VCSLC tag starting at position 109. None of the remaining have the metal binding region starting in the same place, and range from 96 to 149. There is much more of a spread with the cobalt centred sequences if you track the equivalent tag, as the histogram below shows.

Methods of immobilization of NHase, and a new one.

There is a new paper on the immobilization of nitrile hydratase to give greater stability. There are a few previous examples of this topic including:

·         Nitrile hydratase CLEAs: The immobilization and stabilization of an industrially important enzyme from Sander van Pelt, Sandrine Quignard, David Kubac, Dimitry Y. Sorokin, Fred van Rantwijk and Roger A. Sheldon in Green Chemistry in 2008. (DOI: 10.1039/b714258g)

·         Production of Acrylamide using Alginate-Immobilized E. coli Expressing Comamonas testosteroni 5-MGAM-4D Nitrile Hydratase from Lawrence J. Mersinger, Eugenia C. Hann, Frederick B. Cooling, John E. Gavagan, Arie Ben-Bassat, Shijun Wu, Kelly L. Petrillo, Mark S. Payne, and Robert DiCosimo in Advanced Synthesis and Catalysis in 2005. (DOI: 10.1002/adsc.200505039)

·         Biotransformation of nitriles by Rhodococcus equi A4 immobilized in LentiKats from David Kubáč, Alena Čejková, Jan Masák, Vladimír Jirků, Marielle Lemaire, Estelle Gallienne, Jean Bolte, Radek Stloukal, Ludmila Martínková in Journal of Molecular Catalysis B: Enzymatic in 2006. (doi:10.1016/j.molcatb.2006.01.004)

This one is Catalytic Properties of a Nitrile Hydratase Immobilized on Activated Chitosan by Yu. G. Maksimova, T. A. Rogozhnikova, G. V. Ovechkina, A. Yu. Maksimov, and V. A. Demakov in Applied Biochemistry and Microbiology (DOI: 10.1134/S0003683812030076).

They have used a nitrile hydratase isolated from a strain of Rhodococcus ruber gt1 and immobilized it on chitosan activated with 0.1% benzoquinone solution. They show that this immobilized enzyme can be used for 50 consecutive cycles of acrylonitrile transformation with activity holding up well.

They also found that their immobilized nitrile hydratases remain active at pH 3.0–4.0 which usefully extends its effective pH range.

Rotation

The quick and simple pdb2MGIF tool is available again at http://www.glycosciences.de/modeling/pdb2mgif/

Here is the nitrile hydratase from Comamonas tetosteroni Ni1 (4FM4) in two different rotating animations. I have cut the PDB file down so it only shows a single A/B dimer.

Overlaying the NHases in 4FM4 and 2QDY

The iron centred NHase which I have most experience of is the Rhodococcus erythropolis AJ270. I have overlayed it onto the structure for the Comomonas testosteroni Ni1, and it is very similar as you might expect both at the secondary structure level (4FM4 in red, 2QDY in blue and yellow) and as an alignment. (The original paper makes in-depth analysis with 1AHJ from Rhodococcus sp. r312.)

New publications- September 2012

There are two new publications:

• A review on stereoselectivity in nitrile hydratases by Anming Wang and co-workers in the African Journal of Microbiology Research (DOI: 10.5897/AJMR12.101). I am not sure it adds much to previous reviews. 
• A paper called “Biotransformation of benzonitrile herbicides via the nitrile hydratase–amidase pathway in rhodococci” in the Journal of Industrial Microbiology and Biotechnology with Ludmilla Martınkova as corresponding author (DOI: 10.1007/s10295-012-1184-z). It looks at the ability of Rhodococcus rhodochrous PA-34 and Rhodococcus erythropolis A4 to hydrate and hydrolyze chloroxynil, bromoxynil, iodoxynil and dichlorobenil. The NHase seemed to be able to turnover the herbicides more easily than the downstream amidases were able to hydrate the resulting amides.

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…

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.

Data to enzyme

I was talking at the "Leading Industrial Biotechnology: Focus on Biocatalysis" meeting last week in York, and I put up this slide which caused a bit of comment amongst those of a chemistry background in the audience who didn't know the power of state-of-the-art molecular biology for those looking for new enzymes not available from catalogue based suppliers. Moral of the story: find yourself a molecular biologist and you will have more (and more interesting) enzymes available than you can imagine.

The Importance of Oxidation in VCTLCSC

Just before my sabbatical on this blog, I was looking at the length of bonds which linked the metal to the sulphurs of the cysteine residues to test the state of evidence in the small molecule cystallographic record on how oxidation might change such bond lengths. There wasn't much data around for Co and Fe based systems to draw many inferences from. However, a new paper is just out looking at a model system based around Ru which has been synthesized in the various oxidation states of sulphur with the hope of giving some clues on what sulphur oxidation might offer to NHases.

Influence of Sequential Thiolate Oxidation on a Nitrile Hydratase Mimic Probed by Multiedge X-ray Absorption Spectroscopy by Jason Shearer, Paige E. Callan, César A. Masitas, and Craig A. Grapperhaus in Inorganic Chemistry (DOI: 10.1021/ic202453c).

They use a range of techniques including multiedge X-ray absorption spectroscopy and some serious hardcore DFT calculations to look at/predict how ionic and how Lewis acidic the complexed metal becomes as the sulphur ligands are sequentially oxygenated. Basically you get a harder Lewis acid as you move from naked sulphur through to a mixed sulfenato/sulfinato species. As the abstract graphic shows....

In the final few paragraphs, they speculate what this means for the mechanism of nitrile hydratase hydration (they say "hydrolysis" but that means the complete breaking up the whole bond to acid and ammonia in my book, not just breaking a couple of pi bonds with a molecule of water.). They show versions of the two most commonly proposed models- metal-bound hydroxide attacking a free floating nitrile, and metal-bound nitrile getting walloped by a hydroxide. They suggest that increasing hardness of the metal centre should favour the attachment of an oxygen based ligand over the nitrogen of a nitrile, and hence favour the metal bound hydroxide model. They end with the comment that harder Lewis acidity would actually aid NHase catalysis whatever was bound to the metal. 

How widespread are NHases in eukaryotes?

There is a neat diagram summarizing the findings of the paper by Marron, Akram and Walker called "Nitrile Hydratase Genes Are Present in Multiple Eukaryotic Supergroups" in PLoS One to be found here in full. There is quite a lot of legend that goes with it that I recommend you look at the paper for, but crucially asterisk * means evidence of one eukaryotic NHase subunit (and hence ** means evidence of both), and hash # means a dodgy read more suggestive of prokaryotic contamination.

So that's a black mark for any NHase in plants and animals then...

NHase in harmful algal blooms

Last year I put up a blogpost about finding what looked like the alpha chain of a nitrile hydratase in the eukaryote Aurecococcus anophagefferens which is something that makes up brown tide algal blooms. The paper that introduces the genomic data which provided my hit back then is

Christopher Gobler, Dianna Berry, Sonya Dyhrman, and Steven Wilhelm. "Niche of harmful alga Aureococcus anophagefferens revealed through ecogenomics" Proceedings of the National Academy of Sciences of the USA 108.11 (2011): 4352-4357

This little beastie has a whole load of enzymes involved in degrading organic nitrogen containing compounds. The authors of the paper suggest that since it lives in an ecological niche which is typically low in organic nitrogen but high in organic nitrogen so having a suite of enzymes to harvest nitrogen from organics is a strategy to give a competitive advantage over other phytoplankton which don't have this ability. There is a supplemental figure which shows the list of these enzymes and whether competing species have them. A rough cut and paste below shows that it is really out on its own from this data.

This organism also seems to be a Guinness world record holder for the number of selenium containing proteins in its proteome (56). Interesting but not entirely relevant for this blog!

How have the database numbers changed in a year?

Last year I did a quick text search for "nitrile hydratase" as a search term under proteins on the NCBI website. This gave me 2869, of which 1042 were RefSeq data. Today when I checked there are 3573 (+25%), of which 1369 (+31%) were RefSeq.
No new PDB files have been deposited of nitrile hydratases since March 2011 which was the NHase from Pseudomonas putida.

New paper on eukaryotic nitrile hydratases

One of the things that has refocussed me on NHases is the new paper in PLoS ONE on eukaryotic nitrile hydratases. It looks fascinating on a brief flick through.

Nitrile Hydratase Genes Are Present in Multiple Eukaryotic Supergroups by Alan O. Marron, Michael Akam and Giselle Walker in PLoS ONE here.

They have found more single subunit NHases in small eukaryotes, and discount their presence in anything bigger than a protist (like the ones in Ricinus communis) as prokaryotic contamination.

I'll be having a good perusal of this paper.

It's been a while...

I have been concentrating on my other research interests recently (anti-fouling surfaces, paint analysis and mass spectrometry as it happens) so I haven't updated this blog for a couple of months. I have had one student (Hello Luke!) working away on NHases, and he reckons that two of our NHases (including the one from Monosiga brevicollis) can monohydrate a substituted malonitrile.

S-M bond lengths

I was wondering what a "normal" bond length might be for a thiol to iron or cobalt might be, and then what might happen to that bond length might do if that bond was oxidized once and then twice. The best way to do this is to look at the small molecule crystallographic data so I got my friend Ross to comb through the usual database to find examples of the various bonding motifs.

It turns out that there are no examples of 1b in the CCD, and precious few of 1c (only 4), 2b (only 5) and 2c (a more respectable 15). There are loads of examples of 1d (actually 286) and 2d (102).
Anyway, there isnt much of a difference in average bond lengths between these types: 1c- 2.221 angstroms, 1d- 2.276 angstroms, 2b- 2.225 angstroms, 2c- 2.208 angstroms and 2d- 2.252 angstroms.
Here are a few histograms showing the distributions that lie behind these means.