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Friday 23 December 2011

Just Accepted NHase manuscripts

  • Enantioselective biocatalytic hydrolysis of beta-aminonitriles to beta-amino-amides using Rhodococcus rhodochrous ATCC BAA-870, by Varsha Chhiba, Moira L. Bode, Kgama Mathiba, Wendy Kwezi, Dean Brady in Journal of Molecular Catalysis B: Enzymatic [doi:10.1016/j.molcatb.2011.12.005]

“A range of β-aminonitriles (3-amino-3-phenylpropanenitrile and derivatives) were synthesised by reaction of various benzonitriles with acetonitrile and subsequent reduction of the resulting acrylonitrile products. These compounds were hydrolysed to the corresponding amides using the nitrile biocatalytic activity of Rhodococccus rhodocrous ATCC BAA-870. Results showed that the nitrile hydratase enzyme was enantioselective for these compounds, in particular 3-amino-3-p-tolylpropanenitrile and 3-amino-3-(4-methoxyphenyl)propanenitrile and the corresponding amides (up to 85% in one case).” They found interesting levels of steric hindrance (probably) when the amino group was functionalized, and they had most success when the biotransformations were run at pH9 to minimize as much as possible protonation of the amine if it wasn’t derivatized. Obviously it being a Rhodococccus, this is an example of enantioselectivity with an iron-centred NHase.
  • Biotransformation of the Neonicotinoid Insecticide Thiacloprid by Bacterium Variovorax boronicumulans Strain J1 and Mediation of the Major Metabolic Pathway by Nitrile Hydratase by Hui-Juan Zhang , Qian-Wen Zhou , Guang-Can Zhou , Yu-Min Cao , Yi-Jun Dai , Wei-Wei Ji , Guang-Dong Shang , and Sheng Yuan in Journal of Agricultural and Food Chemistry [DOI: 10.1021/jf203232u]
“A neonicotinoid insecticide thiacloprid-degrading bacterium strain J1 was isolated from soil and identified as Variovorax boronicumulans by 16S rRNA gene sequence analysis… A 2.6-kb gene cluster from V. boronicumulans J1 that includes the full length of the nitrile hydratase gene was cloned and investigated by degenerate primer polymerase chain reaction (PCR) and inverse PCR. The nitrile hydratase gene has a length of 1304 bp and codes a cobalt-type nitrile hydratase with an alpha-subunit of 213 amino acids and a beta-subunit of 221 amino acids.” This protein was then expressed in an active form in E. coli BL21. This bacterium is a boron-accumulating microbe isolated from soil.

Wednesday 16 November 2011

Aligning cobalt centre NHases

I have been using the FATCAT protocol to look at structural alignments of differing cobalt centred NHases. This is available as a tool here.
Here is a picture of the alpha chains of 1V29 and 1 IRE aligned.

And here is a picture of the alignment of the beta chains. A bit more variation there.

Thursday 3 November 2011

A new review: Industrial Biotechnology- the future of green chemistry

The most recent issue of Green Chemistry has a 41 page review (DOI: 10.1039/C1GC15579B) by Udo Kragl and his co-workers Stefanie Wenda, Sabine Illner and Annett Mell, entitled  "Industrial Biotechnology- the future for green chemistry" which is a useful overview of where biocatalysis is now. A nice feature it has that I havent seen before is the use of little boxes which are labelled "critical remarks" to discuss highlight problems sometimes in the perception and sometimes in the reality of using biocatalytic process industrially.
As is necessary in this sort of review, there is a discussion (p. 3011) on how good nitrile hydratase is for bulk synthesis of acrylamide though it is interesting to see the scale of the use is "more than 50,000 tons per year" referring back to a viewpoint paper in ChemCatChem authored by Yuryev and Liese which actually says the Mitsubishi Rayon process "runs on scales up to 50,000 tons per year"- whatever that actually means! So it would appear that my suspicion that no one actually knows how successful (in terms of level of adoption) one of the most successfully implemented biocatalytic processes is, has not been contradicted!
Directly following the discussion of acrylamide manufacture is a discussion comparing the chemical and the Lonza chemoenzymatic routes to nicotinamide, with some excellent leading references.

Wednesday 26 October 2011

Polymers and nitrile hydratase activity

There have been a few papers over the years looking at the possibility that nitrile-active enzymes might be able to attack nitrile groups on the surface of nitrile-containing polymers such as polyacrylonitrile.  An early example of this is the paper by Gübitz and co-workers [Nitrile Hydratase and Amidase from Rhodococcus rhodochrous Hydrolyze Acrylic Fibers and Granular Polyacrylonitriles, from Appl Environ Microbiol (2000)] which uses a cell free extract from Rhodococcus rhodochrous NCIMB 11216 to create pendant carboxylate groups on the fibres.

More recently there has been another paper looking at this topic using a different cell-free extract
This uses an extract from Amycolatopsis, and they do show conversion of the surface to carboxylate, by functionally tracking a NHase activity and an amidase activity. I am not sure how they know there isnt a nitrilase in there helping along too (NCBI records currently an Amycolatopsis species nitrilase).

In the light of the congested active site entrance which is generally found with NHase, it is quite interesting this works.

When nitrilases act like nitrile hydratases...

There were other posters than mine at Biotrans2011 which looked at nitrile active enzymes (from the groups of Norbert Klempier and Ludmila Martinkova). One of the issues that came up more than once was how sometimes nitrilases can sometimes stop being nitrile hydrolyzers and become nitrile hydraters (like NHases). This is an interesting topic in itself because, in terms of synthetic chemistry utility, nitrilases are probably more widely applicable than nitrile hydratases, and predicting when a particular nitrilase isnt quite up to the full hydrolysis could be a useful timesaver. In my experience, some IMAC purified nitrilases that we have worked with have upset commercial collaborators in stopping at the primary amide and not producing any carboxylic acid product, and the effect has been reported in the literature before, summarized in Martinkova and Kren 's recent nitrilase review.

Ludmila Martínková, Vladimír Křen, Biotransformations with nitrilases, Current Opinion in Chemical Biology, Volume 14, Issue 2, April 2010, Pages 130-137, 10.1016/j.cbpa.2009.11.018.

Obviously this leads me to the reverse query... Is it possible to get a nitrile hydratase to hydrate a nitrile under extreme/altered conditions?

Thursday 20 October 2011

My poster from Biotrans2011

Here is my poster from Biotrans2011. As you can see quite a lot of what I wrote has appeared on this blog previously.

Friday 30 September 2011

Biotrans 2011

I am off to Biotrans 2011 next week. I am presenting a poster on nitrile hydratases which I will put up here on my return.

Wednesday 28 September 2011

More automated modelling

I had a go at trying to get SWISSmodel to produce a structure prediction for the 3D structure of the single subunit NHase of Monosiga brevicollis. It had a fair stab at it, as I would expect since there are existing PDB files of alpha and beta subunits which match the relevant portions of the chain, but still it isnt right. The placement of the metal ion is the immediate indicator for me that it has gone wrong- I would want the active site in the middle not on an exposed edge.

Considering the similarity of the sequence to the alpha subunit to that in the PDB file 1IRE (left below), and the beta subunit in PDB file 1V29 (middle below), I was expecting something a bit like those to thrown together, with some guess at the histidine-heavy chain linking the two ends (poking out to the right on my guesstimate shown below right).


Friday 23 September 2011

NCBI numbers for September

It has been a while since I had done the usual text search for "nitrile hydratase" under proteins on NCBI's website. Having done the proper filtering job on this numbers in August, I know there is a lot of duplication, error and bizarre search engine behaviour going on but I do think it shows how quickly the number of vaguely applicable sequences is growing. As of today there are 3066 hits for the target phrase (+63 since July), and 1125 RefSeq hits (+20 in the same period).

Disappointingly there was no change in the number of structures listed on the
Protein Data Bank.

Wednesday 14 September 2011

It should be possible to 3D printing of enzyme models... but how?

It sends me slightly square-eyed staring at my PC trying to get an idea of the steric requirements for molecules to pass down portals into the active sites of nitrile hydratase enzymes. As a big fan of actual physical models of chemical structures, it would be nice to be able to produce something you could hold in your hand which would complement the computer based view. In my situation this should be relatively simple... there are some nice structures available as PDB files, and just across the courtyard from me there is an engineering lab which specializes in rapid prototyping using the more than slightly magical Z-Corp 3D printer. But how on earth do you convert a PDB file format to a PLY or VRML file that the printer reads? Perhaps naively I thought there would be a site somewhere which you can plug in your PDB file and it spits out something the printer will read. Use of Google to research this is like a step back in time leading to an infinite loop of pages from the turn of the millenium where the crucial link is no longer live, or another link to the Milwaukee School of Engineering or the Scripps Institute who will print a structure for a nice price. Oh well.

Wednesday 7 September 2011

New patent from Mitsui

A Google alert has led me to a new US patent from Mitsui Chemicals Inc.

The abstract is very abstract: "A nitrile hydratase variant comprising substitution of at least one amino acid with another amino acid to improve two or more properties of nitrile hydratase by substitution of one or more and three or less amino acids".

The objective of the work under the patent is to produce a NHase variant with improved stability and high initial reaction rate.

It appears to be about Pseudonocardia thermophila variants, but I havent read all 77 pages of it since it tends to dwell on the very many changes to the sequence that they are protecting, both at the amino acid and nucleotide level.

Friday 19 August 2011

Hot or not

My August analysis of nitrile hydratase sequences also revealed that 95% of nitrile hydratase alpha chain sequences come from mesophilic organisms. The remaining split pretty much equally between the thermophilic and the psychrophilic. There was nothing that could be termed a hyperthermophile.

Opisthokonts, choanoflagellata and nitrile hydratases

There is an ongoing project called "The Origins of Multicellularity" which involves Gertraud Burger (University of Montreal, Canada), Michael W. Gray (Dalhousie University, Canada), Peter Holland, (University of Oxford, UK), Nicole King (University of California Berkeley, USA), B. Franz Lang (University of Montreal, Canada), Andrew Roger (Dalhousie University, Canada) and Iñaki Ruiz-Trillo (University of Barcelona, Spain) which seeks to investigate commonalities and differences underlying multicellularity in animals and fungi (to use their phrase). As I understand it, the idea is to take a range of simple organisms which go by the name of “opisthokonts” (which sounds quite unsavoury if said in a Dublin Irish accent) and use genomic analysis to track down features of a proposed eukaryotic ancestor, and propose how evolutionary pathways have lead to fungi and animals. These episothokonts are the sort of miniscule organisms the existence of which are not exactly common knowledge, and hence come with names that make bacterial monikers seem easy to remember. Here is a tree of the organisms this project are looking at- those in blue bold type are those which the genomes the project is working on. 
One subset of the opisothokonts are the choanoflagellata, and it contains both Monsiga brevicollis and Salpingoeca rosetta both of which have a single chain NHase.  The genome data from the project is going up online as it is produced so NCBI searching works well for making BLASTp type comparisions. I have had a good go at working through the organisms listed on this tree, BLASTp both the beta/alpha single chain NHase from Monsiga brevicollis and the alpha chain from CGA009, and I cannot find anything meaningful in anything apart from Salpingoeca rosetta, which I found interesting. So there are nothing NHase-like in the fungi taxid, nothing in filasterea or ichthyosporea AND nothing in the genome for Monosiga ovata (which seems to have had a bit of a chequered history with impurity issues). That last fact seems weird to me but I even tried using BLASTp with a section of the beta like chain of Monsiga brevicollis  (just in case there was more exon/intron messing about going on, as the authors of the original Monsiga brevicollis paper identified, that I hadn't picked up on) and still nothing of consequence. (Diagram below from this paper).
It makes me wonder why just these organisms carry something like a functional NHase… there must be some evolutionary reason.

Friday 12 August 2011

Proper NCBI numbers for August

I have been looking for a number for the actual number of proper NHase alpha subunit sequences on record. I have done this by really combing through the NCBI protein records using a BLASTp approach for similarity, and excluding sequences that aren't an appropriate length nor have the usual active site binding motif. I have ignored environmental samples, and dropped out data from PDB files.
As of the start of this week (8th Aug 2011) I reckon there are about 190 NHase alpha sequences. That breaks down as about 25% iron and 75% cobalt centred. There are four from eukaryotic organisms (three marine and one plant), and the rest are... not.

Aureococcus anophagefferens- another eukaryote with a nitrile hydratase alpha chain

Whilst combing the data for new versions of nitrile hydratases which might be worthy of investigation, I came across a protein in the eukaryote alga Aureococcus anophagefferens which looks hugely like the alpha chain of a cobalt centred nitrile hydratase. I havent spotted the beta chain in the genome yet, but it is interesting to see that it doesnt seem to share the single subunit pattern of NHase like Monosiga brevicollis or Salpingoeca.
This phytoplankton is behind algal blooms which give "brown tides" off the eastern seaboard of the USA. (Image of organism below from here)

Monday 1 August 2011

Nitrile hydratases in metagenomes: when is something too different?

Looking for variants of an enzyme in an exotically sourced metagenome is an obvious way to extend the diversity of that enzyme classification. There are a number of these metagenomes now fully searchable but it is a whole order of difficulty greater to do searching in them than in genome sequences (certainly for me as someone who is trained as a chemist, and is learning bioinformatics as I go along). The resource I have been playing with recently is MetaBioME which offers search tools (like this) for investigating almost 50 metagenomes. It doesn't have data which doesn't occur in NCBI resources but it is a different way to look through the data. Maybe there is a quicker way to analyze the data from it than the following, but if you put in a FASTA sequence such as the one for the alpha subunit of Rhodopseudomonas palustris CGA009, it will send you back matching contigs for one of the metagenomes.
Even if you interrogate with a protein sequence, it gives a nucleotide sequence back so for someone like me who likes to "eyeball" their hits, this requires a translation tool like the one at ExPASy which will give you an answer including all three reading frames for both 5'3' and vice versa. I must admit I then use the rather low teach tool of text searching within my browser window for fragments of the classic active site motif to find if there is a classic nitrile hydratase present. That is quite a long sequence of things to do, so I dont think I'll be doing this by hand with, for instance, the 213 hits from the Global Ocean Sampling Expedition metagenome it gave on default settings. However there are metagenomes which sound interesting in the list- I thought "termite gut" sounded worth investigating, and after tweaking the settings (as its default search settings seem rather conservative for nitrile hydratase searching), you do get a top hit which aligns amino acids 25-209 of CGA009 alpha with rather a nice correlation.
The usual success indicator of asterisks, colons and full stops are all over the alignment... but look at the metal binding motif (third row, about a third along)- it all looks the same as a cobalt bound NHase but instead of "VCTLCSC", it has "VCTQCSC". Leucine for glutamine- can that be right? Leucine isnt involved in binding but that is quite a change. I wonder what are the chances this is weird but right or is it just a wrong bit of nucleotide identification/translation. After all the triplets CUA and CUG are very similar to CAA and CAG.

Tuesday 19 July 2011

Nitrile hydration is exothermic

I spotted a reference to the fact that the hydration of a nitrile is an exothermic process, and as such at scale needs some form of refrigeration. Obviously this is an issue in biocatalytic processes where your enzyme might be thermal sensitive but is also true of the plants which operate the copper based chemical process (like the one I was shown round in Bradford then run by Ciba- thanks Yvonne!).
I used Hyperchem running a semiempirical PM3 method to model the process of conversion of acrylonitrile, benzonitrile and nicotinonitrile to the relevant amide and they all give an exotherm of about 20kcal per mole. I repeated one using a moderately intensive DFT expt and it agreed with this figure.

Friday 15 July 2011

Castor oil plant nitrile hydratases

Another of the projects from JCVI is the castor oil plant (Ricinus communis) genome. The auto-annotation has picked up two alpha subunits of nitrile hydratase, and there is at least one thing that looks like a beta in there too.

Thursday 14 July 2011

Nitrile hydratases and the Gordon and Betty Moore Foundation’s Marine Microbiology Initiative

Since the most exciting new source of nitrile hydratase sequences has been marine organisms recently, I thought I would have a quick look at the preponderance of nitrile hydratase sequences in marine bacteria. I decided to look at the Moore Foundation's Marine Microbiology Initiative which was run by JCVI. I used the alpha sequence from the nitrile hydratase from Rhodopseudomonas palustris CGA009 as a probe sequence and BLASTped this sequence against the genomes produced by this initiative. This was a slightly more tedious job than I imagined because the BLAST server at JCVI wasn't working, so I had to work through a NCBI BLASTp input, which involves a fair bit of cutting and pasting, AND the taxonomy of the names listed isn't always the same in both databases. Anyway, of the 177 marine microbes which had their genomes listed, 30 produced sequences which had query coverages of greater than 68%, and all were auto-annotated as nitrile hydratase alpha subunits and contained the appropriate metal binding sequence.

Tuesday 12 July 2011

A predicted nitrile hydratase from a psychrophilic bacterium

I have been scanning the sequences, and by chance I came across the fact that there is a predicted nitrile hydratase from a bacterium called Octadecabacter antarcticus strain 307 which is a marine organism found at the poles. From the looks of the primary amino acid sequence of the alpha subunit (gb|EDY76981.1|), it is a cobalt-centred NHase.

Monday 11 July 2011

Automated Protein Structure prediction... or probably not.

I was browsing through a bioinformatics textbook last week, and read up about the work of Prof  David Baker in the field of protein structure prediction. One of the resources that the Baker Lab offer for free to academic customers is access to their Rosetta methodology via their Robetta structure prediction software hosted on their own computers. It is a really easy to use resource with minimal hassle in terms of registration. Anyway I thought it might be interesting to see what an automated program might be able to make of the single protein NHase of Monosiga brevicollis which is made up of the standard two separate alpha and beta subunit proteins tied together, both of which are well known from 41 (and counting) fairly similar PDB files which have a high degree of similarity. First of all, you submit your FASTA sequence, and it parses it using the Ginzu domain prediction technique.
I was pretty pleased with this start as it spotted the two domains by BLAST, and match them to two PDB files of cobalt containing NHases. I then set it away to do the full structure prediction thinking that knowing those two starting points, most of the heavy lifting in terms of the structure prediction had been done. The prediction said it would take seven days but actually was finished after two (not a busy weekend for their computers it would appear!). Its output is the five best structures...
I have to say I have no confidence in any of those structures... all look a bit long and dangly for me. If you contrast them with a couple of PDB structures of NHases of the usual type below you can see that they tend to be much more compact- look where the two subunits' beta sheets are in the PDBs and in the prediction.

I guess it was asking a lot to get a reasonable guess at the histidine-heavy chain in the middle of the Monosiga NHase, and that is a crucial part of this prediction. No need to welcome our robot overlords just yet, and I need to go and talk to a human homology modeller!

Wednesday 6 July 2011

NCBI numbers for July

The usual text search for "nitrile hydratase" under proteins on NCBI's website shows 3003 hits for the phrase (+134 since May), and 1105 RefSeq hits (+63 in the same period).

No change in the number of structures listed on the
Protein Data Bank.

Comparing the access of the active site of 3QXE and 3HHT

If you orient the pdb file of the Geobacillus which is coded as 3HHT as I have done for 3QXE and 2QDY in the previous posts, you get the following two images.



The cobalt is no longer visible. However if you twirl the structure around you can find a view of it through a different aperture. I wonder how this impacts on the selectivity of this enzyme.



Looking inside for the active site of 3QXE

This is just a couple of rough pictures of 3QXE (the nitrile hydratase from Pseudomonas putida), once again from PyMol, showing a ribbon view of it with the active site cobalt-binding residues shown as CPK spheres, followed by a surface model with some of the active site just visible inside. I have been careful to keep it oriented similarly to the AJ270 nitrile hydratase in the previous post.

Monday 4 July 2011

Looking for the iron in AJ270

 Using PyMol I have been looking to see what the tunnel which connects the iron centre to the outside world looks like. Here are some pictures. Iron is represented as a warmpink sphere, and the pdb structure used is 2QDY.


Or close up as...

Thursday 23 June 2011

A more extreme than usual NHase

There is a cobalt centred nitrile hydratase from Rhodococcus opacus which has been the subject of work by Yasukawa and patents and commercial usage by Degussa. If you BLASTp the alpha chain of this, as NCBI titles it, "unnamed protein product" against their RefSeq collection, you find there are a lot of similar NHases. One that is relatively new comes from the newly released draft genome sequence of the bacterium Caldalkalibacillus thermarum TA2.A1. There is a good clue in its name to the fact that this bacterium grows optimally 65-70 degrees C and pH 9.5. That is pretty hot for a NHase, and with those pH conditions, no surprise it isnt an iron-centred version!

Tuesday 21 June 2011

Microbial production of acrylamide by nitrile hydratase

I have just obtained a copy of the "Acrylamide, Microbial Production by Nitrile Hydratase" by R-C Zheng, Y-G Zheng and Y-C Shen from the Encyclopedia of Industrial Biotechnology: Bioprocess, Bioseparation and Cell Technology, published in 2010; DOI: 10.1002/9780470054581.eib004. It is new, interesting resource for how much large scale use of NHases there is going on, and includes some information I havent seen anywhere else before.
  • It gives a worldwide estimate for the biocatalytic production of acrylamide as 400,000 t/a
  • There are "more than 10 plants" in China running microbial acrylamide production using a Nocardia strain to yield a total production capacity which "has exceeded 200,000 t/a", with plans to increase this to 300,000 t/a
  • Degussa have a plant in Russia with a production capacity of 24,000 t/a using a R. rhodocrous M8 strain.
  • Mitsui Chemical have a JV acrylamide plant in Pusan, Korea.
  • SNF Floerger built a 20,000 t/a plant using the Nitto technology in 1999 (but it doesnt say where), and have plans for a further 5 of the same size.

Wednesday 15 June 2011

More on the industrial exploitation of Nitrile Hydratases

I have just been pointed in the direction of a rather nice summary ("Nitrile hydratases in synthesis") of the state of play for nitrile hydratase commercial exploitation from the May/June 2008 issue of Chemistry Today from Sander van Pelt. It includes more details of the Mitsubishi Rayon, Lonza, and DuPont usage, and shows how SNF Floerger were looking to introduce biocatalytic acrylamide production to Europe.

Monday 13 June 2011

The length of the beta unit in a nitrile hydratase

I have also had a quick look at the length of the beta chain in NHases using much the same method as I did for the alpha chains. I have to say that this time, I did do a bit of manual pruning because there was loads of inappropriate sequences in the list I downloaded. Anywhere here is the answer- about 200 again.

Sunday 12 June 2011

Salpingoeca

There is another marine eukaryote which has single subunit NHase- it is Salpingoeca. If you CLUSTALW it against Monosiga, it looks pretty similar (B subunit comes before A subunit, plenty of stars and colons but not the big section of histidines on the link.)

A better estimate of the number of NHases.

I have spent some time devising a spreadsheet into which you can download a list of FASTA data, and it will scan through looking for the "CTLCSC" fragment which indicates cobalt-centred and "CSLCSC" fragment which indicates iron centring, and total up how many hits there are of each in the list. It is all a bit more definite than just relying on "nitrile hydratase alpha" to give you accurate numbers!
Using the term "nitrile hydratase alpha" and then selecting the RefSeq selections, you get 121 cobalt centred NHases and 18 iron centred NHases. If on the other hand, you use all those sequences which are tagged as "bacteria", you get 278 cobalt-centred NHases and 131 iron-centred NHases. There are a few things to mention- these numbers don't include those sequences which have X in the tag to indicate that the cysteine is oxidized (these tend to be the PDB linked sequences) but do include some sequences which don't start with M and would make a molecular biologist wince, and the list hasn't been checked for duplication.

Friday 10 June 2011

Double length equals a single subunit?

Following on from the last post... it seems reasonable to check that if you get sequences thrown up by a search which are about the double the length of one subunit in a two subunit enzyme that you don't have a situation where the two subunits are actually linked. This is the situation for the nitrile hydratase from Monosiga brevicollis which can be seen to the right of the histogram in the last post. For this reason, it seems like a good idea to check out the two sequences in the n=360 and n=380 boxes. Well the one in the n=380 box is titled "nitrile hydratase regulator [Agrobacterium radiobacter K84]" and doesn't look right at all. Scratch that one. The other one is simply called GK12401 and is from Drosophila willistoni... a eukaryote like Monosiga. Looking at the GenPept file it is easy to see how the thing was given the NHase alpha coding (I have put a green line around the bit that looks like the metal binding bit of a NHase) though I am not convinced it is what it actually is or does (based on trying to align it to Monosiga NHase or an example alpha subunit from AJ270).

The length of the alpha chain in a nitrile hydratase

If you look at number of amino acids in the alpha chain of a nitrile hydratase, I think it is safe to say that you get suspicious of its attribution as NHase if the number was much different from 200. I was wondering if this is actually a reasonable hunch to have so here is a graph.

I got this in the usual not terribly robust way of using a text search for "nitrile hydratase alpha", and then downloading only those which were RefSeq. Today that gives you 174 sequences of which some are definitely bobbins, but I feel bad at hand-pruning datasets if your initial search is a bit of a rough design to start with. The mean length I calculated to be 199, and as you can see the mode in the histogram above is for sequences between 200 and 210. Nice to see that for once, a hunch is backed by some data! Other things of note are that the Monsiga brevicollis NHase is found by this search and is the tiny block to the far right, and a quick scan through the sequences with n=60-140 indicates these are non-NHase waifs and strays picked up by my rough search terms.

Thursday 9 June 2011

Industrial Exploitation of Nitrile Hydratases

Nitrile hydratases are one of the big successes of industrial biocatalysis. An enzymatic way to convert a nitrile to an amide is obviously attractive for its controlability and selective hydration.
There are several companies which use NHase-based nitrile hydration. The original company I think was Nitto Chemical Industries (now Mitsubishi Rayon) who use have used Rhodococcus and Pseudomonas to convert acrylonitrile to acrylamide. Wolfgang Aehle in his 2007 book "Enzymes in Industry: Production and Applications" indicates that this process runs (p281/2 available via a Google Books search) at 30,000 t/a. There are indications behind a paywall that this business may no longer be owned by Mitsubishi but by SNF Floerger Group of France but I don't want to know enough to pay up.
Lonza are another company often quoted as industrial users of NHases but in the conversion of 3-cyanopyridine to nicotinamide. They have a 3000 t/a plant in Guangzhou and it has been reported that they use the Nitto technology for this conversion.
A more recent entrant (2008) into the acrylonitrile to acrylamide bioconversion is Senmin in South Africa who have used Ciba-BASF technology to do the hydration. I dont know the scale of the operation but apparently (slides 37 onwards) they could make 20,000 tons of polyacrylamide polymer from its output.
It is not easy finding verifiable information on industrial usage of biocatalysis- I am going back to reading the academic literature!

Wednesday 18 May 2011

Salt bridges and hydrogen bonding

I have been playing with PISA tool as it offered insight into size and nature of the interface between the two subunits in NHases. I stuck in the PDB data for the most distinct cobalt-centred NHases- 1UGP, 1V29 and 3HHT, and PISA suggested that these have 61, 67 and 65 hydrogen bonds (respectively) between the alpha and beta subunits, and between 26 and 29 salt bridges across this interface. Compare these numbers with that for 2QDY (the iron-centred NHase from AJ270)... 51 hydrogen bonds and 8 salt bridges. That looks quite a difference... sadly things might not be so clear-cut in grouping these enzymes by metal centre- the newest PDB for 3QXE gives an average of 53 hydrogen bonds and 15 salt bridges, and that is cobalt-centred. Furthermore I do wonder, with such a small set of structures, whether we are seeing a distinction based on the fact that the first three cobalt-centred PDBs are of enzymes specifically tagged as "thermophilic", and AJ270 and Pseudomonas putida aren't. [I also looked at 2DPP and got 62 hydrogen bonds and 27 salt bridges].

Tuesday 17 May 2011

Searching with CTLCSC or CSLCSC

If you search the NCBI archives for nitrile hydratase enzymes just using that name you do get a large number of hits. Previous blog posts have recorded the size and rate of change of the number of hits you get if you do simple text searches with the phrase "nitrile hydratase". It is a slightly different story if you look at the RefSeq subset and then search through for the six amino acid sequence which is the metal binding motif for NHases. I am sure there are more elegant ways to do this but the way I did this was to download the relevant search with the sequences in FASTA format, load it up into MS Word and then use the "Find" function to look for each occurrence. [You can use the "reading highlight" tool and it gives you the count in the pop up box immediately].
Anyway the results were
CTLCSC (cobalt centred) -  123 hits
CSLCSC (iron centred) -  19 hits
So there are many more cobalt versions (87%) recorded than iron versions (13%) currently but given the incompleteness of the genome record, I reckon this may represent the current status of what that has been sequenced rather than the relative levels of occurrence in the wild.

Sunday 15 May 2011

Hyperthermophilic nitrile hydratases?

There are plenty of reports of nitrilases which have been cloned from organisms with names that denote obviously heat-loving tendencies- an example is the “Cloning, overexpression, and characterization of a thermoactive nitrilase from the hyperthermophilic archaeon Pyrococcus abyssi” by Mueller, Egorova, Vorgias, Boutou, Trauthwein, Verseck and Antranikian in Protein Expr Purif. 2006, 47(2), 672-81. It is quite noticeable that NHases do not appear to have the same hyperthermophilicity. If you search the translated genomes of the four organisms which produce nitrilases in this paper (Pyrococcus abyssi, Pyrococcus horikoshii, Pyrococcus furiosus, and Aeropyrum pernix) by using BLASTp against the alpha subunit of my favourite cobalt centred NHase CGA009 or the “local” iron centred NHase AJ270, you get just about nothing that shows any similarity.
Something that does stand out from examination of all the current PDB files of NHases that almost everyone says their NHase is thermostable or thermophilic. I get the impression that this labelling of these generally sensitive enzymes in itself tells a story. 

Cobalt binding in 3QXE

Above is an excerpt from Figure 6C in "Evidence for Participation of Remote Residues...etc by Ondrechen and Ringe which very nicely shows how the cobalt ion is bound into a nitrile hydratase.  There are three sulphur based points of attachment- aC112, aC115 and aC117, the latter two as sulfinic acids and two amide nitrogen points of attachment, aS116 and aC117. It is really clearly shown in this view how the arginine bR52 preorganizes the sulfinic cysteines and amide nitrogens into a planar SSNN arrangement.

Friday 6 May 2011

New papers on nitrile hydratase enzymes

A couple of NHase papers, one new and the other upcoming:
  • Biosynthesis of 2-amino-2,3-dimethylbutyramide by nitrile hydratase from a newly isolated cyanide-resistant strain of Rhodococcus qingshengii  by  Zhi-Jian Lin, Ren-Chao Zheng, Yu-Guo Zheng and Yin-Chu Shen in Biotechnology Letters, DOI: 10.1007/s10529-011-0623-7.
This paper reports a Rhodoccocus which can do the usual nitrile hydration reaction but can do it in the presence of significant quantities of cyanide. The paper describes whole-cell biotransformation conditions but I am guessing from the species this is an iron-centred NHase. We have found that iron-centred NHases tend to be a bit more tolerant of cyanide than the cobalt-centred ones which sounds hopeful if you want to have a play with getting some dynamic kinetic resolution of mandelonitrile type structures but they also tend to be a lot less chirally selective too! There is no chiral analysis in this paper, and obviously you don’t know if the cyanide resistance is due to cell structure or enzyme specific with this whole cell biotransformation.
  • Biotransformation of nitriles to hydroxamic acids via a nitrile hydratase-amidase cascade reaction by Vojtěch Vejvoda, Ludmila Martínková, Alicja B. Veselá, Ondřej Kaplan, Sabine Lutz-Wahl, Lutz Fischer and Bronislava Uhnáková in  Journal of Molecular Catalysis B: Enzymatic,  article in press at doi:10.1016/j.molcatb.2011.03.008
This paper describes the use of a two enzyme system to hydrate a number of alkyl and aryl nitriles to the related hydroxamic acid via a NHase-produced amide. Hydroxamic acids are an interesting endpoint because they form a coloured complex with ferric ions so there is leeway here to make a screen for enzyme activity. Interestingly, Martínková’s team develop a system using either the NHase from Rhodoccocus erythropolis A4 (as a cell free extract) or cell free extracts from E. coli clones bearing the NHases from either a strain of Raoultella terrigena or Klebsiella oxytoca. Obviously the Rhodoccocus is iron centred, but the other two are cobalt-centred, and the latter pair appear to be more robust towards hydroxylamine which is a nice contrast to the usual cyanide sensitivity. When I first started looking at nitrile-active enzymes a few years back, I came across a paper by Dadd (Biotechnology Letters 23; 221-225, 2001) showing how a whole cell preparation of Rhodoccocus rhodocrous LL100-21 could be used to this transformation though the authors ascribe the reaction to the nitrilase onboard this strain- interesting to see that this transformation works with both flavours of NHase too.

Tuesday 3 May 2011

Du Pont and NHases

One of the interesting things about the paper "Evidence for Participation of Remote Residues...etc" by Ondrechen and Ringe is that the source of the recombinant NHase is Du Pont. Du Pont have quite a history of applying this class of enzyme to chemical problems. A search of the chemical or patent literature for the names "Robert Di Cosimo" (sometimes DiCosimo on Espacenet), "Mark S Payne" and "Robert Fallon" pulls up a range of uses of NHases.

For instance, WO 2006049618 which protects the NHase and amidase from Comamonas testosteroni 5-MGMA-4D from both authors, or the 1997 paper in Applied Microbiology and Biotechnology from Fallon, Stieglitz and Turner called "A Pseudomonas putida capable of stereoselective hydrolysis of nitriles" which describes work on the strain NRRL-18668.

NCBI numbers for May

A quick text search for "nitrile hydratase" under proteins on NCBI's website shows 2869 (+42 this month) hits for the phrase, and 1042 (+19 in the last month) RefSeq hits. It describes on the NCBI website how the RefSeq collection is their gold standard selection, so there has been high proportion of good stuff uploaded in the last month.

No change in the number of structures listed on the Protein Data Bank.

Thursday 14 April 2011

The paper that goes with 3QXE

The paper which describes the work that lead to the pdb file 3QXE and its mutant brethren has just been put up as an accepted manuscript for the ACS journal, Biochemistry. Its corresponding authors are Mary Jo Ondrechen and Dagmar Ringe, and it goes by the inviting title of "Evidence for Participation of Remote Residues in the catalytic activity of Co-type Nitrile hydratase from Pseudomonas putida". They have been looking at which residues in the outer shells around the active site influence activity.

Tuesday 12 April 2011

PDBs on a phylogenetic tree

There is a figure of a phylogenetic tree in the Prasad Balla review from 2010 which gives a separation of the different currently known sequences. I have very roughly stitched the tree together from the two parts given on two pages of the review and then I have dropped on top the rough positions of the three most distinct cobalt centred PDB files (1V29, 1UGP and 3QXE) and one of the rhodococcus iron centred files (1AHJ). It is interesting that workers seem to be producing PDB files solely from the right hand half of this tree.

3QXE- the new NHase pdb file- from Pseudomonas putida

I thought I'd have a look to see how different the new pdb file was from those already in the public domain.
First of all a ClustalW alignment of the alpha chains shows that it has a score of 52 with 1V29 and a score of 54 with 1UGP. (For reference, 1V29 and 1UGP have a score of 59 between them). The numbers might not be completely robust because I note that the modified cysteines are not dealt with the same in the three amino acid sequences. The central portions of the sequence are very similar, with most of the diversity at the ends (see below: 1V29 then 1UGP and finally 3QXE)
Using DeepView's RMS measure of 3D similarity after an Iterative Magic Fit of  the new pdb with the files for 1V29 and 1UGP enzymes you get two numbers of approximately 1-1.05 (for reference between 1V29 and 1UGP the value is 1.15). This all suggests that this new file describes a new distinct structural arrangement.

Monday 11 April 2011

But which is more popular nitrilase or nitrile hydratase?

Google have a rather entertaining Google Labs product called the NGram viewer where they allow you to check the ranking of the number of times that books published in a specific year use words which you define. Their examples are like "nursery school" versus "kindergarten". I thought it might be rather more relevant for this blog to try "nitrilase" against "nitrile hydratase".

NGram viewer- nitrilase v nitrile hydratase

Basically nitrilase has been more popular apart from a burst of enthusiasm for NHase between 1988 and 1994, and a photofinish victory in 2003.

Thursday 7 April 2011

NCBI- April numbers for NHase

A quick text search for "nitrile hydratase" under proteins on NCBI's website shows 2827 (+150 in a month!) hits for the phrase, and 1023 (+35 since the start of March) RefSeq hits. The number of PDB hits is up 5 to 41, as a new crystal structure from the cobalt centred NHase from Pseudomona putida has been uploaded along with the data for four point mutants of it.

Monday 4 April 2011

Looking at the NHase from Monosiga brevicollis

I have been looking at the sequence for the single subunit NHase from the eukaryote Monosiga brevicollis. There are four things that spring to mind of interest:
1. The thing has joined the two prokaryotic subunits together in the fashion B to A.
2. The usual signature bit of the sequence says it is a cobalt centred NHase, and the alpha subunit is very similar to that shown by the two cobalt centred NHases with pdb files 1V29 and 1UGP.
 3. The joint between the alpha and beta subunits has a LOT of histidines in it, which explains why it took a long time to come off the nickel affinity column!

4. The surface loop which is nice differentiating feature between cobalt-centred and iron centred NHase (which dont have it) is very different with this beast. The picture below is of 1V29, with the loop indicated.