Enzymatic cascade synthesis of (S)-2-hydroxycarboxylic amides and acids using a hydroxynitrile lyase, nitrile-active enzymes and an amidase

This review (Journal of Molecular Catalysis B: Enzymatic, 114, 2015, 25–30) by van Rantwijk and Stolz covers the bienzymatic conversion of aldehydes into enantiomerically pure hydroxycarboxylic acids and amides via an enzymatic cascade of hydrocyanation and nitrile hydration/hydrolysis. It compares results obtained via cross-linked enzyme aggregates (CLEAs) as well as whole-cell Escherichia coli expressing two enzymes. It highlights these methods’ potential for yielding near-quantitative yield and ee at synthetically relevant concentrations.

NCBI Sequence numbers for nitrile hydratase and nitrilase to 27/2.16

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 10224 hits, of which 4117 were RefSeq data. 

There are 80688 sequences labelled as "nitrilase" (not sure how robust that is currently), of which 20872 are pegged as RefSeq data.

The Alpha Subunit of Nitrile Hydratase Is Sufficient for Catalytic Activity

That’s a title which is going to catch the eye.

Nitrile hydratases have two subunits, the alpha and beta, they zip together like the two bits of rubber that make a tennis ball and the active site sits in between the two, protecting the weird metallic centre from the all the life can throw at it. Here's a crystal structure (1UGP) with one subunit in light blue, the other in dark blue and the seam marked by red and green,

A new paper in Biochemistry (DOI: 10.1021/bi500260j) by Bandarian and co-workers describe a toyocamycin nitrile hydratase from Streptomyces rimosus which has three subunits. Toyocamycin is a pyrrolopyrimidine compound. Of the three subunits, one is similar to your usual alpha unit (ToyJ), one (ToyL) is similar to the front half of the beta subunit and the final bit (ToyK) is similar to the end of the beta subunit (diagram below from the paper using 1IRE as scaffold). These subunits were all cloned into E. coli and they produce pure recombinant ToyJKL which is orange and is shown to be a cobalt centred NHase. The amazing bit that happens after that is that they get ToyJ to express well by itself and able to hold the cobalt needed in the active site. They then show that this active protein can turn over its desired substrate nitrile (admittedly not as well as the full complex), and also 3-cyanopyridine. The authors speculate that the ToyKL bits might add substrate specificity, but as you might imagine this early in this research there is no structural data on the enzyme complex.

 

Nitrilase and nitrile hydratase from Pseudomonas sp. UW4

Having just finished a project where we looked at a range of nitrilases and what their preferred substrates are, it is always interesting to ponder what the bacterium actually wanted the enzyme for (as compared to the host of xenobiotics you threw at it). We have often had the situation where we have an enzyme which we reckon ought to be active but doesn't seem interested in any of the forty or so compounds we have in our simple screen.
There is a recent paper in Applied and Environmental Microbiology by Duca, Rose and Glick which is concerned with investigating the biosynthesis of indoleacetic acid (IAA), which is a plant growth hormone. This compound comes from indoleacetonitrile (IAN) and there are two obvious pathways to get from there to IAA- via the NHase and via a nitrilase. These workers cloned both enzymes in to E. coli, and then looked at their level of interest in IAN. Interestingly the nitrilase had a habit of producing a proportion of amide as well as the usual acid. Also of interest is that the enzymes have different pH and temperature optima (Nase like 50 degrees C and pH6, the iron-centred NHase likes 4 degrees C and pH7.5), though I wonder if the lower temperature for the NHase is due to the fundamental lack of stability of iron NHases rather than an adaption. Additionally, the authors use some bioinformatics to confirm their experimental findings that this is an aromatic nitrile active system.

Nitrile hydratase in March's Advanced Organic Chemistry

As a graduate student, March's Advanced Organic Chemistry was my "go-to" text for leading references for synthetic chemistry questions. It was an incredibly thick book with an extraordinary number of references, and it has only increased in size since my days in TCD, and it is now in its seventh edition.
As my indexing system these days needs more key strokes to navigate than is needed to find things on the Web using Google, I decided to find my first paper on nitrile hydratase online to see which substrates I should have in my "interesting nitriles" cupboard which were ortho-substituted. Sure enough the paper was there but so also was a link to that paper as a reference in March. Yes, it's reference 103 on page 1080 of the seventh edition, as a reference to consult about amide formation though the reason for its inclusion is a bit weird. Anyway, I am very pleased!

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.

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

Vikash Babu, Bijan Choudhury, Competitive adsorptions of nitrile hydratase and amidase on polyacrylonitrile and its effect on surface modification, Colloids and Surfaces B: Biointerfaces, Volume 89, 1 January 2012, Pages 277-282, 10.1016/j.colsurfb.2011.08.025.

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?

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.

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.

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.

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.

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.

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.

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!

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. 

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.

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.

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.