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 hydratases and amidases from Rhodococcus erythropolis strains

Antonie van Leeuwenhoek has a paper (Expression control of nitrile hydratase and amidase genes in Rhodococcus erythropolis and substrate specificities of the enzymes) from Nesvera and co-workers at the Institute of Microbiology in Prague which looks at the expression of the aldoxime dehydratase/nitrile hydratase/amidase pathway in two different strains of NHase from Rhodococcus erythropolis- A4 and CCM2595. It also looks at some of the selectivity of the constituent enzymes in this pathway towards cyanohydrins. They make some comparisons to the AJ270 strain which we  published on in 2011. Rather like us, they find that Rhodococcus erythropolis strains are not very enantioselective NHases for mandelonitrile derivatives- our paper reports E=2 for unadorned mandelonitrile.

 

PVA-chitosan based immobilization of NHase for dynamic kinetic resolution of rac-mandelonitrile

A recently published paper by Pawar and Yadav in Industrial and Engineering Chemistry Research entitled “Enantioselective Enzymatic Hydrolysis ofrac-Mandelonitrile to R-Mandelamide by Nitrile Hydratase Immobilized onPoly(vinyl alcohol)/Chitosan–Glutaraldehyde Support” describes the improvement in performance the nitrile hydratase from Rhodococcus rhodocrous ATCC BAA-870 exhibits when immobilized on PVA/chitosan. The reaction being tested is the DKR of rac-mandelonitrile.

New patent on NHase from Mitsubishi Rayon

Mitsubishi Rayon have published a new US patent (20140120588) protecting a specific set of primary sequences of NHase.

Enhancement of NHase stability with self assembling peptides

There is an in-press paper available online entitled “Enhancement ofthermo-stability and product tolerance of Pseudomonasputida nitrile hydratase by fusing with self-assembling peptide” in the Journal of Bioscience and Bioengineering by Zhemin Zhou and co-workers. They describe how they have used some self-assembling peptide based tags appended to the beta subunit to enhance thermal stability and substrate tolerance.

A mechanism model for NHase using a cyclic and a disulfide intermediate.

There is a very interesting paper proposing a new mechanism for the hydration reaction of iron-centred NHases published in Inorganic Chemistry by Kathrin Hopmann. The paper, entitled "Full Reaction Mechanism of Nitrile Hydratase: A Cyclic Intermediate and an Unexpected Disulfide Switch" sides with the opinion that the nitrile ligates to the iron centre. Hopmann suggests that the oxygen of the cysteine post-translationally modified to a sulfenic acid then acts as the nucleophile attached the C-N bond.

 

This oxygen is the one that becomes the oxygen in the nascent amide carbonyl, with the two cysteine sulfurs combining to form a disulfide link.

After loss of the amide from the iron centre, the cysteine is oxidized again using an oxygen from water. My suspicion with all these mechanistic investigation is that it may be that a lot of different mechanisms may operate with the dominant one being very tied to the specific sequence/space properties of each enzyme. I have been a fan of nitrile-bound mechanisms solely on the basis that they seem much more likely to render chiral selectivity which we know is a possibility for some enzymes and some substrates.

I also wonder how the numbers from the calculations reported in this paper change for cobalt centre NHases.

Two publications on the nitrilase from Sphingomonas wittichii RW1

Sphingomonas wittichii RW1 is a bacterium which has been shown to have the ability to degrade polychlorinated dioxins, and hence has had its genome fully sequenced. It has two different nitrilases in its genome.

YP_001264656.1

YP_001261492.1

Two papers just released with the corresponding authors Er-Zheng Su and Dong-Zhi Wei report on the nitrilase with the accession code YP_001264656. These authors and enzyme have previously been mentioned on this blog- I complained there was no information on the enzyme discovery... here it is!

The first entitled “Cloning, Overexpression, andCharacterization of a High Enantioselective Nitrilase from Sphingomonaswittichii RW1 for Asymmetric Synthesis of (R)-Phenylglycine” and published in Applied Biochemistry and Biotechnology: Part A: Enzyme Engineering and Biotechnology describes the construction of a mini-library of nitrilase enzymes chosen by the similarity to a nitrilase from Pseudomonas fluorescens EBC191 (GenBank accession no. AAW79573) which has been shown to be highly activity toward phenylglycinonitrile. The nitrilase from Sphingomonas wittichii RW1 was shown to be most promising nitrilase to achieve chiral synthesis of R-phenylglycine in a kinetic resolution mode.

The second entitled “Efficient asymmetric synthesis ofD-N-formyl-phenylglycine via cross-linked nitrilase aggregates catalyzeddynamic kinetic resolution” and published in Catalysis Communications describes the preparation of cross-linked enzyme aggregates (CLEAs) of nitrilase from Sphingomonas wittichii RW1, and then its use to perform dynamic kinetic resolution to make D-N-formyl-phenylglycine. The paper also demonstrates that the CLEAs have improved stability over non-immobilized enzyme and are reuseable up to six times whilst retaining 70% initial activity.

NHase expression in Corynebacterium glutamicum

Industrial scale biocatalytic production of acrylamide relies on the use of engineered strains of Rhodococcus. There is a newly accepted manuscript in Applied Microbiology and Biotechnology from three Korean groups lead by J-H Lee and H-S Kim where the NHase from a Rhodococcus strain is expressed in Corynebacterium glutamicum (a cell factory already used commercially in amino acid biosynthesis) and tested for its ability to hydrate acrylonitrile. Whilst it didnt have the same activity as the NHase in the homologous system, the advantage the authors propose is that the rate of growth and hence enzyme production is higher with C. glutamicum.

 

Chiral intermediate for cilastatin by nitrilase hydratase/amidase combo

There is a new accepted manuscript in the Journal of Molecular Catalysis B from Yu-Guo Zheng and co-workers entitled "Industrial production of chiral intermediate of cilastatin by nitrile hydratase and amidase catalyzed one-pot, two-step biotransformation" which describes their research into using a chiral nitrile to carboxylic acid conversion which results from a cascade of NHase from (Rhodococcus boritolerans FW815) then amidase (from Delftia tsuruhatensis ZJB-05174). The compound they are aiming to hydrolyze is rac 2,2-dimethylcyclopropanecarbonitrile to the S-2,2-dimethylcyclopropanecarboxamide which is an intermediate in the synthesis of cilastatin (a booster component to some antibiotics which prolongs their activity by blocking the kidney’s dehydropeptidase activity). This is achieved using a NHase which is notably active but not chirally selective, and an amidase which is only avid for the R enantiomer of the amide. The R acid and S amide are separated using macroporous resin adsorption chromatography and the R acid is converted to the acid chloride. This can than have its chirality scrambled with a bit of heat such that its conversion to amide by addition of ammonia gives a fresh racemic batch of amide to challenge the R selective amidase with. The conditions described are to get it to work on the 100kg scale. Considering that this is a report on a process which has been undergoing continuous optimization since 2005, I guess the choice of amidase to do the chiral resolution was “baked in” before there was literature evidence of significantly stereoselective nitrile hydratases which might have prompted an enzyme discovery effort in that direction. Having said that racemization using the wrong acid chloride and heat rather than on the wrong isomer of the nitrile is a neat touch.

Nitrilase from sequence to 50 litre scale

We have been working on developing a specific nitrilase reaction in a consortium with Chemoxy and Biocatalysts Ltd with funding assistance from the UK Technology Strategy Board. In nine months, we have gone from the selection of amino acid sequences to expressed active proteins to assessment of substrate preference using a novel greener assay method which works with cell-free extracts to chemistry on the one litre scale, and finally at the end of last month to doing the reaction in the 50 litre plant at CPI's National Industrial Biotechnology Facility. Just to finish the boasting, the outcome of that reaction was over a kilo of product with great conversion.

We are now going into a TSB-funded collaborative research and development project with the same partners to really scale this process and bring it nearer to market by optimizing the enzyme, its use and reuse. It's going to be fun.

Dynamic Kinetic Resolution in alpha aminonitrile hydrolysis

The use of an equilibrium between enantiomers of your starting material to enable hydrolysis of more than 50% of a racemic mixture has been a point of keen interest in nitrilase research. The hurdle to it becoming widespread  has tended to be that the pH at which racemization occurs at a useful rate tends not to be one that your standard biocatalyst is happy operating at. A newly accepted manuscript into Tetrahedron Letters called “High yield synthesis of D-phenylglycine and its derivatives bynitrilase mediated dynamic kinetic resolution in aqueous-1-octanol biphasicsystem” by Jian Qiua, Erzheng Su, Wei Wang, and Dongzhi Wei is a useful addition to this literature. They use a nitrilase (after citing unpublished data on its enantioselectivity… why unpublished? It looks an interesting nitrilase!) from Sphingomonas wittichii RW1 to get DKR in a biphasic system (buffer/octanol).

How does the nitrile hydratase activator protein work?

This is a question which is still up for debate. It would appear to be involved with incorporation of the cobalt ion in those NHases which are cobalt-centred. In a newly accepted manuscript of FEMS Microbiology Letters entitled “The effect of flexibility and positive charge of the C‐terminal domain on the activator P14K function for nitrile hydratase in Pseudomonas putida” by Zhemin Zhou and co-workers, mutants of the relevant proteins were modelled and made, and then tested in the hydration of 3-cyanopyridine.

 

Supporting a nitrile hydratase for better performance

There is a new accepted manuscript for the Journal of Molecular Catalysis B: Enzymatic which adds to the literature on the support of a nitrile hydratase to increase this notoriously sensitive enzyme’s robustness.  PVA/Chitosan-glutaraldehyde cross-linkednitrile hydratase as reusable biocatalyst for conversion of nitriles to amides by SV Pawar and GD Yadav  describes how to prepare a cross linked assembly containing the NHase from Rhodococcus rhodochrous ATCC BAA-870. When they test this immobilized enzyme in the hydration of 3-cyanopyridine, they find it is able to withstand a higher temperature and more basic conditions. They also show that their preparation is a reusable format which retains 50% of activity after 8 batches.

The figure below from the paper shows relative activity of free and immobilized NHase over time at 45 degrees C in 0.1M phosphate buffer. The upper line is the immobilized form.