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.