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.