Connecting B to A

Looking back on the bioinformatic analysis of eukaryotic nitrile hydratases in Marron, Akam and Walker's PLOS One paper to be found here, I thought it would be interesting to look at the link between the two segments which are separate subunits in prokaryotic NHases.
I mocked up this model of where the link had to go from two prokaryotic subunits which had most similarity to the Monosiga brevicollis NHase. The dangling ends of the right hand side of the structure indicate where the linker has to go.

As the easiest to access, I chose to align the protein sequences suggested by the transcriptions of ESTs from the four organisms held within the Broad Institute's Origins of Multicellularity project, namely M. brevicollis, Thecamonas trahens, Salpingoeca rosetta and Sphaeroforma arctica. (NB order in alignment: MB, SR, TT and last SA)

As Marron's paper comments:
"This histidine-rich region is prominent in T. trahens (12 residues), S. arctica (11 residues) and M. brevicollis (17 residues), but shorter in [...] S. rosetta (2 residues)."
That seems quite a lot of variation with only M. brevicollis having a string of pure histidines. In contrast the sequences are around the metal binding segment are quite similar.

Data to enzyme

I was talking at the "Leading Industrial Biotechnology: Focus on Biocatalysis" meeting last week in York, and I put up this slide which caused a bit of comment amongst those of a chemistry background in the audience who didn't know the power of state-of-the-art molecular biology for those looking for new enzymes not available from catalogue based suppliers. Moral of the story: find yourself a molecular biologist and you will have more (and more interesting) enzymes available than you can imagine.

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).



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)