NHase in halophilic archaeon DL31

I notice that both an alpha chain and a beta chain of a cobalt centred NHase have been recently annotated (14 June 2012) in the genome of the rather unprepossessingly named "halophilic archaeon DL31". It must be quite an elusive beast because Google Images doesn't deliver a picture of anything suitable.
Halophilicity quite often comes with tolerance to alkaline conditions, which can mean all sorts of utility in the world of synthetic biocatalysis. As I mentioned in the "No extremes" post we published with Roger Sheldon's group on the synthetic potential of one of these halophilic NHases, but this is a good example of a genome-sourced potential halophilic NHase. BLASTing both the alpha and beta chains against prokaryotic genomes shows that the beta chain is most similar to beta chain of Caldalkalibacillus thermarum TA2.A1 (I think we can guess the proclivities of this microbe) with 3 of the next 4 best hits being the sequences from the crystal structures 3HHT, 2DPP and 1V29 all of which are moderately thermophilic.

Interestingly there is a slightly different aspect to the alpha chain BLAST: Pseudonocardia dioxanivorans and Rhodopseudomonas palustris make up the top hits before Caldalkalibacillus thermarum TA2.A1 turns up.

Salt bridges and hydrogen bonding

I have been playing with PISA tool as it offered insight into size and nature of the interface between the two subunits in NHases. I stuck in the PDB data for the most distinct cobalt-centred NHases- 1UGP, 1V29 and 3HHT, and PISA suggested that these have 61, 67 and 65 hydrogen bonds (respectively) between the alpha and beta subunits, and between 26 and 29 salt bridges across this interface. Compare these numbers with that for 2QDY (the iron-centred NHase from AJ270)... 51 hydrogen bonds and 8 salt bridges. That looks quite a difference... sadly things might not be so clear-cut in grouping these enzymes by metal centre- the newest PDB for 3QXE gives an average of 53 hydrogen bonds and 15 salt bridges, and that is cobalt-centred. Furthermore I do wonder, with such a small set of structures, whether we are seeing a distinction based on the fact that the first three cobalt-centred PDBs are of enzymes specifically tagged as "thermophilic", and AJ270 and Pseudomonas putida aren't. [I also looked at 2DPP and got 62 hydrogen bonds and 27 salt bridges].

How many clearly different structures of NHase are reported as pdb files?

Another helpful thing that the Iterative Magic Fit function within DeepView can do is provide a numerical value (based on a RMS calculation) for the similarity of the three dimensionality of the enzymes being overlaid. After concentrating on enzymes which are clearly NHases, discarding structures which are clearly mutants of an existing structure (which is either obvious from the title or the combination of author and date of submission), you get left with 13 distinct pdb files [1AHJ, 1IRE, 1UGP, 1V29, 2AHJ, 2CYZ, 2CZ6, 2D0Q, 2DPP, 2QDY, 2ZPB, 3A8O and 3HHT]. I then IMFed every one of these against each other to get a matrix of values for similarity. Basically if they had a value of less than 0.5, they look pretty much the same, greater than 1 than they look noticeably different.
The results of this showed that ALL the iron centred enzymes have RMS of <0.5 with each other which is not entirely surprising since they are all from Rhodococcus species. They all have RMS of greater than 1 for all the cobalt centred enzymes. There are two distinctly different cobalt centred arrangements: the Pseudonocardia thermophila pair of 1IRE and IUGP, and the trio of Bacillus structures of 1V29, 2DPP and 3HHT. Within these groupings they have RMS of less than 0.5, and between the groupings RMS of greater than 1.
Effectively this says to me that there are three basic structures for NHase known currently- one iron based and two cobalt based. Below is an excerpt from a table holding all the results from these calculations which shows this clustering.

PDB ID	1AHJ	1IRE
1AHJ		1.06
1IRE	1.06
1UGP	1.06	0.07
1V29	1.19	1.14
2AHJ	0.45	1.09
2CYZ	0.44	1.07
2CZ6	0.47	1.09
2D0Q	0.45	1.09
2DPP	1.22	1.16
2QDY	0.46	1.09
2ZPB	0.44	1.08
3A8O	0.47	1.11
3HHT	1.22	1.15