New thermophilic NHase sequence

I have the NCBI database set up to send me a weekly email digest of newly uploaded nitrile hydratase sequences. One of the ones which appeared this week is from the proteome of Mycobacterium hassiacum DSM 44199. This organism is one of the Mycobacterium which use humanity as its primary habitat, and this particular one was isolated from a urine sample collected in Germany in 1995. The literature reports that this organism is comfortable with temperatures up to 65 degrees C, and can deal with up to 5% salt, all of which might well offer typical "moderately thermophilic" properties to its nitrile hydratase. The alpha chain shows a "CTLCSC" sequence suggesting it is cobalt-centred, and BLASTing this sequence shows that 7 other Mycobacterium species are between 89-85% sequence similar, with non-Mycobacterium sequences being significantly different (starting below 55% similar). Interestingly most of the other Mycobacteria which are sources of these sequences are from environmental samples with much lower temperature ranges. I cant find any reference to a paper where a Mycobacterium has been exploited for its nitrile hydratase activity either in the Prasad and Bhalla review from 2010 or from a Google Scholar search. Perhaps here's one to start with.

PDBs on a phylogenetic tree

There is a figure of a phylogenetic tree in the Prasad Balla review from 2010 which gives a separation of the different currently known sequences. I have very roughly stitched the tree together from the two parts given on two pages of the review and then I have dropped on top the rough positions of the three most distinct cobalt centred PDB files (1V29, 1UGP and 3QXE) and one of the rhodococcus iron centred files (1AHJ). It is interesting that workers seem to be producing PDB files solely from the right hand half of this tree.

NHases in organisms that aren't bacterial

A protein search amongst NCBI or SwissProt's data for NHases that arent in bacteria has been a pretty fruitless task. There are two notable exceptions to the "NHases are in bacteria, period" rule. The paper [Foerstner KU, Doerks T, Muller J, Raes J, Bork P. A nitrile hydratase in the eukaryote Monosiga brevicollis. PLoS ONE 2008;3:e3976.] showed that there is something in an aquatic metazoan Monsiga brevicollis which is combines the alpha and beta subunit in one protein chain. That sounded interesting so we have done some molecular biology magic and put that sequence in E. coli, and it expresses OK, though we havent had the time/manpower to play with it yet. The other outlier is pointed out in Prasad and Balla's Biotech Advances review of last year which points out that the plant Ricinus communis has also got the primary sequence for a NHasetoo. The usual tag sequence (VCTLCSC) in the alpha subunit suggests it is a cobalt centred one and if you ClustalW that with the cobalt-centred NHase which is featured in pdb file 1V29 (from Bacillus smithii) you get an alignment score of 54. Interesting.

General reviews about nitrile-active enzymes

A couple of new general reviews of NHase activity within the context of the whole gamut of nitrile-converting enzymes were published in 2010.

*Developments in nitrile and amide biotransformation processes- Velankar, Clarke, du Preez, Cowan and Burton, in Trends in Biotechnology (2010), doi:10.1016/j.tibtech.2010.08.004

This review is more from a microbiology perspective (not a chemical structure in sight) but with plenty of chemical detail none the less. It also includes a table I rather appreciate- Table 2 lists published examples of nitrilases, NHases and the related amidases which have been successfully recombinantly expressed in E.coli. Thanks very much for this info... now all we need is a list of the ones that don't successfully express this way!

*Nitrile hydratases (NHases): At the interface of academia and industry-  Prasad and Bhalla in Biotechnology Advances (2010), doi:10.1016/j.biotechadv.2010.05.020

This review appears to be the modern successor of the Banerjee review on nitrile active enzymes from 2002, but concentrates on NHase. It covers the usual material, and has discussion on mechanism and phylogeny. It also has some molecular modelling of the various structural intricacies.