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].

Searching with CTLCSC or CSLCSC

If you search the NCBI archives for nitrile hydratase enzymes just using that name you do get a large number of hits. Previous blog posts have recorded the size and rate of change of the number of hits you get if you do simple text searches with the phrase "nitrile hydratase". It is a slightly different story if you look at the RefSeq subset and then search through for the six amino acid sequence which is the metal binding motif for NHases. I am sure there are more elegant ways to do this but the way I did this was to download the relevant search with the sequences in FASTA format, load it up into MS Word and then use the "Find" function to look for each occurrence. [You can use the "reading highlight" tool and it gives you the count in the pop up box immediately].
Anyway the results were
CTLCSC (cobalt centred) -  123 hits
CSLCSC (iron centred) -  19 hits
So there are many more cobalt versions (87%) recorded than iron versions (13%) currently but given the incompleteness of the genome record, I reckon this may represent the current status of what that has been sequenced rather than the relative levels of occurrence in the wild.

Hyperthermophilic nitrile hydratases?

There are plenty of reports of nitrilases which have been cloned from organisms with names that denote obviously heat-loving tendencies- an example is the “Cloning, overexpression, and characterization of a thermoactive nitrilase from the hyperthermophilic archaeon Pyrococcus abyssi” by Mueller, Egorova, Vorgias, Boutou, Trauthwein, Verseck and Antranikian in Protein Expr Purif. 2006, 47(2), 672-81. It is quite noticeable that NHases do not appear to have the same hyperthermophilicity. If you search the translated genomes of the four organisms which produce nitrilases in this paper (Pyrococcus abyssi, Pyrococcus horikoshii, Pyrococcus furiosus, and Aeropyrum pernix) by using BLASTp against the alpha subunit of my favourite cobalt centred NHase CGA009 or the “local” iron centred NHase AJ270, you get just about nothing that shows any similarity.

Something that does stand out from examination of all the current PDB files of NHases that almost everyone says their NHase is thermostable or thermophilic. I get the impression that this labelling of these generally sensitive enzymes in itself tells a story. 

Cobalt binding in 3QXE

Above is an excerpt from Figure 6C in "Evidence for Participation of Remote Residues...etc by Ondrechen and Ringe which very nicely shows how the cobalt ion is bound into a nitrile hydratase.  There are three sulphur based points of attachment- aC112, aC115 and aC117, the latter two as sulfinic acids and two amide nitrogen points of attachment, aS116 and aC117. It is really clearly shown in this view how the arginine bR52 preorganizes the sulfinic cysteines and amide nitrogens into a planar SSNN arrangement.

New papers on nitrile hydratase enzymes

A couple of NHase papers, one new and the other upcoming:

• Biosynthesis of 2-amino-2,3-dimethylbutyramide by nitrile hydratase from a newly isolated cyanide-resistant strain of Rhodococcus qingshengii  by  Zhi-Jian Lin, Ren-Chao Zheng, Yu-Guo Zheng and Yin-Chu Shen in Biotechnology Letters, DOI: 10.1007/s10529-011-0623-7.

This paper reports a Rhodoccocus which can do the usual nitrile hydration reaction but can do it in the presence of significant quantities of cyanide. The paper describes whole-cell biotransformation conditions but I am guessing from the species this is an iron-centred NHase. We have found that iron-centred NHases tend to be a bit more tolerant of cyanide than the cobalt-centred ones which sounds hopeful if you want to have a play with getting some dynamic kinetic resolution of mandelonitrile type structures but they also tend to be a lot less chirally selective too! There is no chiral analysis in this paper, and obviously you don’t know if the cyanide resistance is due to cell structure or enzyme specific with this whole cell biotransformation.

• Biotransformation of nitriles to hydroxamic acids via a nitrile hydratase-amidase cascade reaction by Vojtěch Vejvoda, Ludmila Martínková, Alicja B. Veselá, Ondřej Kaplan, Sabine Lutz-Wahl, Lutz Fischer and Bronislava Uhnáková in  Journal of Molecular Catalysis B: Enzymatic,  article in press at doi:10.1016/j.molcatb.2011.03.008

This paper describes the use of a two enzyme system to hydrate a number of alkyl and aryl nitriles to the related hydroxamic acid via a NHase-produced amide. Hydroxamic acids are an interesting endpoint because they form a coloured complex with ferric ions so there is leeway here to make a screen for enzyme activity. Interestingly, Martínková’s team develop a system using either the NHase from Rhodoccocus erythropolis A4 (as a cell free extract) or cell free extracts from E. coli clones bearing the NHases from either a strain of Raoultella terrigena or Klebsiella oxytoca. Obviously the Rhodoccocus is iron centred, but the other two are cobalt-centred, and the latter pair appear to be more robust towards hydroxylamine which is a nice contrast to the usual cyanide sensitivity. When I first started looking at nitrile-active enzymes a few years back, I came across a paper by Dadd (Biotechnology Letters 23; 221-225, 2001) showing how a whole cell preparation of Rhodoccocus rhodocrous LL100-21 could be used to this transformation though the authors ascribe the reaction to the nitrilase onboard this strain- interesting to see that this transformation works with both flavours of NHase too.

Du Pont and NHases

One of the interesting things about the paper "Evidence for Participation of Remote Residues...etc" by Ondrechen and Ringe is that the source of the recombinant NHase is Du Pont. Du Pont have quite a history of applying this class of enzyme to chemical problems. A search of the chemical or patent literature for the names "Robert Di Cosimo" (sometimes DiCosimo on Espacenet), "Mark S Payne" and "Robert Fallon" pulls up a range of uses of NHases.

For instance, WO 2006049618 which protects the NHase and amidase from Comamonas testosteroni 5-MGMA-4D from both authors, or the 1997 paper in Applied Microbiology and Biotechnology from Fallon, Stieglitz and Turner called "A Pseudomonas putida capable of stereoselective hydrolysis of nitriles" which describes work on the strain NRRL-18668.

NCBI numbers for May

A quick text search for "nitrile hydratase" under proteins on NCBI's website shows 2869 (+42 this month) hits for the phrase, and 1042 (+19 in the last month) RefSeq hits. It describes on the NCBI website how the RefSeq collection is their gold standard selection, so there has been high proportion of good stuff uploaded in the last month.

No change in the number of structures listed on the Protein Data Bank.