A crystal structure of nitrilase Nit6803 from Syechocystis sp. PCC6803

PDB: 3WUY_A

>gi|742261201|pdb|3WUY|A Chain A, Crystal Structure Of Nit6803
GSHMLGKIMLNYTKNIRAAAAQISPVLFSQQGTMEKVLDAIANAAKKGVELIVFPETFVPYYPYFSFVEP
PVLMGKSHLKLYQEAVTVPGKVTQAIAQAAKTHGMVVVLGVNEREEGSLYNTQLIFDADGALVLKRRKIT
PTYHERMVWGQGDGAGLRTVDTTVGRLGALACWEHYNPLARYALMAQHEQIHCGQFPGSMVGQIFADQME
VTMRHHALESGCFVINATGWLTAEQKLQITTDEKMHQALSGGCYTAIISPEGKHLCEPIAEGEGLAIADL
DFSLIAKRKRMMDSVGHYARPDLLQLTLNNQPWSALEANPVTPNAIPAVSDPELTETIEALPNNPIFSH

PDB: 3WUY_B

>gi|742261202|pdb|3WUY|B Chain B, Crystal Structure Of Nit6803
GSHMLGKIMLNYTKNIRAAAAQISPVLFSQQGTMEKVLDAIANAAKKGVELIVFPETFVPYYPYFSFVEP
PVLMGKSHLKLYQEAVTVPGKVTQAIAQAAKTHGMVVVLGVNEREEGSLYNTQLIFDADGALVLKRRKIT
PTYHERMVWGQGDGAGLRTVDTTVGRLGALACWEHYNPLARYALMAQHEQIHCGQFPGSMVGQIFADQME
VTMRHHALESGCFVINATGWLTAEQKLQITTDEKMHQALSGGCYTAIISPEGKHLCEPIAEGEGLAIADL
DFSLIAKRKRMMDSVGHYARPDLLQLTLNNQPWSALEANPVTPNAIPAVSDPELTETIEALPNNPIFSH

Making an approximate 3D model of an enzyme

Sometimes we want to check out how an amino acid sequence from a database might translate into a 3D dimensional structure. This is straightforward enough if there exists an x-ray crystal structure for that exact enzyme in the Protein Data Bank or, like often happens in the nitrile hydratase class, there is very limited variation within sequences so overlaying a short portion of the sequence "by eye" on an existing enzyme is possible.
We are currently working on nitrilases, and despite increasing interest in their use as biocatalysts, the number of nitrilases (i.e. enzymes that convert nitriles to carboxylic acids not the other looser biochemical definition) which exist as structures in the PDB s precisely one. It is from Pyrococcus abyssi and is pretty much restricted in substrate tolerance to small aliphatic nitriles like fumaronitrile. It should not be a surprise that it isnt a great model for many other nitrilases.
If the PDB has come up short we tend to use a link out of the Uniprot database to give a prediction of 3D structure. So if you want to see an estimate of what the nitrilase from Aurantimonas manganoxydans looks like, than the link labelled "ModBase" under the subtitle "3D structure databases" leads you to a page of predictions and further tools. For this specific enzyme it is suggested that a mouse nitrilase superfamily structure contains the best 3D match.

3D structure of Wild Type Fe-type Nitrile Hydratase from Comamonas testosteroni Ni1

There is a new NHase PDB file available. With the code 4FM4, it refers to the NHase from the recent Holz paper.

Wild Type Fe-type Nitrile Hydratase from Comamonas testosteroni Ni1

The PDB from Comamonas testosteroni Ni1 is currently waiting (3/7.12) to be processed at the Protein Data Bank but will be called 4FM4.

NCBI numbers for September

It has been a while since I had done the usual text search for "nitrile hydratase" under proteins on NCBI's website. Having done the proper filtering job on this numbers in August, I know there is a lot of duplication, error and bizarre search engine behaviour going on but I do think it shows how quickly the number of vaguely applicable sequences is growing. As of today there are 3066 hits for the target phrase (+63 since July), and 1125 RefSeq hits (+20 in the same period).

Disappointingly there was no change in the number of structures listed on the Protein Data Bank.

Automated Protein Structure prediction... or probably not.

I was browsing through a bioinformatics textbook last week, and read up about the work of Prof  David Baker in the field of protein structure prediction. One of the resources that the Baker Lab offer for free to academic customers is access to their Rosetta methodology via their Robetta structure prediction software hosted on their own computers. It is a really easy to use resource with minimal hassle in terms of registration. Anyway I thought it might be interesting to see what an automated program might be able to make of the single protein NHase of Monosiga brevicollis which is made up of the standard two separate alpha and beta subunit proteins tied together, both of which are well known from 41 (and counting) fairly similar PDB files which have a high degree of similarity. First of all, you submit your FASTA sequence, and it parses it using the Ginzu domain prediction technique.

I was pretty pleased with this start as it spotted the two domains by BLAST, and match them to two PDB files of cobalt containing NHases. I then set it away to do the full structure prediction thinking that knowing those two starting points, most of the heavy lifting in terms of the structure prediction had been done. The prediction said it would take seven days but actually was finished after two (not a busy weekend for their computers it would appear!). Its output is the five best structures...

I have to say I have no confidence in any of those structures... all look a bit long and dangly for me. If you contrast them with a couple of PDB structures of NHases of the usual type below you can see that they tend to be much more compact- look where the two subunits' beta sheets are in the PDBs and in the prediction.

I guess it was asking a lot to get a reasonable guess at the histidine-heavy chain in the middle of the Monosiga NHase, and that is a crucial part of this prediction. No need to welcome our robot overlords just yet, and I need to go and talk to a human homology modeller!

3QXE- the new NHase pdb file- from Pseudomonas putida

I thought I'd have a look to see how different the new pdb file was from those already in the public domain.
First of all a ClustalW alignment of the alpha chains shows that it has a score of 52 with 1V29 and a score of 54 with 1UGP. (For reference, 1V29 and 1UGP have a score of 59 between them). The numbers might not be completely robust because I note that the modified cysteines are not dealt with the same in the three amino acid sequences. The central portions of the sequence are very similar, with most of the diversity at the ends (see below: 1V29 then 1UGP and finally 3QXE)

Using DeepView's RMS measure of 3D similarity after an Iterative Magic Fit of  the new pdb with the files for 1V29 and 1UGP enzymes you get two numbers of approximately 1-1.05 (for reference between 1V29 and 1UGP the value is 1.15). This all suggests that this new file describes a new distinct structural arrangement.

ClustalW analysis of pdb sequences

Following on from looking at the similarity of the pdb files, I thought I would run a ClustalW analysis of the primary sequences I used for that analysis. A lot are very similar (>98%) as you might expect, and once again it is obvious these files actually represent one iron centred and two cobalt centred templates. The iron centred template has scores between 40 and 50% for the cobalt centred pair, and the score between the two cobalt centred templates is about 60%.

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

Current PDB files for nitrile hydratase

PDB ID	Structure Title	Structure Author
1AHJ	NITRILE HYDRATASE	Huang, W., Schneider, G., Lindqvist, Y.
1IRE	Crystal Structure of Co-type nitrile hydratase from Pseudonocardia thermophila	Miyanaga, A., Fushinobu, S., Ito, K., Wakagi, T.
1UGP	Crystal structure of Co-type nitrile hydratase complexed with n-butyric acid	Miyanaga, A., Fushinobu, S., Ito, K., Shoun, H., Wakagi, T.
1UGQ	Crystal structure of apoenzyme of Co-type nitrile hydratase	Miyanaga, A., Fushinobu, S., Ito, K., Shoun, H., Wakagi, T.
1UGR	Crystal structure of aT109S mutant of Co-type nitrile hydratase	Miyanaga, A., Fushinobu, S., Ito, K., Shoun, H., Wakagi, T.
1UGS	Crystal structure of aY114T mutant of Co-type nitrile hydratase	Miyanaga, A., Fushinobu, S., Ito, K., Shoun, H., Wakagi, T.
1V29	Crystal structure of Nitrile hydratase from a thermophile Bacillus smithii	Hourai, S., Miki, M., Takashima, Y., Mitsuda, S., Yanagi, K.
2AHJ	NITRILE HYDRATASE COMPLEXED WITH NITRIC OXIDE	Nagashima, S., Nakasako, M., Dohmae, N., Tsujimura, M., Takio, K., Odaka, M., Yohda, M., Kamiya, N., Endo, I.
2CYZ	photo-activation state of Fe-type NHase in anaerobic condition	Kawano, Y., Hashimoto, K., Odaka, M., Nakayama, H., Takio, K., Endo, I., Kamiya, N., RIKEN Structural Genomics/Proteomics Initiative (RSGI)
2CZ0	photo-activation state of Fe-type NHase in aerobic condition	Kawano, Y., Hashimoto, K., Odaka, M., Nakayama, H., Takio, K., Endo, I., Kamiya, N., RIKEN Structural Genomics/Proteomics Initiative (RSGI)
2CZ1	photo-activation state of Fe-type NHase with n-BA in anaerobic condition	Kawano, Y., Hashimoto, K., Odaka, M., Nakayama, H., Takio, K., Endo, I., Kamiya, N., RIKEN Structural Genomics/Proteomics Initiative (RSGI)
2CZ6	Complex of Inactive Fe-type NHase with Cyclohexyl isocyanide	Nojiri, M., Kawano, Y., Hashimoto, K., Kamiya, N., RIKEN Structural Genomics/Proteomics Initiative (RSGI)
2CZ7	Fe-type NHase photo-activated for 75min at 105K	Nojiri, M., Kawano, Y., Hashimoto, K., Kamiya, N., RIKEN Structural Genomics/Proteomics Initiative (RSGI)
2D0Q	Complex of Fe-type NHase with Cyclohexyl isocyanide, photo-activated for 1hr at 277K	Nojiri, M., Kawano, Y., Hashimoto, K., Kamiya, N., RIKEN Structural Genomics/Proteomics Initiative (RSGI)
2DPP	Crystal structure of thermostable Bacillus sp. RAPc8 nitrile hydratase	Tsekoa, T.L., Tastan-Bishop, A.O., Cameron, R.A., Sewell, B.T., Sayed, M.F., Cowan, D.A.
2QDY	Crystal Structure of Fe-type NHase from Rhodococcus erythropolis AJ270	Song, L., Shi, J., Xue, Z., Wang, M.-X., Qian, S.
2ZCF	Mutational study on Alpha-Gln90 of Fe-type nitrile hydratase from Rhodococcus sp. N771	Takarada, H., Kawano, Y., Hashimoto, K., Nakayama, H., Ueda, S., Yohda, M., Kamiya, N., Dohmae, N., Maeda, M., Odaka, M., RIKEN Structural Genomics/Proteomics Initiative (RSGI)
2ZPB	nitrosylated Fe-type nitrile hydratase	Hashimoto, K., Suzuki, H., Taniguchi, K., Noguchi, T., Yohda, M., Odaka, M.
2ZPE	nitrosylated Fe-type nitrile hydratase with tert-butylisonitrile	Hashimoto, K., Suzuki, H., Taniguchi, K., Noguchi, T., Yohda, M., Odaka, M.
2ZPF	Complex of Fe-type nitrile hydratase with tert-butylisonitrile, photo-activated for 18min at 293K	Hashimoto, K., Suzuki, H., Taniguchi, K., Noguchi, T., Yohda, M., Odaka, M.
2ZPG	Complex of Fe-type nitrile hydratase with tert-butylisonitrile, photo-activated for 120min at 293K	Hashimoto, K., Suzuki, H., Taniguchi, K., Noguchi, T., Yohda, M., Odaka, M.
2ZPH	Complex of Fe-type nitrile hydratase with tert-butylisonitrile, photo-activated for 340min at 293K	Hashimoto, K., Suzuki, H., Taniguchi, K., Noguchi, T., Yohda, M., Odaka, M.
2ZPI	Complex of Fe-type nitrile hydratase with tert-butylisonitrile, photo-activated for 440min at 293K	Hashimoto, K., Suzuki, H., Taniguchi, K., Noguchi, T., Yohda, M., Odaka, M.
3A8G	Crystal structure of Nitrile Hydratase mutant S113A complexed with Trimethylacetonitrile	Yamanaka, Y., Hashimoto, K., Ohtaki, A., Noguchi, K., Yohda, M., Odaka, M.
3A8H	Crystal structure of Nitrile Hydratase mutant S113A complexed with Trimethylacetamide	Yamanaka, Y., Hashimoto, K., Ohtaki, A., Noguchi, K., Yohda, M., Odaka, M.
3A8L	Crystal structure of photo-activation state of Nitrile Hydratase mutant S113A	Yamanaka, Y., Hashimoto, K., Ohtaki, A., Noguchi, K., Yohda, M., Odaka, M.
3A8M	Crystal structure of Nitrile Hydratase mutant Y72F complexed with Trimethylacetonitrile	Yamanaka, Y., Hashimoto, K., Ohtaki, A., Noguchi, K., Yohda, M., Odaka, M.
3A8O	Crystal structure of Nitrile Hydratase complexed with Trimethylacetamide	Yamanaka, Y., Hashimoto, K., Ohtaki, A., Noguchi, K., Yohda, M., Odaka, M.
3HHT	A mutant of the nitrile hydratase from Geobacillus pallidus having enhanced thermostability	Van Wyk, J.C., Sewell, B.T., Cowan, D.A., Sayed, M.F., Tsekoa, T.L., Tastan Bishop, A.O.

3D structure of AJ270 NHase

We have worked with the nitrile hydratase from Rhodococcus erythropolis AJ270 which was a bacterium which was collected on the banks of the river Tyne in Newcastle. The key authors who have worked with this bacterium in synthesis over the years are Otto Meth Cohn (at Sunderland University) and Mei-Xiang Wang  (originally working with OMC but now at the Chinese Academy of Sciences, Beijing). M-X W has published a crystal structure of the nitrile hydratase from AJ270, and it can be found on PDB as 2QDY here. A little bit of playing with the data using PyMol and you can see how the two subunits fit together, and how there is a small channel from the exterior of the enzyme down into the active site.

NCBI

Every so often I do a quick and dirty search for the text string "nitrile hydratase" on the NCBI website under the "proteins" database tab. Just now there were 2672 proteins annotated thus, of which 985 were RefSeq. Obviously this double counts the number of enzymes (at least) because NHase is two subunit enzyme and this search doesnt distinguish between alpha and beta chains, and there are always some misannotated things... but maybe there are 400-1000 different nitrile hydratase sequences out there?
A similar text search of PDB gives 36 structures. Some are pretty similar and are plus/minus something in the active site. A rough trawl suggests that's about a dozen distinct structures.