Comparing 3D structures of a Fe-centred NHase and a Co-centred NHase

Using DeepView, I have used their "Iterative Magic Fit" to compare the AJ270 structure (Fe-centred) with the cobalt-centred structure reported as 3HHT by Van Wyk, Sewell, Cowan, Sayed, Tsekoa and Tastan Bishop.
The top picture shows how much homology there is between a cobalt centred NHase and an iron-centred NHase which I essentially chose at random [blue indicates similar, grading to yellow for dissimilar]. I have labelled the position of the metals in the centre of the overlayed proteins, and the text is unreadable because they fall on top of each other with this fit (which is based on aligning the carbon backbone)

The second picture is an attempt to show this metal-containing middle portion in greater detail.

This final picture above shows the 3D structure window and the primary sequence alignment window together. The lower sequence (Co-centred 3HHT) shows an extra section which isnt in the upper sequence (Fe-centred  AJ270), and corresponds to the alpha helix which is coloured yellow and lies roughly horizontally across the middle of the enzyme structure. This is a bit of structure which has been suggested as making a crucial difference in terms of extra stability.

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.

3A8O vs 2QDY

I have been playing with Deepview which offers a handy function where you can overlay pdb files and the resulting structure is colour coded for alignment diversity. Blue indicates really not much at all. As I have mentioned before the pdb for the Rhodococcus species AJ270 is coded as 2QDY and that for Rhodococcus species N-771 is 3A8O. Overlaying these two shows almost everything is dark blue, apart from differences for the two post-translationally modified cysteines in the active site and one bit dangling off the end. So that would be very similar then. You can see the same sort of picture if you overlay these with the 2CYZ too.

Unravelling the mechanism of NHase hydration

This paper (corresponding author, Masafumi Odaka) develops the knowledge around how a nitrile hydratase actually manages to stuff a water into a rather stable carbon-nitrogen triple bond. It also resulted in a rather nice suite of crystal structures- one parent structure (in pdb as 3A8O) and four single point mutants.

*Kinetic and structural studies on roles of the serine ligand and a strictly conserved tyrosine residue in nitrile hydratase - Yamanaka, Hashimoto, Ohtaki, Noguchi, Yohda and Odaka, in Journal of Biological Inorganic Chemistry (2010), doi: 10.1007/s00775-010-0632-3

This is an image produced using DeepView of 3A8O with the position of the iron labelled.

When expression doesn't work...

In the light of my comment in the last blog post commending the Clarke review for its table of successfully expressed enzymes, I thought I would put up here the following information... two genome sourced sequences, which in E.coli, gave inactive protein.

 >gi|50084777|ref|YP_046287.1| along with its beta subunit from Acinetobacter sp. ADP1 which we predicted would be an iron centred NHase
>gi|27379609|ref|NP_771138.1| along with its beta subunit from Bradyrhizobium japonicum USDA 110 which you would predict from its sequence to be a cobalt centred NHase.

Not sure why these didn't work... but with the wealth of sequences out there, there is always another one to aim at!

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.

Rotating image

There used to be a rather neat website (sadly since departed) which convert pdb files to animated GIFs which you could use to create slightly rough and ready rotating models of enzymes. This rotating model of a NHase was produced this way.

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.

Common features of cobalt and iron based NHases

With a couple of local colleagues, I have a paper in press with the International Journal of Data Mining and Bioinformatics. It is the eventual result of Joe, a mathematician asking if I had any high dimensional data sets that needed interrogating. In terms of this blog, it has a section looking at what are the main primary sequence features (using a set of one hundred NHases) which are important in distinguishing cobalt centred NHases from iron centred ones. Thanks to Marcus for all the 3D structure analysis, and spotting the hinge/loop differences first suggested as relevant to differential stability by Miyanaga in 2001

http://northumbria.academia.edu/JoeFaith/Papers/320396/Predicting_Functional_Residues_of_Protein_Sequence_Alignments_as_a_Feature_Selection_Task

Two recent articles on NHases

*Unique Biogenesis of High-Molecular Mass Multimeric Metalloenzyme Nitrile Hydratase: Intermediates and a Proposed Mechanism for Self-Subunit Swapping Maturation-

Zhou, Hashimoto, Cui, Washizawa, Mino, and Kobayashi in Biochemistry (2010)

DOI: 10.1021/bi100651v

An investigation of the subunit structure of the NHases in Rhodococcus rhodochrous J1.

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*High-yield continuous production of nicotinic acid via nitrile hydratase-amidase cascade reactions using cascade CSMRs 

Cantarella, Gallifuoco, Malandra, Martinkova, Spera, and Cantarella in Enzyme and Microbrial Technology(article in press, 2010)

DOI: 10.1016/j.enzmictec.2010.12.010

This looks at using the NHase/amidase system from Microbacterium imperiale CBS 498-74 in a bioreactor to convert 3-cyanopyridine to nicotinic acid.

Recent patent from Mitsubishi Rayon

Google alerts tells me that Mitsubishi Rayon have published a patent for a heat resistant nitrile hydratase which works on aromatic nitriles.

US Patent 7803578 http://www.freepatentsonline.com/7803578.html

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.

First Post

The point of this blog is to follow what is going with one particular class of enzyme, nitrile hydratase. I may stray the odd time into posting about nitrilases or amidase but my research interest is more on NHases.

First up, let me give a couple of references to NHase papers where I am corresponding author:

Biotransformation of nitriles using the solvent-tolerant nitrile hydratase from Rhodopseudomonas palustris CGA009- Black, Gregson, McPake, Perry and Zhang in Tetrahedron Letters (2010) doi:10.1016/j.tetlet.2009.12.094

Probing the Enantioselectivity of a Diverse Group of Purified Cobalt-Centred Nitrile Hydratases- van Pelt, Zhang, Otten, Holt, Sorokin, Rantwijk, Black, Perry and Sheldon (accepted manuscript, 2011) DOI: 10.1039/C0OB01067G

The recombinant enzymes mentioned in these papers are available for purchase through a few companies, namely Prozomix, Almac and CLEA Technologies.

If you are a UK/EU based academic, and interested in working with our enzymes, feel free to contact me.