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Various nitrilase structure projects

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Introduction

The study of the function and structure of nitrilases is in its infancy. Most work to date has been done by biotechnologists who have sought to exploit the versatile lys-cys-glu catalytic triad for the manufacture of acids of industrial importance from nitriles or for the detoxification of cyanide. Our contribution to date has been the determination of the quaternary structure of two cyanide dihydratases, from Bacillus pumilus and from Pseudomonas stutzeri by a combination of 3D EM, homology modelling and fitting. The structures have a novel, defined length, spiral form comprising 18 and 14 subunits respectively. We have proposed a model for the termination of the spiral based on our structure and have made predictions about the existence and nature of a new inter subunit contact which gives rise to the spiral.

The structural work we have done is not of sufficiently high resolution to visualise the interface or indeed the active site at atomic resolution – but it does give insight into the reasons for the persistent failure to crystallize these enzymes and suggests strategies to overcome this bottleneck. One goal of our future work is to address these aspects, however our structure also suggests that the nitrilase may be the core of a larger multienzyme complex and pursuing this line might give insight into the biological role of these fascinating enzymes.

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What we know about nitrilases

1. Structure

Knowledge at the atomic level is based on the crystal structures of some distantly related superfamily members. Our own papers describe the interesting quaternary structure of these enzymes.

2. Occurrence

By far the majority of papers simply note the occurrence of nitrilases in different organisms.

3. Sequences

It is not easy to find all the nitrilase superfamily sequences in the database. A comprehensive search was compiled by Pace and Brenner which is slightly out of date.

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Summary of proposed projects

Four different projects are proposed:

1. The structural effects of mutations on nitrilase homologues

The model proposed by Sewell et al (2003) makes certain specific predictions about the location and role of various parts of these enzymes with respect to the formation of spiral structures. It is proposed that a series of mutations be made to test the model. It is envisaged that this approach will give insight into the details of the structure of the spiral and the interaction between the quaternary structure and activity of these enzymes. Furthermore, it is hoped that one of the smaller complexes will crystallize allowing the visualization of the active site and the predicted, spiral forming “C” surface.

2. Details of the structural transition in Bacillus pumilus cyanide dihydratase

This nitrilase forms defined length (16-18 sbunit) spirals in the pH range 6-8. However at pH 5.4 it forms long helical rods. It is proposed that the structural change is driven by the interaction of a charged histidine group with a carboxyl group. The goal of the project is the identification of the charged groups involved and the characterization of the molecular re-arrangements that accompany the gross structural changes.

3. Structure and identification of large multi-enzyme complexes involving nitrilases

4. The structure of the nitrilase from Rhodococcus rhodochrous J1.

Nagasawa et al (2000) have found that isolated dimers of the related nitrilase from Rhodococcus rhodochrous J1 are inactive. However in the presence of certain substrates they assemble to form an active decamer. ( A decamer is required to produce one turn of the spiral.) This behaviour is commonly observed in nitrilases from Rhodococcus sp. and is highly suggestive that the formation of the quaternary structure alters the active site. Crystals of J1 nitrilase in the absence of substrate have previously been obtained. The goal of this project would be to obtain an atomic structure in the absence of substrate and a 3DEM reconstruction of the oligomer in the presence of substrate.

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1. The structural effects of mutations on nitrilase homologues.

We have suggested an alignment of three cyanide degrading nitrilases to the crystallographically determined structures of Nit and DCase which was primarily created using GenTHREADER.

We have also aligned the structures of Nit and DCase using ALIGN. (see superpose.pdb)

From this we have concluded that insertions and deletions in the cyanide degrading enzymes occur in externally located loops and that there are two major insertions, one major deletion and a substantial C-terminal extension.

An important difference between our enzymes, and indeed the majority of nitrilases, and the solved structures of superfamily members is the formation of spiral oligomers comprising specific numbers of subunits – but different numbers of subunits (ranging from 10-18) in different enzymes. We have postulated that the interaction that leads to the formation of the oligomer is due to an 15 amino acid insertion found in the loop between the beta strands labelled NS9 and NS10 in Nit. This, we postulate, leads to the formation of an interface (which we have called the C surface) which has pseudo two-fold symmetry. Indeed based on our model the insertion between NS2 and NH2 may also contribute to the interface and the deletion between NS5b and NS6a is essential as it would result in a clash preventing the formation of the C surface.

Our next step is to try and visualize the postulated C surface and to establish whether our postulates are correct. We have made (or intend making) a series of mutants in both the P. stutzeri and B. pumilus enzymes that:

  • disrupt the A surface – the point being to make dimers which preserve the C surface and crystallize.
  • remove the 15 amino acid insertion which we beleive is responsible for the C surface and verify that “Nit-like” dimers are formed
  • truncate the C-terminal extension so that its structural or other purpose can be determined.

To date the following mutants have been made and are ready for expression from pET expression vectors.

Enzyme

mutation

activity

# oligomers
P. stutzeri Y285stop No ?
P. stutzeri Q296stop No ?
P. stutzeri R303stop No ?
P. stutzeri K304stop Yes ?
P. stutzeri S310stop No ?
P. stutzeri Y241D + C244D No ?
P. stutzeri Del M260-F274 No ?
B. pumilus Y245stop No ?
B. pumilus M293stop Yes ?
B. pumilus K303stop Yes ?
G. sorghi Y217D ? ?
G. sorghi Y217E ? ?

The task of the student will be to express and purify the proteins, assess their oligomerization status by column chromatography and/or EM, set up crystallizations of any dimers or tetramers and solve the structure of one of the successful crystallizations. Much of the work will involve collaboration with Professor Michael Benedik at the University of Houston. Professor Benedik will be visiting in January 2004.

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3. Identification and structure of large multi-enzyme complexes involving nitrilases

or “What protein is associated with the cyanide degrading nitrilase from Bacillus pumilus?”

Cyanide degrading nitrilases occur in a number of prokaryotic and eukaryotic microorganisms. Their biological role is generally unknown. They have attracted interest as agents for bioremediation of cyanide spills. There is also substantial interest in the use of closely related enzymes in biological catalysts. There is indirect evidence that the nitrilase plays a role in sporulation in the case of B. pumilus.

We have studied the structure of the nitrilase and have discovered that it has an 18 subunit spiral arrangement. This is the first report of this type of quaternary structure which has the effect of concentrating the nitrilase in a specific location and of creating a scaffold to which other proteins can attach.

The aim of this project is to find whether another protein is complexed with the nitrilase and if so to find out as much as possible about this protein.

There are a number of leads that point in the direction of there being such a protein:

  • We have seen vestiges of density at specific locations on EM reconstructions of nitrilase prepared from the native organism (but not on the recombinant protein). This indicates low occupancy of attached proteins.
  • In C. elegans and Drosophila the nitrilase exists as a fusion protein.
  • In C. elegans the nitrilase is a tetramer associating via a beta sheet. This beta sheet interface is exposed on the outside of the quaternary helix in our model and is this free to associate with other proteins. The location of the beta sheet corresponds with the observed vestigial density.
  • In the case of Bacillus sp. strain OxB-1 the gene for phenylacetaldoxime dehydratase (which cleaves an aldoxime to make a nitrile) is in the same gene cluster as the nitrilase which is a close homologue of ours. This genetic relationship is not the case in B. pumilus but there is the suggestion that two enzymes which could form part of a pathway are in close association.

Our initial plan is to try and pull intact complexes out of the native organism using immuno-affinity chromatography. But a number of other strategies are possible including affinity chromatography using immobilised purified recombinant enzyme.

The initial plan calls for:

  1. Attachment of chicken antibodies prepared against recombinant B. pumilus to a CL Sepharose matrix and using this as the basis of an affinity column.
  2. Preparation of a culture of B. pumilus, homogenization of this culture and passing it over the column.
  3. Analysis of high affinity entities to see if any contain the nitrilase and hopefully another protein.
  4. Visualization of the attached entity in the electron microscope.
  5. Characterization of the other protein by, SDS electrophoresis, MALDI, trypsin cleavage + MALDI and database searching.
  6. Analysis of the details of binding using BIACORE.

There is a great deal that may go wrong with this plan but there are a variety of alternative sub projects including, for example, using different species of nitrilase containing bacteria, using different affinity matrices, using electron microscopy to debug the various stages. And there are alternative routes depending on the specific interests of the student – for example – three dimensional reconstruction of the complex rather than the BIACORE experiments.

We have prepared the following resources for a student selecting this project:

  • Overexpression vectors for three different nitrilases (both with and without his tags).
  • Antibodies from chickens
  • Eggs from which further antibodies may be extracted.
  • Two species of bacteria (B. pumilus and Pseudomonas stutzeri)

Techniques

Antibody characterization, preparation and use of immunoaffinity columns, SDS gel electrophoresis, electron microscopy, preparation of bacterial cultures, preparation of protein from overexpression systems, MALDI, working with enzymes, using bioinformatics databases, BIACORE etc.

Literature

  1. Pace, H.C., Hodawadekar, S.C., Draganescu, A., Huang, J., Bieganowski, P., Pekarsky, Y., Croce, C.M. and Brenner, C.: Crystal Structure of the Worm NitFhit Rosetta Stone Protein Reveals a Nit Tetramer Binding Two Fhit Dimers. Current Biology, 10: 607-617, 2000.
  2. H.C. Pace & C. Brenner: The Nitrilase Superfamily: Classification, Structure and Function. Genome Biology, 2: reviews 0001.1-0001.9, 2001
  3. C. Brenner, “Catalysis in the Nitrilase Superfamily,” Current Opinion in Structural Biology, 6: 775-782, 2002.
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