Nitrilases

1. Nitrilases Self-terminating, homo-oliogomeric spirals with industrial applications Trevor Sewell University of Cape Town with lots of help from: Mark Berman (Cape Town) Paul Chang (Cape Town) Dakshina M. Jandhyala and Michael Benedik (Houston) Paul Meyers (Cape Town) Ed Egelman (Virginia) Dennis Burford (Cape Town) Helen Saibil (London) and the EMU at UCT: Mohamed Jaffer Brendon Price Miranda Waldron James Duncan William Williams The Wellcome Trust

2. Establishing the principles underlying the oligomeric structure of the nitrilases.

3. Insights into the structures of nitrilases and GroEL from 3D electron microscopy Trevor Sewell with lots of help from: Mark Berman (Cape Town) Dakshina M. Jandhyala and Michael Benedik (Houston) Paul Meyers (Cape Town) Ed Egelman (Virginia) Dennis Burford (Cape Town) Helen Saibil (London) and the EMU at UCT: Mohamed Jaffer Brendon Price Miranda Waldron James Duncan William Williams The Wellcome Trust

4. Why nitrilases are interesting: • Cleave non-peptide C-N bonds • Used in industrial processes e.g. manufacture of acrylic acid - efficient and environmentally friendly • Detoxification of cyanide waste - bioremediation • Role in plants - in synthesis of auxin - is one of few biological roles properly documented • Variety of different reported sizes of apparently homogeneous material • Apparent link between quaternary structure and activity in some enzymes

5. What we know: • Cysteine, lysine and glutamic acid at active site • pH optimum 7.6 - 8.0 • Molecular weight of subunit = 37kD • Close relatives all have large molecular weights - reported number of subnits varies in different species from monomers and dimers, to tens and occasionally hundreds. • Sequences of over 400 members of the nitrilase superfamily • Atomic structure of two (now four) distant members of the superfamily. • The B. pumilus enzyme complex measures 10nm x 10nm x 20nm

6. The Structure of Nitrilases Self-terminating, homo-oliogomeric spirals with industrial applications Trevor Sewell, Biotechnology Department UWC and EMU, University of Cape Town Ndoriah Thuku (UWC) Margot Scheffer(UCT) Mark Berman (UCT) Paul Chang (UCT) Dakshina M. Jandhyala(Houston) Xing Zhang (Houston) Michael Benedik (Tamu) Paul Meyers (Cape Town) Ed Egelman (Virginia) Arvind Varsani(Cape Town) Helen Saibil (London) and the EMU at UCT: Mohamed Jaffer Brandon Weber Brendon Price Miranda Waldron James Duncan Sean Karriem The Wellcome Trust Carnegie Corporation

7. Useful Industrial Enzymes Nicotinic Acid Mandelic Acid Ibuprophen Detoxification of cyanide

8. Reactions catalysed Nitrilase - cyanide dihydratase - B. pumilus, P.stutzeri Cyanide hydratase - G. sorghi

9. Nit active site

10. Putative catalytic mechanism

13. Topology diagram of the a-b-b-a-a-b-b-a dimer structure found in both DCase and Nit. Nit labelling. Pace et al (2000) To Fhit domain To Fhit domain

14. Location of the active site

16. Two questions concerning the quaternary structure : • Homologous nitrilases have subunit molecular weights around 40 kDa but are generally reported to occur in complexes with 2 - 18 subunits. Why is this? • Nitrilases from several Rhodococcus species are inactive as dimers but form active decamers or dodecamers on incubation with substrate. Why is this?

17. What we did: • Reconstructed a 3D map from negatively stained images to a resolution of 2.5nm using SPIDER • Located homologues in the PDB and aligned them to our sequences with GENthreader. • Developed a dimer model for our enzymes based on the non-spiral forming homologues. • Located the dimer model within the density with CoLoRes in SITUS and O.

18. The Process • Negative stain (uranyl acetate on carbon film) • Image using low dose • Digitize film • Select images • Classify images • Starting model using a common-lines based method • Match images to projections of model • Reconstruct » new model • Check resolution of structure iterate

19. Negatively stained native B. pumilus nitrilase, pH8

20. Multi-reference alignment Iterative 3D reconstruction

21. Averages of the 84 image sets used in the reconstriction of the cyanide dihydratase from P. stutzeri AK61

22. The refinement of the structure of the nitrilase from Pseudomonas stutzeri (7008 images) video made by Paul Chang

23. The refinement of the structure of the nitrilase from Bacillus pumilus (11661 images) video made by Paul Chang

24. B. pumilus nitrilase (pH 6) bulge ridge P. stutzeri nitrilase (pH 8)

25. Evidence for the global dyad: Reconstruction with no imposed symmetry

26. Cylindrical projection of P. stutzeri nitrilase 32 1.6 nm vertical displacement between local two fold axes z (nm) 0 -180 180 0 f (°) 96.5 76.5 70.5 70.5 76.5 96.5 Angular offset between local two-fold axes (°)

27. The cylindrical projection shows that successive local two fold axes are separated by increasing angular rotations but a constant shift along the helix axis. The projections of the subunits also appear increasingly elongated along v, because they are closer to the helix axis.

28. We know the sequences of the B. pumilus enzyme, thanks to Michael Benedik and Dakshina Jandhyala at the University of Houston, and the P. stutzeri enzyme due to Atsushi Watanabe et al, (1998) BBA, 1382, 1-4. They have 70% sequence homology. A search for structurally homologous enzymes in the Protein Data Bank using GenTHREADER produced two enzymes: Nit and DCase. These have less than 20% sequence homology to our enzymes.

29. Two family members are tetramers Nit DCase

30. In the tetramer there are two interacting surfaces almost at right angles to one another Surface A alpha helix Surface B beta sheet Nit DCase

31. Topology diagram of the a-b-b-a-a-b-b-a dimer structure found in both DCase and Nit. Nit labelling. Pace et al (2000) To Fhit domain To Fhit domain

32. Superposition of the alpha carbons of DCase and Nit DCase Nit cys 169, lys 127, glu 54 catalytic triad

33. An alignment of the nitrilase sequences with Nit and DCase by GenTHREADER

34. From the sequence comparisons we conclude that: • The insertions and deletions in our enzymes relative to NIT and DCase are in outer loops and will not impinge on the tertiary structure that is crucial to the fold. • A major difference between our enzymes and the tetramers is the existence two significant insertions and the C-terminal extension.

35. Need to fit model into density The two fold axes must coincide

36. Dimer with A surface associating modeled on residues 10-291 of Nit Surface A Surface B C - terminal C - terminal Surface B

37. Dimer with B surface associating modeled on residues 10-280 of Nit Surface A C - terminal C - terminal Surface B Surface A

38. 4 ways to align global dyad to dimer axis A surface mating B surface mating This was repeated for the other handedness

39. What is wrong with the B surface models? Steric clash between NH5 and NS13 and NH3 in the neighbouring dimer Poor fits Unexplainable gaps in density

40. The final, left-handed, 14-subunit model

41. Termination of the helix • The C surface is flexible and operates as a hinge between the subunits. • As subunits are added at terminus of the spiral new opportunities arise for interactions across the groove. • The addition of a further subunit will occur if the energetic considerations favour this in preference to interactions across the groove which result in steric hindrance which would prevent the addition of a further subunit.

42. Contacts a and b result in the terminal dimer having an inwards tilt of 12 degrees thus preventing the addition of a further dimer. . I B a

43. Contacts c and d are between helices NH2. The contact area has a local pseudo-dyad axis. M d d K glu 82 D c c B lys 86

44. N L b J d H c M F a K D b I B d G c a E C A (a) Cylindrical projection 32 z (nm) 0 -147 244 -320 -71 0 71 147 320 -244 f (°) (b)

45. Superposition of the P. stutzeri nitrilase dimer model onto the A surface Nit dimer Insertions thought to be responsible for the C surface interactions Deletion: causes steric hindrance and would prevent C surface interactions

46. A prominent ridge on the outer surface was not filled by the initial model. A four stranded segment of sheet from bovine superoxide dismutase fills the density has the correct number of residues and mates with the ends in left handed models only.

47. 10x(?) 8x(?) Crosslinking with glutaraldehyde: 6x(?) 4x the protein from the column was diluted 32 fold and crosslinked with the glutaraldehyde concentration indicated for 1.25 hrs. 3x 2x nitrilase monomer { Incompletely unfolded conformational isomers? 0 .002 .005 .01 .02 .05 .1 .2%

48. The flexible C surface

49. The location of the active site and B surface

50. Does the quaternary structure have functional significance? 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.) We do not yet know whether this occurs in our case as we don't yet know how not to produce the spiral in our enzymes.