Nitrilases

Trevor SewellUniversity of Cape Town

with lots of help from:Mark Berman (Cape Town)Paul Chang (Cape Town)Dakshina M. Jandhyala andMichael Benedik (Houston)Paul Meyers (Cape Town)Ed Egelman (Virginia)Dennis Burford (Cape Town)Helen Saibil (London)

and the EMU at UCT:Mohamed JafferBrendon PriceMiranda WaldronJames DuncanWilliam Williams

The Wellcome Trust

Self-terminating, homo-oliogomeric spirals with industrial applications

Establishing the principles underlying the oligomeric structure of the nitrilases.

Insights into the structures of nitrilases and GroEL from 3D

electron microscopyTrevor Sewell

with lots of help from:Mark Berman (Cape Town)Dakshina M. Jandhyala andMichael Benedik (Houston)Paul Meyers (Cape Town)Ed Egelman (Virginia)Dennis Burford (Cape Town)Helen Saibil (London)

and the EMU at UCT:Mohamed JafferBrendon PriceMiranda WaldronJames DuncanWilliam Williams

The Wellcome Trust

Why nitrilases are interesting:

Cleave non-peptide C-N bondsUsed in industrial processes e.g. manufacture of acrylic acid - efficient and environmentally friendly

Detoxification of cyanide waste - bioremediationRole 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

Cysteine, lysine and glutamic acid at active sitepH optimum 7.6 - 8.0Molecular weight of subunit = 37kDClose 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

What we know:

The Structure of Nitrilases

Trevor Sewell, Biotechnology Department UWC and

EMU, University of Cape TownNdoriah 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 JafferBrandon WeberBrendon PriceMiranda WaldronJames DuncanSean Karriem

The Wellcome TrustCarnegie Corporation

Self-terminating, homo-oliogomeric spirals with industrial applications

Useful Industrial Enzymes

Nicotinic AcidMandelic AcidIbuprophenDetoxification of cyanide

Reactions catalysed

Nitrilase - cyanide dihydratase - B. pumilus, P.stutzeri

Cyanide hydratase - G. sorghi

Nit active site

Putative catalytic mechanism

To Fhit domain

To Fhit domain

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)

Location of the active site

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?

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.

Negative stain (uranyl acetate on carbon film)Image using low dose Digitize filmSelect imagesClassify imagesStarting model using a common-lines based methodMatch images to projections of modelReconstruct » new modelCheck resolution of structure

iterate

The Process

Negatively stained native B. pumilus nitrilase, pH8

Multi-reference alignment

Iterative 3D reconstruction

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

The refinement of the structure of the nitrilase from Pseudomonas stutzeri

(7008 images)

video made by Paul Chang

The refinement of the structure of the nitrilase from Bacillus pumilus

(11661 images)

video made by Paul Chang

B. pumilus nitrilase (pH 6)

P. stutzeri nitrilase (pH 8)

ridge

bulge

Evidence for the global dyad:Reconstruction with no imposed symmetry

Cylindrical projection of P. stutzeri nitrilase

z (nm)

0

32

-180 0 180

f (°)

Angular offset between local two-fold axes (°)

70.5 70.5 76.576.596.5 96.5

1.6 nm vertical displacement between local two fold axes

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.

We know the sequences of the B. pumilusenzyme, 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 enzymesin the Protein Data Bank using GenTHREADERproduced two enzymes: Nit and DCase.

These have less than 20% sequence homologyto our enzymes.

Nit DCase

Two family members are tetramers

In the tetramer there are two interacting surfacesalmost at right angles to one another

Surface Aalpha helix

Surface Bbeta sheet

Nit DCase

To Fhit domain To Fhit domain

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)

Superposition of the alpha carbons of DCase and Nit

DCase

Nit

cys 169, lys 127, glu 54 catalytic triad

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

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.

Need to fit model into density

The two fold axes must coincide

Surface B

C - terminal

Surface A

C - terminal

Surface B

Dimer with A surface associating modeled on residues 10-291 of Nit

C - terminal

C - terminal

Surface BSurface A

Surface A

Dimer with B surface associating modeled on residues 10-280 of Nit

4 ways to align global dyad to dimer axis

A surface mating

B surface mating

This was repeated for the other handedness

What is wrong with the B surface models?

Unexplainable gaps in density

Steric clash between NH5 and NS13 and NH3 in the neighbouring dimer

Poor fits

The final, left-handed, 14-subunit model

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.

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

aB

I

Contacts c and d are between helices NH2. The contact area has a local pseudo-dyad axis.

d

cc

d

B

KD

M

glu 82

lys 86

(a)

(b)

A

B

C

D

E

F

GI

J

K

L

M

N

a

a

b

b

c

cd

d

H

-71 71 147-147-244 244

z (nm)

0

32

-320 3200

f (°)

Cylindrical projection

Superposition of the P. stutzeri nitrilase dimer modelonto 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

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.

Crosslinking withglutaraldehyde:

the protein from the column was diluted 32 fold and crosslinkedwith the glutaraldehydeconcentration indicated for 1.25 hrs.

0 .002.005 .01 .02 .05 .1 .2%

nitrilasemonomer

2x

3x

4x

6x(?)

8x(?)10x(?)

Incompletely unfolded conformational isomers?

{

The flexible C surface

The location of the active site and B surface

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.

The enzyme from B. pumilus forms long fibres at pH 5.4

Unidirectional shadowing shows that the long helicesare left handed.

The handedness of the spiral

Defined length oligomers from B. pumilus form long helices at pH 5.4. These are shown by shadowing to be left handed.

Our dimer model fits better into left handed spirals than right handed spirals as shown by SITUS correlation co-efficients.

Only in left handed spirals is there empty space in the map to accommodate the insertions relative to non spiral-forming homologues.

What came out of the study?

A new, defined size, short, spiral, homo-oligomeric quaternary structure

The handedness of the spiralThe conserved interface (A surface)The residues involved in a previously undiscovered interface (C surface)

A model of this interface which would explain its flexibility

A reason for the termination of the spiralA reason for the variety of subunit sizes in the enzymes

Structural transitions in B. pumilus nitrilase

pH 8 pH 6

pH 5.4

The transitions between pH 6 and pH 5.4 may involve the titration of a histidine.

The drop in pH from 8 to 6 results in reduced occupancy of the terminal subunits.

Regular helix having 9.4 residues per turn ( for dimer model: Dv=76.7 , Dz=1.58 nm )

B. pumilusP. stutzeriG. sorghi

Potential for two salt bridges in pumilusRepulsion in stutzeri - no long fibresOne salt bridge in sorghi - always fibres

Activity increases when structural transition occurs.Could this mean that 2 extra sites per 18mer become active?

Mutant Surface Change and location Activity

B pumilus 1. Delta 303 A Vgtg->stop Full

activity2. Delta 293 A Matg->stop Partial

activity3. Delta 279 A Ytat->stop Inactive4. Y201D/A204D

A Ytat->Dgac, Agcg->Dgac Inactive

5. Delta 219-233

C MKEMICLTQEQRDYF was deleted. 235 Egaa->Naac

Inactive

6. 90 D EAAKRNE->AAARKNK Full activity

P stutzeri 7. Delta 310 A Sagt->stop Inactive8. Delta 302 A Vgtg->stop Inactive9. Delta 296 A Qcag->stop Inactive10. Delta 285 A Ytat->stop Inactive11. Delta 276 A Kaaa->stop Inactive12. Y200D/C203D

A Ytac->Dgac, Ctgc->Dgac Inactive

13. Delta 220-234

C MKDMLCETQEERDYF deleted. Inactive

Hybrids 14. Pum – Stu A Residues 1-286 from B. pumilus, 287-

end from P. stutzeri Full Activity

15. Stu – Pum A Residues 1-286 from P. stutzeri, 287-end from B. pumilus

Inactive

The Effect of Surface Mutations on Activity

The only histidines in pumilus that are not in stutzeri.

The ATCC pumilus has no histidines in the tail - its properties are being studied

Negatively stained fibres of J1 nitrilase (0.45mg/ml) buffered in 20mM KH2PO4, 50mM NaCl at pH 7.8. Magnification 50000x

20nm

Rhodococcus rhodochrous J1

WT1 (film) WT2 (CCD) Mutant R87Q(CCD)

G. sorghi CHT reconstructions

Gloeocercospora sorghi cyanide hydratase

Surprise! Quaternary helix is right handed

What's empty?

C surface linker as before

C terminal extension

B. pumilusP. stutzeriG. sorghi

What interactions stabilize the spiral?

noY217Ea-surface

noY217Da-surface

yesD92Q-

noR91Q+

noE82V-

activemutantcharge

E82V

R91Q

We think we know where all the bits of the molecule are at coarse resolution.

We think we know what stabilizes the spiral and causes its termination.

We think that the spiral is essential for activity.

Biotechnological uses?

Can the knowlege we have gained be used to enhance:StabilityActivityEase of PurificationEase of Immobilization????

B. pumilus has a complex internal structure which changes during its life cycle. It is therefore relevant to ask where the nitrilase is located in the hope that it may give a clue to its function.