Helical structure of unidirectionally shadowed metal replicas of cyanide hydratase from Gloeocercospora sorghi

https://doi.org/10.1016/j.jsb.2007.09.019Get rights and content

Abstract

The helical filaments of the cyanide hydratase from Gloeocercospora sorghi have been reconstructed in three dimensions from freeze dried, unidirectionally shadowed specimens using iterative real-space helical reconstruction. The average power spectrum of all selected images has three clear reflections on different layer lines. The reconstruction is complicated by the fact that three possible indexing schemes are possible and reconstructions using the starting symmetries based on each of these indexing schemes converge on three-dimensional volumes which appear plausible. Because only one side is visible in shadowed specimens, it is necessary to examine the phases from a single filament by cryo-electron microscopy in order to make an unequivocal assignment of the symmetry. Because of the novel nature of the reconstruction method used here, conventional cryo-EM methods were also used to determine a second reconstruction, allowing us to make comparisons between the two. The filament is shown to have a left-handed one-start helix with D1 symmetry, 5.46 dimers per turn and a pitch of 7.15 nm. The reconstruction suggests the presence of an interaction across the groove not previously seen in nitrilase helical fibres.

Introduction

Cyanide hydratase (CHT) is a substrate-specific member of the nitrilase family of enzymes. Nitrilases catalyse the hydrolysis of nitriles to amides or to acids and ammonia with a broad range of specificities ranging from hydrogen cyanide to various aliphatic and aromatic nitriles (Pace and Brenner, 2001). In general, their natural substrates have not been identified. Because of the inherent enantio-specific nature and selectivity of enzymatic catalysis, nitrilases are used in a variety of industrial processes (Brady et al., 2004). For instance, nitrilases are used to manufacture the active enantiomers, (R)-mandelic acid, (R)-3-chloromandelic acid (Brady et al., 2004), (S)-phenyllactic acid and (R)-3-hydroxy-4-cyano-butyric acid, an important intermediate in the synthesis of the cholesterol-lowering drug atorvastatin calcium (Banerjee et al., 2002, O’Reilly and Turner, 2003). Although microbial treatment of toxic industrial effluent is hampered by the presence of conditions that inhibit microbial growth (reviewed by Baxter and Cummings, 2006), the potential exists for the use of purified microbial nitrilases for on-site cyanide remediation since they may be engineered to tolerate varying levels of pH and temperature, which the organisms themselves may be sensitive to (Jandhyala et al., 2003). CHTs have been identified in a variety of fungal species, but appear not to occur in bacteria or plants (Wang et al., 1999). They catalyse the conversion of cyanide to formamide with a small fraction (up to 0.4%, Nolan et al., 2003) being converted to formic acid. The different reaction pathways result from the breakage of a different bond in the tetrahedral thioimidate intermediate formed with the active site cysteine (Jandhyala et al., 2003; reviewed by O’Reilly and Turner, 2003).

Gloeocercospora sorghi is a fungus that infects sorghum. When lesions are formed on sorghum, cyanoglycocides degrade to form hydrogen cyanide (HCN). Expression of CHT is induced in G. sorghi by HCN and this enzyme can account for up to 18% of the protein in the organism (Wang et al., 1992). It was therefore postulated that the purpose of the enzyme was to protect the pathogen in a high cyanide environment; however, expression of CHT is not necessary for infectivity and thus its role in the cell remains uncertain (Wang et al., 1999). G. sorghi CHT has about 30% identity to the bacterial cyanide dihydratases (cynD) from Pseudomonas stutzeri AK61 and Bacillus pumilus C1. Both of these enzymes form self-terminating, homo-oligomeric spirals of 14 and 18 subunits, respectively (Sewell et al., 2005). However, the CHT from G. sorghi has a significantly higher molecular weight (2–10 MDa) (Fry and Millar, 1972), despite having a similar subunit molecular weight of ∼40.9 kDa (Wang et al., 1992).

Freeze-drying and unidirectional heavy metal shadowing are an established procedure for investigating biological macro-molecular surface structure (Gross et al., 1990) allowing for unambiguous determination of the handedness. Micrographs of samples prepared using these methods display excellent signal to noise ratio, allowing direct observation of structures at resolutions down to ∼20 Å (2 nm) (Hoenger et al., 2000) without making replicas. The Midilab instrument in which the freeze-drying and shadowing occur in a chamber mounted on the microscope column allows optimal preservation of structural detail by maintaining the sample under vacuum and cryo-conditions during shadowing, transfer into the microscope and viewing (Gross et al., 1990).

Micrographs of unidirectionally shadowed material contain three-dimensional information in the form of pixel density, which is dependent on the angle between the sample surface normal, and the shadowing direction. The thickness of the metal film deposited is directly related to the topography of the sample when viewed from a direction other than the original evaporation source (Gross, 1987). The contrast in the resulting micrograph is therefore related to the surface topography (Guckenberger, 1985). This information, combined with image processing methods, has been used to derive three-dimensional surface reconstructions in the form of digital elevation maps (DEMs) by the method of surface relief reconstruction (Smith and Kistler, 1977, Smith and Ivanov, 1980).

The limitation of these methods is that the resulting map consists of a 2.5-dimensional (2.5D) representation of a three-dimensional object. This limitation has been overcome by applying conventional Fourier–Bessel methods to rotary shadowed filaments which produces a three-dimensional reconstruction of the metal cast, from which the filament can be extracted (Morris et al., 1994). More recently reconstructions have been produced by applying tomographic and single-particle approaches to rotationally shadowed samples (Lanzavecchia et al., 1998, Lanzavecchia et al., 2005, Lupetti et al., 2005). This allows a complete reconstruction of the metal cast surrounding the object to be made. The advantage of this approach is that there is no topological constraint on the resulting reconstruction.

Although several superb visualizations of helical objects have been made with the Midilab instrument, no attempt has been made to reconstruct such images in three dimensions. Such reconstruction would require that the metal coating be sufficiently thin to allow proportionality between the thickness of the coating and the projected density. The coating applied in the Midilab instrument has a thickness of 0.3–0.4 nm and is thus thin enough to allow reconstruction as the linear relationship between the metal thickness and the absorbance is maintained. Since the shadowing is unidirectional, the amplitudes of the various layer lines are dependent on the orientation of the fibre relative to the shadowing direction. This means that Fourier–Bessel reconstruction cannot be used. However, since the fibres lie at all angles to the shadowing direction a reconstruction method based on averaging techniques could conceivably be used and this should result in a reconstruction of the metal component of the specimen. The ability to average successfully would require that there is sufficient overlap of information in fibres shadowed from different directions to allow for accurate alignment.

We have applied the iterative helical real-space reconstruction method (IHRSR) (Egelman, 2000) here to micrographs of unidirectionally shadowed G. sorghi filaments. IHRSR allows the determination of helical symmetry by iterative refinement. This method applies a single-particle approach, including reference-based alignment and back-projection to boxed segments of the continuous extended fibres. The advantage of IHRSR is that the method can deal with disordered or heterogeneous filaments and is unaffected by the problem of Bessel overlap (Egelman, 2000). Applying IHRSR to metal-shadowed data resulted in the determination of the low-resolution structure of G. sorghi CHT. We show here that the CH from G. sorghi forms extended left-handed helices, built on similar principles to those described for the cyanide dihydratases (Sewell et al., 2005). In parallel, for the purposes of comparison, we have used IHRSR to produce a low-resolution reconstruction from images of the CHT embedded in ice and recorded on a CCD camera. Comparisons between the two reconstructions give considerable insight into the nature of images made with Midilab.

Section snippets

Expression and purification

The cyanide hydratase was recombinantly expressed in Escherichia coli BL21 pLysS by IPTG induction from the plasmid MB2313 (Jandhyala et al., 2005). The cells were pelleted by centrifugation at 4000g at 4 °C for 20 min and resuspended in 50 mM Tris–HCl, pH 8.0 containing a protease inhibitor cocktail (Roche). Cells were disrupted by sonication (Misonix 3000, US) and the soluble fraction was clarified by centrifugation at 20,000g at 4 °C for 30 min. The soluble lysate was subjected to ammonium

Results

The unprocessed, unidirectionally shadowed micrographs of G. sorghi nitrilase reveal that the enzyme forms a filament with a diameter of ∼12 nm. The height of the fibre can be approximated from the length of the shadow cast by the fibre and the shadowing angle (Fig. 1a). The height is similar to the estimated diameter, suggesting that the fibre is not flattened. Prominent one-start left-handed helical striations as well as right-handed apparently four-start helical striations are visible in

Reconstruction strategies

Surface relief reconstruction (Guckenberger, 1985) provides a 2.5D digital elevation map of the structure of metal-shadowed surfaces. This method has been successfully applied (e.g. Rockel et al., 2000, Walz et al., 1996, Dimmeler et al., 2001) to specimens of appropriate topology. However, the majority of biological structure is incompatible with the digital elevation map (Lupetti et al., 2005). The alternate method is to treat metal-shadowed surfaces as two-dimensional projections of the

Conclusions

The helical structure of G. sorghi CHT has been determined at a resolution of ∼33 Å by applying the IHRSR algorithm to freeze-dried unidirectionally shadowed filaments. These filaments have been unambiguously determined to be left-handed. The reconstruction is plausible, but so are those which converge on the incorrect helical symmetry. Therefore considerable care needs to be taken when indexing the power spectrum. Surprisingly, the resulting reconstruction closely resembles that obtained using

Acknowledgments

We thank Heinz Gross and Peter Tittmann for generously giving us access to the Midilab instrument; Edward H. Egelman for his considerable assistance with IHRSR; the National Research Foundation and the Carnegie Corporation of New York for their financial support.

References (34)

Cited by (17)

  • Symmetry-restrained flexible fitting for symmetric em maps

    2011, Structure
    Citation Excerpt :

    In particular, the nitrilase from R. rhodochrous J1 was found to be inactive in its dimer form, but active in its helical-fiber form (Thuku et al., 2007). Negative stain and cryo-EM have been used to resolve the 3D structure of these helical fibers for native nitrilases as well as mutants (Thuku et al., 2009; Woodward et al., 2008), in order to understand the relationship between function and spiral quaternary structure formation in the nitrilase family. We applied MDFF to fit a two-turn-helix model of R. rhodochrous J1 nitrilase, consisting of nine dimers, into a low resolution (18 Å) negative stain EM map of a long helix fiber (EMD-1313) (Thuku et al., 2007).

  • Reconstruction of helical filaments and tubes

    2010, Methods in Enzymology
    Citation Excerpt :

    In this case, there would be n = + 5 on ll = 97, rather than n = − 5 as shown. Information would need to be obtained, such as by tilting the sample in the EM (Finch, 1972) or by metal shadowing (Woodward et al., 2008), to determine the hand of the helix. Because of the projection theorem (relating the image to the projection of the three-dimensional density distribution onto two-dimensions), any information about hand has been simply lost, whether one is using cryo-EM of unstained, frozen-hydrated samples, or conventional EM of negatively stained samples (Egelman and Amos, 2009).

View all citing articles on Scopus
View full text