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BIOLOGICAL CRYSTALLOGRAPHY |
The atomic resolution structure of methanol dehydrogenase from Methylobacterium extorquens
aSchool of Biological Sciences, University of Southampton, Bassett Crescent East, Southampton
SO16 7PX, England
*Correspondence e-mail: [email protected]
The of methanol dehydrogenase (MDH) from Methylobacterium extorquens has been refined without stereochemical restraints at a resolution of 1.2 Å. The high-resolution data have defined the conformation of the tricyclic pyrroloquinoline quinone (PQQ) cofactor ring as entirely planar. The detailed definition of the active-site geometry has shown many features that are similar to the quinohaemoprotein alcohol dehydrogenases from Comamonas testosteroni and Pseudomonas putida, both of which possess MDH-like and cytochrome c-like domains. Conserved features between the two types of PQQ-containing enzyme suggest a common pathway for between MDH and its physiological cytochrome cL. A pathway for proton transfer from the active site to the bulk solvent is also suggested.
3D view: 1w6s
PDB reference: methanol dehydrogenase, 1w6s, r1w6ssf
1. Introduction
Methanol dehydrogenase (MDH) is a pyrroloquinoline quinone-containing protein found
in the periplasm of many Gram-negative bacteria. This enzyme, which oxidizes methanol
to formaldehyde, participates in a short electron-transfer chain (Anthony, 1992![[Anthony, C. (1992). Biochim. Biophys. Acta, 1099, 1-15.]](https://journals.iucr.org/logos/arrows/d_arr.gif "Anthony, C. (1992). Biochim. Biophys. Acta, 1099, 1-15.")
, 2004). The reduced pyrroloquinoline quinone (PQQ) transfers two electrons in single electron-transfer
steps to cytochrome cL, creating a semiquinone form of the after the first (Dijkstra et al., 1989). Cytochrome cL is subsequently oxidized by a small class I c-type cytochrome, cytochrome cH (Read et al., 1999), which is in turn oxidized by the membrane oxidase cytochrome aa3. This pathway produces a proton motive force sufficient to drive the production of
just under one molecule of ATP per molecule of methanol oxidized.
MDH is a large tetrameric protein with an α2β2 subunit composition. The large α-subunit (66 kDa) consists of a single domain with a β-propeller fold. The propeller fold is composed of eight four-stranded β-sheets arranged about an eightfold axis; PQQ is situated at the centre of the molecule on the eightfold axis (Fig. 1). This β-propeller fold has been identified in an increasing number of unrelated proteins,
with a range of four to eight `propeller blades'. In the case of MDH the propeller
fold is stabilized by regularly spaced tryptophan residues, which form hydrophobic
and polar interactions with residues in the same and in adjacent β-sheets; this arrangement is termed the tryptophan-docking motif (Ghosh et al., 1995). The smaller β-subunit (8 kDa) forms a mainly α-helical structure, which wraps around the outside of the α-subunit (Fig. 1), although it is not obvious what (if any) function it serves.
![[Figure 1]](https://journals.iucr.org/10.1107/S0907444904026964/tm5012fig1thm.gif) |
Figure 1 An αβ unit from methanol dehydrogenase. The large α-subunit (shown in green) is folded into a β-propeller structure, with the PQQ and calcium ion (green sphere) at the centre of this fold; the small β-subunit (yellow) wraps around the side of the α-subunit. |
The α-subunit harbours the active site, which consists of the PQQ a calcium ion and the catalytic amino acid Asp303 (Afolabi et al., 2001). As shown in Fig. 2, PQQ forms many hydrogen bonds to protein atoms in the active site and also provides
three of the six atoms coordinated to the calcium ion. In addition to these polar
interactions, PQQ forms hydrophobic contacts with a vicinal disulfide bond formed
between two adjacent cysteines (Cys103 and Cys104) and the indole ring of Trp243.
![[Figure 2]](https://journals.iucr.org/10.1107/S0907444904026964/tm5012fig2thm.gif) |
Figure 2 The equatorial interactions of PQQ in the active site and the dative bonds that coordinate the calcium. The lengths of the bonds to the Ca2+ ion are all between 2.4 and 2.8 Å. The orthoquinone group is formed by the central six-membered ring with two O-atom substituents. The vicinal disulfide bond formed by cysteines 103 and 104, which packs against the PQQ ring, has been omitted for clarity. |
The structure of MDH has been solved previously from the bacteria Methylobacterium extorquens and Methylophilus methylotrophus W3A1 to a resolution of 1.94 and 1.90 Å, respectively (Ghosh et al., 1995; Xia et al., 1999). In both of these analyses there appeared to be uncertainty over the conformation
of PQQ, in particular the orthoquinone moiety (Fig. 2) formed by the two O-atom substituents of the central six-membered ring. The structure
from Mb. extorquens suggested that the C-4 carbonyl atom was not in the expected planar conformation
but instead exhibited a distortion from the plane of the ring towards the Trp243 indole
ring. In contrast, the structure of MDH from Mp. methylotrophus showed that the C-5 atom was in a tetrahedral conformation, causing the C-5 atom
to be raised above the PQQ tricyclic plane towards the vicinal disulfide bridge.
In this communication, we present an atomic resolution structure of MDH refined to
1.2 Å from the facultative methylotroph Mb. extorquens. The high-resolution X-ray diffraction data allowed the structure to be refined with
anisotropic B factors and without any restraints imposed on the protein or the Under these conditions, the tricyclic ring of the PQQ is planar. The higher accuracy of the 1.2 Å resolution structure has led us to revise
the coordination geometry of the active-site calcium ion. Finally, the high resolution
of the data set has allowed an extensive comparison of the active-site composition
with that of the quinohaemoprotein alcohol dehydrogenases from Comamonas testosteroni (Oubrie et al., 2002) and Pseudomonas putida (Chen et al., 2002). This has allowed putative electron- and proton-transfer routes from PQQ to the
soluble cytochrome and periplasm, respectively, to be postulated.
2. Materials and methods
2.1. Protein crystallization
The methanol dehydrogenase crystals were grown by the hanging-drop vapour-diffusion method, in which 3 µl volumes of 15 mg ml−1 protein solution at pH 8.0 in 20 mM Tris buffer were placed on siliconized cover slips and then mixed with equal volumes of the well solution. Each cover slip was then sealed with high-vacuum grease over a well containing 1 ml 20% PEG 8000 pH 9.0. Large crystals appeared after two weeks.
2.2. Data collection and refinement
Diffraction data were collected at the ESRF, Grenoble (beamline ID-29). A high-resolution
pass of 185° was collected with an oscillation angle of 1° and a crystal-to-detector
distance of 100 mm. A low-resolution pass of 185° was also collected with the same
oscillation angle and crystal-to-detector distance using an attenuated beam and shorter
exposures in an effort to measure reflections that might have been overloaded in the
high-resolution pass. The crystals were found to belong to the triclinic P1 and have unit-cell parameters a = 61.0, b = 71.7, c = 85.6 Å, α = 86.2, β = 104.4, γ = 109.9°. The data were processed using MOSFLM (Leslie, 1992) and scaled with SCALA (Collaborative Computational Project, Number 4, 1994). The resulting data set contained 373 341 unique reflections with an Rmerge of 10.3%. A set of reflections representing 5% of the unique data was chosen at random
for inclusion in an Rfree set (Brünger, 1992). The program MOLREP (Vagin & Teplyakov, 1997) was used to obtain initial phases. The search model was an α2β2 tetramer from a previously solved MDH structure (Afolabi et al., 2001) with the PQQ cofactor and metal ions removed. Two significant peaks of 47.45σ and 47.21σ, which corresponded to the two equivalent orientations of the search model, were
found when running the cross-rotation function. Since the is P1, the position of the molecule is arbitrary and a translation function was not required.
An initial rigid-body of the single α2β2 tetramer in the crystallographic was performed with SHELX (Sheldrick & Schneider, 1997), after which a number of amino-acid side chains were rebuilt using TURBO FRODO with σA-weighted maps (Read, 1986). In further rounds of two PQQ molecules, two calcium ions and 1405 water molecules were modelled into the
electron density. The introduction of anisotropic displacement parameters and the
use of riding H atoms using SHELX reduced the R factor and Rfree to 15.8% and 19.6%, respectively. The final and structure-quality statistics are shown in Table 1. The Ramachandran plot of the final structure shows that 86.6% of the residues are
in the region most favoured by the PROCHECK criteria of Laskowski et al. (1993), with another 12.3% in the additionally allowed region, leaving four residues from
the α2β2 tetramer in disallowed regions; these were Lys19 and Asp105 from both of the α-subunits.
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3. Results and discussion
The overall structure of MDH from Mb. extorquens refined at 1.2 Å resolution is, as expected, very similar to the previously reported
MDH structure, which was solved at an appreciably lower resolution (Ghosh et al., 1995). The α2β2 tetramer is arranged into two αβ units, with the α-subunit exhibiting the eight-bladed β-propeller fold and the β-subunit wrapping around one side of the structure; the PQQ molecule is located at
the centre of the β-propeller in each of the α-subunits. The improved resolution of the new data set allowed many surface polar
side chains that had previously been poorly defined by the electron density to be
positioned.
The atomic resolution data have allowed the PQQ conformation to be determined with
no stereochemical restraints imposed during the This analysis has shown that the bound PQQ is a completely planar group and is probably
in the oxidized orthoquinone form (Figs. 3 and 4). In contrast, the previously reported structure of MDH from Mb. extorquens possessed PQQ in the semiquinone (Ghosh et al., 1995). In this structure, the C-4 carbonyl atom of the orthoquinone moiety was distorted
from planarity, exhibiting a `kink' from the tricyclic plane of 40°. In spite of this
difference, the hydrogen-bonding pattern of the PQQ moiety in the 1.2 Å structure
is essentially the same as that in the previously reported analysis, showing only
slight alterations in the geometry of the interactions. The planarity of the PQQ is consistent with a number of other recently solved quinohaemoprotein dehydrogenase
structures (Chen et al., 2002; Oubrie et al., 2002).
![[Figure 3]](https://journals.iucr.org/10.1107/S0907444904026964/tm5012fig3thm.gif) |
Figure 3 The interactions made by the active-site Ca2+ ion. The length of the interaction between Asn261 and the Ca2+ ion is 3.18 Å, which is too long to be considered a coordinated bond. The 1.2 Å resolution 2Fo − Fc map contoured at the 1.0σ level is shown in grey/blue lines. |
![[Figure 4]](https://journals.iucr.org/10.1107/S0907444904026964/tm5012fig4thm.gif) |
Figure 4 Proposed pathways for electron transfer from PQQ to cytochrome cL (shown in yellow) and proton translocation from PQQ to the periplasm (shown in red). Both pathways are predicted on the basis of the structures of the quinohaemoprotein alcohol dehydrogenase from the bacterium C. testosteroni (Oubrie et al., 2002 ). The electrons originating from oxidation of methanol would travel from the PQQ
cofactor into the adjacent disulfide and then towards Asn52 or the water molecules
599 and 904 and thence to cytochrome cL. The putative proton-transfer pathway involves the side chains of a number of acidic
and basic residues as well as active-site water molecules. |
The accuracy of the data has allowed a critical assessment of the nature of the metal
ion in the active site. The calcium ion bound here forms an unusual coordination sphere
in the active site. Three PQQ atoms coordinate to the calcium ion: these are the C-7
carboxylate, C-5 carbonyl O and N-6 quinoline N atoms. The remaining three calcium
ligands are provided by both carboxylate O atoms from Glu177 and the O atom from the
side chain of Asn261 (Fig. 3). The distances between the calcium ion and its coordinating atoms were reported
to be between 2.3 and 2.8 Å in the earlier 1.9 Å resolution structure (Ghosh et al., 1995). In the 1.2 Å resolution structure of MDH reported here, the bond length from the
calcium ion to Asn261 is 3.18 Å (Fig. 3), suggesting that this side chain does not actually coordinate to the metal ion.
However, Asn261 does hydrogen bond to the catalytic base Asp303 through its side-chain
O atom (Afolabi et al., 2001). This hydrogen bond serves to orient the Asp303 carboxylate group for an efficient
proton abstraction from the substrate hydroxyl group in the initial step of the reaction.
Closer inspection of the 2Fo − Fc electron density occupied by the Ca2+ ion showed it to be oval rather than the expected spherical shape. Furthermore, some
significant features of difference Fo − Fc electron density were present. The enzyme for this study was prepared from a methanol
oxidation mutant that was defective in one of several processing enzymes that are
required to either add the Ca2+ ion into the active site of MDH or remove an alternative metal ion in order to allow
the Ca2+ ion to bind (Toyama et al., 1998). Consequently, it was thought that the MDH preparation from this methanol oxidation
mutant might contain Mg2+ (a far more abundant metal ion in cells) instead of Ca2+ in the active site. An Mg2+ ion was fitted to the electron-density maps for each of the two α-subunits in the and the resulting model was refined using anisotropic B factors. Unfortunately, the yielded very unsatisfactory electron density for the metal ions once Mg2+ was added into the structure. This result suggests that Mg2+ ions do not occupy the substrate-binding site.
Following this finding, a Ca2+ ion was re-inserted to replace the Mg2+ ion and the occupancy of the Ca2+ ion was refined during cycles of anisotropic B-factor The occupancy of the Ca2+ ion refined to 69% (the average of both α-subunits in the asymmetric unit) and its isotropic B factor was reduced from 30 to 20 Å2. More importantly, the electron density surrounding the Ca2+ ion was more uniform and lacked any significant Fo − Fc contribution. This fact led to the conclusion that in the crystals of MDH from the methanol oxidation mutant not all of the active sites are occupied by Ca2+ ions, suggesting a deficiency in the mechanism of metal insertion in these mutant bacteria.
The unusual disulfide bridge formed between adjacent cysteine residues Cys103 and
Cys104 has recently been implicated in intramolecular electron-transfer events in
the quinohaemoprotein alcohol dehydrogenases from C. testosteroni (Chen et al., 2002) and P. putida (Oubrie et al., 2002). Quinohaemoprotein alcohol dehydrogenases are type II PQQ-containing alcohol dehydrogenases.
This class of redox proteins are periplasmic proteins that contain both a PQQ-containing
domain, folded into a β-propeller fold, and a smaller cytochrome c domain, which is analogous to a typical class I c-type cytochrome. These two domains are connected via a proline-rich linker region, which lacks any secondary structure, and we have used
these structures to model the electron-transfer complex formed by MDH and cytochrome
cL.
The X-ray crystal structures of the quinohaemoprotein dehydrogenases (Chen et al., 2002; Oubrie et al., 2002) have allowed speculative electron- and proton-transfer pathways from the PQQ domain
to the cytochrome c domain to be postulated. These pathways have highlighted various water molecules
and protein residues as being crucial to this process. After comparing the active
site of MDH with the quinohaemoproteins, it was apparent that the majority of the
components of these postulated pathways for proton and were conserved, making the quinohaemoprotein alcohol dehydrogenases a good model
for the MDH–cytochrome cL interaction. Amongst these features were the disulfide bond formed between Cys103
and Cys104, Asp105 and a channel of water molecules, which constitute a solvent channel
leading from the PQQ molecule to the surface of MDH, where a cytochrome cL molecule could bind and accept an electron. A potential pathway for the electron
to reach the protein exterior is shown in Fig. 4 (yellow arrows). The release of protons from the catalytic site to the surrounding
periplasm establishes a proton gradient across the periplasmic membrane. A number
of residues that are likely proton-transfer candidates are shown in Fig. 4 (red arrows). The proton-transfer pathway is thought to involve the side chains of
a number of acidic and basic residues, as well as water molecules held within the
active-site cleft. In conclusion, we believe that this is an interesting example of
how knowledge of the structure of a two-domain protein can be used to model the functional
complex formed by two proteins, each homologous with one domain of the `parent' structure.
Acknowledgements
The authors gratefully acknowledge the Biotechnology and Biological Sciences Research Council (UK) for project grant support and a research studentship (to PAW). The authors also thank the European Synchrotron Radiation Facility (ESRF, Grenoble) for provision of beam time and associated travel support.
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