Journal of Molecular Biology
Volume 286, Issue 3, 26 February 1999, Pages 899-914
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Formaldehyde ferredoxin oxidoreductase from Pyrococcus furiosus: the 1.85 Å resolution crystal structure and its mechanistic implications1

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Abstract

Crystal structures of formaldehyde ferredoxin oxidoreductase (FOR), a tungstopterin-containing protein from the hyperthermophilic archaeon Pyrococcus furiosus, have been determined in the native state and as a complex with the inhibitor glutarate at 1.85 Å and 2.4 Å resolution, respectively. The native structure was solved by molecular replacement using the structure of the homologous P. furiosus aldehyde ferredoxin oxidoreductase (AOR) as the initial model. Residues are identified in FOR that may be involved in either the catalytic mechanism or in determining substrate specificity. The binding site on FOR for the physiological electron acceptor, P. furiosus ferredoxin (Fd), has been established from an FOR-Fd cocrystal structure. Based on the arrangement of redox centers in this structure, an electron transfer pathway is proposed that begins at the tungsten center, leads to the (4Fe:4S) cluster of FOR via one of the two pterins that coordinate the tungsten, and ends at the (4Fe:4S) cluster of ferredoxin. This pathway includes two residues that coordinate the (4Fe:4S) clusters, Cys287 of FOR and Asp14 of ferredoxin. Similarities in the active site structures between FOR and the unrelated molybdoenzyme aldehyde oxidoreductase from Desulfovibrio gigas suggest that both enzymes utilize a common mechanism for aldehyde oxidation.

Introduction

The possibility of a biological function for the element tungsten (W) was established in the early 1970s (Andreesen & Ljungdahl, 1973), and the first tungsten-containing protein, a formate dehydrogenase, was purified in 1983 (Yamamoto et al., 1983). At present, 14 tungstoenzymes have been purified, all from anaerobic microorganisms, most of which are thermophilic to a greater or lesser extent Adams 1994, Johnson et al 1996. These enzymes can be subdivided into those that oxidize aldehydes of various types, and those that activate CO2. Most of the former type are classified within the AOR family, named after the aldehyde ferredoxin oxidoreductase (AOR) from the hyperthermophilic archaeon Pyrococcus furiosus, an organism that grows optimally at 100°C Johnson et al 1996, Mukund and Adams 1991. All members of the AOR family have a subunit of ∼65 kDa which contains a single W atom and at least four iron atoms. Although these enzymes also contain a pterin cofactor, they share no sequence or structural similarities with molybdenum-pterin cofactor-containing enzymes (Johnson et al., 1996).

Three members of the AOR family have been purified from P. furiosus (Pf) . They all catalyze the oxidation of aldehydes to corresponding carboxylic acids, and use the redox protein ferredoxin (Fd) as their physiological electron acceptor. The oxidation of the reduced Fd is coupled to the production of H2, or, if S0is present, to the production of H2S. The three enzymes are AOR, formaldehyde Fd oxidoreductase (FOR) and glyceraldehyde-3-phosphate Fd oxidoreductase (GAPOR). The amino acid sequences of all three are known Kletzin et al 1995, Roy et al 1999, as is that of the FOR from the hyperthermophilic archaeon Thermococcus litoralis (Kletzin et al., 1995; Figure 1). In addition, the Pf genome contains two additional genes that encode AOR-like enzymes of unknown function (Roy et al., 1998). Moreover, the genomes of two related hyperthermophilic archaea, the fermentative Pyrococcus horikoshii(Kawarabayasi et al., 1998) and the sulfate-reducing Archaeoglobus fulgidus(Klenk et al., 1997), contain six and four AOR-like genes, respectively, some (but not all) of which are clearly of the AOR, FOR or GAPOR type (Roy et al., 1998).

The best characterized tungstoenzyme is Pf AOR, the crystal structure of which has been determined to 2.3 Å resolution (Chan et al., 1995). This structure revealed the polypeptide fold of the AOR family and demonstrated that the tungsten atom is coordinated by the dithiolene sulfur atoms of two pterin cofactors. The AOR structure also established the tricyclic nature of the pterin cofactor, which has subsequently been observed in all presently characterized enzymes that utilize both the tungsten and molybdenum forms of this cofactor (Kisker et al., 1997). AOR oxidizes a broad range of aliphatic and aromatic aldehydes that are derived ultimately from amino acid residues, such as alanine, phenylalanine, and tryptophan. During peptide fermentation by Pf, amino acids are converted to 2-keto acids by transamination, and the 2-keto acids are substrates for 2-keto acid oxidoreductases. Depending on the cellular redox potential, these enzymes either oxidize 2-keto acids to their CoA-derivatives or decarboxylate them to the corresponding aldehydes Heider et al 1995, Ma et al 1997. The latter are potentially toxic and are oxidized to the acid form by AOR. However, despite the availability of a crystal structure, spectroscopic analyses have shown that the W site in AOR is heterogeneous (Koehler et al., 1996), and its precise mechanism of catalysis is not known.

Relative to AOR, much less is known about the structural and enzymatic properties of Pf FOR (Roy et al., 1998). The two enzymes are clearly closely related as their subunits are of similar size and show 40 % amino acid sequence identity (61 % similarity). In addition, based on elemental and spectroscopic analyses, they have a similar W and Fe content, although FOR, but not AOR, also contains calcium (∼1 Ca2+per subunit). Residues of functional importance in AOR that are conserved in FOR include the four cysteine residues that coordinate the single (4Fe:4S) cluster and the first of two pterin-binding motifs, although the second pterin-binding motif is not conserved in FOR. In addition, these two proteins differ in oligomerization status, since FOR exists as a homotetramer, while AOR is a dimer. This suggests that the interactions supporting FOR oligomerization must differ from those of AOR, reflecting the replacement in FOR of the Glu and His residues that coordinate a metal ion at the dimer interface in AOR. As its name implies, FOR was purified by its ability to oxidize formaldehyde and it will also utilize C2single bondC4 aliphatic aldehydes. These are unlikely to be of physiological significance, however, as the enzyme has a very low degree of affinity for such compounds (Km⩾25 mM). FOR does oxidize glutaric dialdehyde with a higher degree of affinity (Km = 800 μM), but this is not part of a known biochemical pathway and the true substrate is probably a related C5 or C6 semi- or dialdehyde (Roy et al., 1998).

To provide further insight into the structure and enzymatic mechanism of the AOR family of enzymes, we have determined the crystal structures of Pf FOR and of its complexes with glutarate and Fd, at resolution of 1.85 Å, 2.4 Å and 2.15 Å, respectively. Based on these structural analyses of FOR and previous data on the AOR structure, a mechanism for aldehyde oxidation by this family is proposed that reflects active site similarities between FOR and the unrelated molybdoenzyme, aldehyde oxidoreductase from Desulfovibrio gigas(Romão et al., 1997).

Section snippets

Folding and overall structure

The subunits in the FOR tetramer are related by 222-molecular symmetry that generates a relatively flat, plate-like arrangement approximately 115 Å on each side with a thickness of 50 Å (Figure 2). A channel of ∼27 Å diameter passes through the center of the tetramer, which encompasses the molecular 2-fold axis oriented along the short dimension. One consequence of this arrangement is that each subunit contacts only two of the other three subunits in the tetramer. The four subunits will be

Structure of the tungstopterin and its protein environment

The tungstopterin moiety of FOR consists of two pterin molecules and one tungsten atom (Figure 5). They are designated as the first and the second pterin, respectively, according to the order their binding motifs occur in FOR sequence. Like those found in other pterin-containing protein structures (Rees et al., 1997), the pterins in FOR are tricyclic, formed from the fusion of pterin and pyran ring systems. The tungsten atom is coordinated by all four dithiolene sulfur atoms present in the two

Iron-sulfur cluster

The (4Fe:4S) cluster is located ∼10 Å from the W atom, and is buried ∼6 Å below the protein surface. As anticipated, it is coordinated by the Sγatoms of Cys284, Cys287, Cys291, and Cys491. The (4Fe:4S) cluster is surrounded by hydrophobic side-chains from residues Trp235, Met289, Pro290, Leu493, and Pro494. The cluster is buried inside the protein structure, but a water molecule is found in the environment of the cluster and is hydrogen-bonded to one of the inorganic sulfur atoms and the amide

Calcium binding site

In the process of refinement, a relatively electron-dense feature was found in all four subunits near the O4 of one of the pterin molecules that refined to very low B-factors for a water molecule. The density level and the geometry suggest that it is a cation, most likely calcium or potassium, with the former consistent with the elemental analysis of FOR (Roy et al., 1998). Calcium ions were modeled into each site, and they all refined to reasonable B-factors ranging from 21.5 Å2to 25.6 Å2.

Active site cavity

A channel connecting the tungsten site and the protein surface was observed at the interface of domains 2 and 3 in the AOR structure that was proposed to permit substrates and products to enter and leave the active site, respectively (Chan et al., 1995). In FOR, a cavity, rather than an open channel, is found at the same position (Figure 8). The volume of this cavity is calculated as 1500 Å3by the program VOIDOO (Kleywegt & Jones, 1994), using a 1.2 Å probe radius (Hubbard & Argos, 1995). This

Glutarate-FOR interactions

In the native FOR structure, the active site cavity is occupied by uninterpretable density, with the highest peak near the side-chains of Arg481 and Arg492. The density was approximately the size of a citrate molecule, which is present in the crystallization solution. Since glutaric dialdehyde has the lowest Kmfor any characterized substrate, the FOR crystal was soaked with the oxidation product, glutarate. In the crystal structure of FOR-glutarate complex, a glutarate molecule clearly shows up

FOR-Fd complex and docking areas

The three tungstoenzymes that have been purified from hyperthermophilic archaea all use Fd as their physiological electron carrier Heider et al 1995, Mukund and Adams 1995. Pf Fd is a small protein of 66 residues and one (4Fe:4S) cluster (Busse et al., 1992). With an apparent Kmvalue of 100 μM (Roy et al., 1998), its interaction with Pf FOR does not appear to be as strong as it is with AOR and GAPOR, where the Kmvalues are <10 μM.

The docking regions on both molecules are clearly identified in

Structural comparisons with AOR and homologous enzymes

FOR and AOR monomers have similar folds and can be superimposed with an rms deviation of 1.5 Å, based on 576 Cαatoms from 15 segments of both molecules. This rms deviation is consistent with the estimated value (1.2 Å) expected from the degree of sequence identity (40 %) between these two proteins (Chothia & Lesk, 1986). In agreement with the observation that domain 1 is most highly conserved between AOR and FOR (Kletzin et al., 1995), the Cαatoms of the 208 residues which comprise this domain

Crystallization

Pf FOR was purified (Roy et al., 1998) and crystallized at room temperature under an argon atmosphere using a modification of the melting-point capillary method (Georgiadis et al., 1992); 10μl of precipitant solution was introduced into the capillary, and separated by 2-3 mm from a 50:50 mixture of protein and precipitant solutions. The protein solution contained 55–65 mg/ml of FOR, 50 mM Tris (pH 8.0), 2.0 mM dithionite, 2 mM DTT, 0.2 M KCl. The precipitant solution contained 30 % (v/v)

Acknowledgements

We thank Dr Michael H. B. Stowell and Dr John W. Peters for assistance with data collection. This project is supported by USPHS grants GM50775 (D.C.R.) and GM45587 (M.W.W.A.) and by the US Department of Energy grant FG05-95ER20175 (M.W.W.A.). The rotation camera facility at SSRL is supported by DOE and NIH.

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    Edited by I. A. Wilson

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    Present address: Department of Chemistry and Biochemistry and Laboratory of Structural Biology and Molecular Medicine, University of California, Los Angeles, Los Angeles, CA 90095, USA.

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