Crystallization and preliminary X-ray analysis of the tungsten-dependent acetylene hydratase from<i>Pelobacter acetylenicus</i>

Einsle, Oliver; Niessen, Holger; Abt, Dietmar J.; Seiffert, Grażyna Bernadeta; Schink, Bernhard; Huber, Robert; Messerschmidt, Albrecht; Kroneck, Peter M. H.

doi:10.1107/s174430910500374x

Cited by 15 publications

(10 citation statements)

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“…Crystals of P. acetylenicus AH were grown under strict exclusion of oxygen, as described in ref. 21, and transferred into a buffer containing the mother liquor plus 15% (vol/vol) of 2-methyl-2,5-pentanediol before flash-cooling in liquid nitrogen. Diffraction data were collected at beam lines BW6 (Max-Planck Unit for Structural Molecular Biology) and X11 (European Molecular Biology Laboratory) at Deutsches Elektronen Synchrotron, Hamburg, Germany.…”

Section: Methodsmentioning

confidence: 99%

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Structure of the non-redox-active tungsten/[4Fe:4S] enzyme acetylene hydratase

Seiffert

Ullmann

Messerschmidt

et al. 2007

Proc. Natl. Acad. Sci. U.S.A.

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140

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The tungsten-iron-sulfur enzyme acetylene hydratase stands out from its class because it catalyzes a nonredox reaction, the hydration of acetylene to acetaldehyde. Sequence comparisons group the protein into the dimethyl sulfoxide reductase family, and it contains a bis-molybdopterin guanine dinucleotide-ligated tungsten atom and a cubane-type [4Fe:4S] cluster. The crystal structure of acetylene hydratase at 1.26 Å now shows that the tungsten center binds a water molecule that is activated by an adjacent aspartate residue, enabling it to attack acetylene bound in a distinct, hydrophobic pocket. This mechanism requires a strong shift of pK a of the aspartate, caused by a nearby low-potential [4Fe:4S] cluster. To access this previously unrecognized W-Asp active site, the protein evolved a new substrate channel distant from where it is found in other molybdenum and tungsten enzymes.acetylene reduction ͉ metalloproteins ͉ tungsten enzymes A n estimated one-third of all proteins contain metal ions or metal-containing cofactors, and their overwhelming majority is involved in either electron transfer or the catalysis of redox reactions (1). Different metal centers typically take on specific functional roles, and although their respective substrates can vary significantly, they commonly catalyze similar kinds of chemical reactions. Molybdenum and tungsten are the only known second-and third-row transition metals to occur in biomolecules, and they are almost exclusively coordinated by the organic cofactor molybdopterin (2, 3). Mo/W proteins play important metabolic roles in all kingdoms of organisms and include prominent enzymes, such as nitrate reductase (4, 5), formate dehydrogenase (6), sulfite oxidase (7), or xanthine oxidase (8). They are involved either in oxygen atom transfer reactions or oxidative hydroxylations, whereby the metal undergoes two-electron oxidation/reduction between the states ϩIV and ϩVI (9). An exception to this rule was recently discovered for the pyrogallol:phloroglucinol hydroxyltransferase of Pelobacter acidigallici (10) that catalyzes a net nonredox reaction, but closer inspection reveals a reductive dehydroxylation and an oxidative hydroxylation as separate, consecutive events.A true nonredox reaction has been described for the tungsteniron-sulfur enzyme acetylene hydratase (AH) from Pelobacter acetylenicus (11), a member of the DMSO reductase family of molybdenum and tungsten enzymes (2, 9). It catalyzes the hydration of acetylene to acetaldehyde (see Eq. 1) as part of an anaerobic degradation pathway of unsaturated hydrocarbons (12).Chemically, the mercuric ion, Hg 2ϩ , will catalyze the addition of water to alkynes through the formation of a cyclic mercurinium ion. In a consecutive step, water attacks the most substituted carbon atom of this cyclic intermediate followed by the formation of a mercuric enol, which then rearranges to the corresponding ketone (13, 14). In the living world, AH is the only enzyme known to be able to convert acetylene other than nitrogenase (15, 16), for which,...

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Section: Methodsmentioning

confidence: 99%

“…AH has been purified and crystallized under the strict exclusion of dioxygen in its active, reduced state (21). Here we present the crystal structure determined to a resolution of 1.26 Å by single-wavelength anomalous dispersion methods using the anomalous signal from iron and tungsten at an x-ray energy above the K-edge of iron.…”

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confidence: 99%

Structure of the non-redox-active tungsten/[4Fe:4S] enzyme acetylene hydratase

Seiffert

Ullmann

Messerschmidt

et al. 2007

Proc. Natl. Acad. Sci. U.S.A.

Self Cite

140

169

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“…The crystallization of ACH was achieved under a N 2 /H 2 (94/6% v/v) atmosphere at 20 ° C using the sitting drop vapor diffusion method [Einsle et al, 2005]. Despite the minor differences in the circular dichroism spectroscopy data of ACH-W and ACH-Mo, only the former formed protein crystals suitable for X-ray analysis.…”

Section: Structural Properties Of Achmentioning

confidence: 99%

Structure and Function of the Unusual Tungsten Enzymes Acetylene Hydratase and Class II Benzoyl-Coenzyme A Reductase

et al. 2016

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In biology, tungsten (W) is exclusively found in microbial enzymes bound to a bis-pyranopterin cofactor (bis-WPT). Previously known W enzymes catalyze redox oxo/hydroxyl transfer reactions by directly coordinating their substrates or products to the metal. They comprise the W-containing formate/formylmethanofuran dehydrogenases belonging to the dimethyl sulfoxide reductase (DMSOR) family and the aldehyde:ferredoxin oxidoreductase (AOR) families, which form a separate enzyme family within the Mo/W enzymes. In the last decade, initial insights into the structure and function of two unprecedented W enzymes were obtained: the acetaldehyde forming acetylene hydratase (ACH) belongs to the DMSOR and the class II benzoyl-coenzyme A (CoA) reductase (BCR) to the AOR family. The latter catalyzes the reductive dearomatization of benzoyl-CoA to a cyclic diene. Both are key enzymes in the degradation of acetylene (ACH) or aromatic compounds (BCR) in strictly anaerobic bacteria. They are unusual in either catalyzing a nonredox reaction (ACH) or a redox reaction without coordinating the substrate or product to the metal (BCR). In organic chemical synthesis, analogous reactions require totally nonphysiological conditions depending on Hg²⁺ (acetylene hydration) or alkali metals (benzene ring reduction). The structural insights obtained pave the way for biological or biomimetic approaches to basic reactions in organic chemistry.

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“…The observed variation could reflect changes in the iron content during protein purification or crystallization; as Fe16 is in an approximate octahedral geometry with O–only ligands, the observed large variation in the edge jumps cannot be explained by the polarization effect. [6] Substoichiometric occupancies of metal binding sites are not unprecedented and have been noted in several metalloenzymes, such as the copper containing particulate methane monooxygenase, [7] the tungsten–iron–sulfur enzyme acetylene hydratase, [8] acetyl–CoA synthase/carbon monoxide dehydrogenase, [9] and in the ferrous form of superoxide reductase, [10] as well as for copper–bound metallochaperones. [11] …”

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confidence: 99%