Purification and characterization of acetylene hydratase of Pelobacter acetylenicus, a tungsten iron-sulfur protein

Rosner, Bettina; Schink, Bernhard

doi:10.1128/jb.177.20.5767-5772.1995

Cited by 123 publications

(145 citation statements)

References 53 publications

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“…Although the F(M)DH family is clearly distinct from the AOR family, sequence similarities have been detected between the F(M)DH family and the DMSO reductase family, indicating that the F(M)DH family may be included within this class of Mo-co-containing enzymes. Recently, a possible third class of tungsten enzymes was identified, with the isolation of acetylene hydratase from the acetylene-utilizing anaerobe Pelobacter acetylenicus (29). Unlike any other known tungstoenzymes or molybdoenzymes, this enzyme catalyzes a hydration, not an oxidation-reduction reaction.…”

Section: Enzyme Families Containing the Molybdenum-cofactormentioning

confidence: 99%

MOLYBDENUM-COFACTOR–CONTAINING ENZYMES: Structure and Mechanism

Kisker

Schindelin

Rees

1997

Annu. Rev. Biochem.

488

370

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Molybdenum-containing enzymes catalyze basic metabolic reactions in the nitrogen, sulfur, and carbon cycles. With the exception of the nitrogenase cofactor, molybdenum is incorporated into proteins as the molybdenum cofactor that contains a mononuclear molybdenum atom coordinated to the sulfur atoms of a pterin derivative named molybdopterin. Certain microorganisms can also utilize tungsten in a similar fashion. Molybdenum-cofactor-containing enzymes catalyze the transfer of an oxygen atom, ultimately derived from or incorporated into water, to or from a substrate in a two-electron redox reaction. On the basis of sequence alignments and spectroscopic properties, four families of molybdenum-cofactorcontaining enzymes have been identified. The available crystallographic structures for members of these families are discussed within the framework of the active site structure and catalytic mechanisms of molybdenum-cofactor-containing enzymes. Although the function of the molybdopterin ligand has not yet been conclusively established, interactions of this ligand with the coordinated metal are sensitive to the oxidation state, indicating that the molybdopterin may be directly involved in the enzymatic mechanism. CONTENTS

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Section: Enzyme Families Containing the Molybdenum-cofactormentioning

confidence: 99%

MOLYBDENUM-COFACTOR–CONTAINING ENZYMES: Structure and Mechanism

Kisker

Schindelin

Rees

1997

Annu. Rev. Biochem.

488

370

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“…Energy minimization and a 50 ps molecular dynamics (MD) simulation at 320 K (optimum temperature for activity at 50°C 55 ) were performed using the CHARMM force field 56 as implemented in the CHARMM program. 50 A stochastic boundary potential 57 was applied to maintain the overall structure of the water sphere, and all nonwater molecules were kept frozen during both the energy minimization and equilibration.…”

Section: Introductionmentioning

confidence: 99%

Comparison of QM-Only and QM/MM Models for the Mechanism of Tungsten-Dependent Acetylene Hydratase

Liao

Thiel

2012

J. Chem. Theory Comput.

127

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We report a comparison of QM-only and QM/MM approaches for the modeling of enzymatic reactions. For this purpose, we present a QM/MM case study on the formation of vinyl alcohol in the catalytic cycle of tungsten-dependent acetylene hydratase. Three different QM regions ranging from 32 to 157 atoms are designed for the reinvestigation of the previously suggested one-water attack mechanism. The QM/MM calculations with the minimal QM region M1 (32 atoms) yield a two-step reaction profile, with an initial nucleophilic attack followed by the protonation of the formed vinyl anion intermediate, as previously proposed on the basis of QM-only calculations on cluster model M2 (116 atoms); however, the overall QM/MM barrier with M1 is much too high, mainly due to an overestimate of the QM/MM electrostatic repulsions. QM/MM calculations with QM region M2 (116 atoms) fail to reproduce the published QM-only results, giving a one-step profile with a very high barrier. This is traced back to the strong electrostatic influence of the two neighboring diphosphate groups that were neglected in the QM-only work but are present at the QM/MM level. These diphosphate groups and other electrostatically important nearby residues are included in QM region M3 (157 atoms). QM/MM calculations with M3 recover the two-step mechanism and yield a reasonable overall barrier of 16.7 kcal/mol at the B3LYP/MM level. They thus lead to a similar overall mechanistic scenario as the previous QM-only calculations, but there are also some important variations. Most notably, the initial nucleophilic attack becomes rate limiting at the QM/MM level. A modified two-water attack mechanism is also considered but is found to be less favorable than the previously proposed one-water attack mechanism. Detailed residue interaction analyses and comparisons between QM/MM results with electronic and mechanical embedding and QM-only results without and with continuum solvation show that the protein environment plays a key role in determining the mechanistic preferences in acetylene hydratase. The combined use of QM-only and QM/MM methods provides a powerful approach for the modeling of enzyme catalysis.

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“…Among tungstoenzymes, AH constitutes a distinct class beside the tungsten aldehyde: ferredoxin oxidoreductases (18) and the formyl methanofuran dehydrogenases (3,19,20). Its reaction does not involve electron transfer, and neither its W atom nor the [4Fe:4S] cluster change their oxidation states during catalysis (12,17). However, AH activity requires activation by a strong reductant, indicating that the active form is W(IV) (17).…”

mentioning

confidence: 99%

“…It catalyzes the hydration of acetylene to acetaldehyde (see Eq. 1) as part of an anaerobic degradation pathway of unsaturated hydrocarbons (12).…”

mentioning

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.

136

169

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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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Purification and characterization of acetylene hydratase of Pelobacter acetylenicus, a tungsten iron-sulfur protein

Cited by 123 publications

References 53 publications

MOLYBDENUM-COFACTOR–CONTAINING ENZYMES: Structure and Mechanism

MOLYBDENUM-COFACTOR–CONTAINING ENZYMES: Structure and Mechanism

Comparison of QM-Only and QM/MM Models for the Mechanism of Tungsten-Dependent Acetylene Hydratase

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

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