an aldehyde + H2O + 2 oxidized ferredoxin ⇌ an acid + 3 H+ + 2 reduced ferredoxin
This enzyme belongs to the family of oxidoreductases, specifically those acting on the aldehyde or oxo group of donor with an iron-sulfur protein as acceptor. The systematic name of this enzyme class is aldehyde:ferredoxin oxidoreductase. This enzyme is also called AOR. It is a relatively rare example of a tungsten-containing protein.[1]
Occurrence
The active site of the AOR family feature an oxo-tungsten center bound to a pair of molybdopterincofactors (which does not contain molybdenum) and an 4Fe-4S cluster.[2][3] This family includes AOR, formaldehyde ferredoxin oxidoreductase (FOR), glyceraldehyde-3-phosphate ferredoxin oxidoreductase (GAPOR), all isolated from hyperthermophilic archea;[2]carboxylic acidreductase found in clostridia;[4] and hydroxycarboxylate viologen oxidoreductase from Proteus vulgaris, the sole member of the AOR family containing molybdenum.[5] GAPOR may be involved in glycolysis,[6] but the functions of the other proteins are not yet clear. AOR has been proposed to be the primary enzyme responsible for oxidising the aldehydes that are produced by the 2-keto acid oxidoreductases.[7]
AOR is found in hyperthermophillic archaea, Pyrococcus furiosus.[1] The archaeons Pyrococcus ES-4 strain and Thermococcus ES-1 strain differ by their substrate specificity: AFOs show a broader size range of its aldehyde substrates. Its primary role is to oxidize aldehyde coming derived from the metabolism of amino acids and glucoses.[8] Aldehyde Ferredoxin Oxidoreductase is a member of an AOR family, which includes glyceraldehyde-3-phosphate ferredoxin oxidoreductase (GAPOR) and Formaldehyde Ferredoxin Oxidoreductase.[3]
Function
AOR functions at high temperature conditions (~80 degrees Celsius) at an optimal pH of 8-9. It is oxygen-sensitive as it loses bulk of its activity from oxygen exposure and works in the cytoplasm where it is a reducing environment. Thus, either exposure to oxygen or lowering of the temperature causes an irreversible loss of its catalytic properties. Also, as a result of oxygen sensitivity of AOR, purification of the enzyme is done under anoxic environments.[8]
It is proposed that AOR has a role in the Entner-Doudoroff pathway (glucose degradation) due to its increased activity with maltose incorporation.[3] However, other proposals include its role in oxidation of amino acid metabolism aldehyde side products coming from de-aminated 2-ketoacids. The main substrates for aldehyde ferredoxin oxidoreductase are acetaldehyde, phenylacetaldehyde, and isovalerdehyde, which is a metabolic product from common amino acids and glucose.[8] For example, acetaldehyde reaches its kcat/KM value up to 22.0 μM-1s-1.[8] In fact, some microorganisms only make use of amino acids as a carbon source, such as Thermococcus strain ES1; thus, they utilize aldehyde ferredoxin oxidoreductase to metabolize the amino acid carbon source.[8]
AOR is homodimeric. Each 67kDa subunit contains 1 tungsten and 4-5 Iron atoms.[3] The two subunits are bridged by a low spin Iron center. It is believed that the two subunits function independently.[3]
Tungsten-pterin
Tungsten in the active site of AOR adopts a distorted square pyramidal geometry bound an oxo/hydroxo ligand and the dithiolene substituents of two molybdopterin cofactors.[3]
Molybdopterin cofactor, shown in the dithiol protonation state.
Two molybdopterin cofactors bind tungsten,[9] as observed in many related enzymes.[9] Tungsten is not bonded directly to the protein.[9] Phosphate centers pendant on the cofactor are bound to a Mg2+, which is also bound by Asn93 and Ala183 to complete its octahedral coordination sphere.[3][9] Thus, pterin and Tungsten atoms are connected to the AOR enzyme primarily through pterin's Hydrogen bonding networks with the amino acid residues.[3] In addition, two water ligands that occupy the octahedral geometry take part in hydrogen bonding networks with pterin, phosphate, and Mg2+.[9] While [Fe4S4] cluster is bound by four Cys ligands, Pterin - rich in amino and ether linkages - interacts with the Asp-X-X-Gly-Leu-(Cys/Asp) sequences in the AOR enzyme.[3] In such sequence, Cys494 residue is also hydrogen bonded to the [Fe4S4] cluster.[3] This indicates that Cys494 residue connects the Tungsten site and the [Fe4S4] cluster site in the enzyme.[3] Iron atom in the cluster is additionally bound by three other Cystein ligands: .[9] Also, another linker amino acid residue between ferredoxin cluster and pterin is the Arg76, which hydrogen bonds to both pterin and ferredoxin.[3] It is proposed that such hydrogen bonding interactions imply pterin cyclic ring system as an electron carrier.[3] Additionally the C=O center of the pterin binds Na+.[8] The W=O center is proposed, not verified crystallographically.[9]
AOR consists of three domains, domain 1, 2, and 3.[8] While domain 1 contains pterin bound to tungsten, the other two domains provide a channel from tungsten to protein's surface (15 Angstroms in length) in order to allow specific substrates to enter the enzyme through its channel.[8] In the active site, this pterin molecules is in a saddle-like conformation (500 to the normal plane) to “sit” on the domain 1 which also takes on a form with beta sheets to accommodate the Tungsten-Pterin site.[8]
Iron
The iron center in between the two subunits serve a structural role in AOR.[8] Iron metal atoms takes on a tetrahedral conformation while the ligand coordination comes from two histidines and glutamic acids.[8] This is not known to have any functional role in the redox activity of the protein.[8]
Fe4S4 centre
[Fe4S4] cluster in AOR is different in some aspects to other ferredoxin molecules.[3] EPR measurements confirm that it serves as a one-electron shuttle.[3]
Aldehyde ferredoxin oxidoreductase mechanism
In the catalytic cycle, W(VI) (tungsten "six") converts to W(IV) concomitant with oxidation of the aldehyde to a carboxylic acid (equivalently, a carboxylate).[3] A W(V) intermediate can be detected by EPR spectroscopy.[3][8]
The redox equivalents are provided by the 4Fe-4S cluster.
A tyrosine residue is proposed to activate the electrophilic centre of aldehydes by H-bonding to the carbonyl oxygen atom, coordinated to the W centre.[10] A glutamic acid residue near the active site activates a water molecule for a nucleophilic attack on aldehyde carbonyl center.[10] After nucleophilic attack by water, hydride is transferred to oxo-tungsten sie thus, .[10] Subsequently, W(VI) is regenerated by electron transfer to the 4Fe-4S center.[10] With formaldehyde ferredoxin oxidoreductase, Glu308 and Tyr 416 would be involved while Glu313 and His448 is shown to be present in AOR active site.[9][10]
Related Research Articles
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References
1 2 Majumdar A, Sarkar S (May 2011). "Bioinorganic chemistry of molybdenum and tungsten enzymes: A structural–functional modeling approach". Coordination Chemistry Reviews. 255 (9–10): 1039–1054. doi:10.1016/j.ccr.2010.11.027.
↑ White H, Strobl G, Feicht R, Simon H (September 1989). "Carboxylic acid reductase: a new tungsten enzyme catalyses the reduction of non-activated carboxylic acids to aldehydes". Eur. J. Biochem. 184 (1): 89–96. doi:10.1111/j.1432-1033.1989.tb14993.x. PMID2550230.
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