The name for cupin proteins is derived from the Latin term for small barrel, ‘Cupa’. Proteins that belong to the group of cupins adopt a barrel-like structure (Dunwell et al., 2001). According to the database of Structural Classification of Proteins (SCOP) (Murzin et al., 1995), the cupin proteins have been classified as members of ‘RmlC-like Cupins’ superfamily in the ‘Double Stranded Beta Helix’ (DSBH) fold, however, the nomenclature employed in the literature is somewhat ambiguous since JmjC transcription factors (Clissold & Ponting, 2001) display many features typical of the DSBH fold. These common characteristics for the DSBH fold include a pair of four-stranded antiparallel β-sheets constituting up to eight β strands which form the typical β-sandwich structure. The superfamily comprises of 20 families with members performing diverse functions ranging from enzymatic activities like dioxygenases, hydrolases, decarboxylases, epimerases and isomerases to non-enzymatic functions such as binding to auxin, seed storage, and nuclear transcription factors (Dunwell, 2001, 2004). The nature of substrates used in various enzymatic reactions differs in chemical types, size, and structure. The sequence identity is low among the members of this superfamily. The functional site of members of this superfamily is generally located at the center of a conserved barrel. The cupin domain usually consists of two sequence motifs, each corresponding to two β-strands. A less conserved region separates these motifs. The conserved motifs, GX5HXHX3,4EX6G and GX5PXGX2HXX3N, together contain the residues involved in metal ion binding at the active site, that is known to play a functional role (Dunwell, 2001, 2004). It has been indicated that 10,346 cupin sequences (Finn et al., 2008) have been identified in 843 species that belong to eukaryotes, prokaryotes, archaebacteria and viruses. In some plant species like
Although metal binding cupins were first noted to possess ligands consisting of 3-His-1-carboxylate, it is now apparent that variations in the consensus sequence consisting of either deletions or substitutions of these ligands can produce alternate metal binding sites (Table 1). Representative examples for metal binding sites are included in Fig. 1. This article will focus on structurally characterized cupin proteins possessing metal binding sites consisting of 2-His, 2-His-1-Gln, 2-His-1-Glu-1-X (X = Gln, Tyr), 3-His, 3-His-1-Glu, 3-His-1-Gln, and 4-His. Efforts to model the active sites for these cupin proteins using synthetic compounds will be discussed and areas which would benefit from future studies will be included.
2.1. SyrB2 (PDB:2FCT)
The biosynthesis of halogenated products is common in many microorganisms (van Pée, 1996). While incorporation of halogens into aromatic and heteroaromatic moieties is usually catalyzed by FAD-dependent enzymes, the more challenging formation of aliphatic carbon-chlorine bonds is generally accomplished by α-ketoglutarate-dependent dioxygenases (Chang et al., 2002; Guenzi et al., 1998). SyrB2 belongs to this group of oxygenation enzymes (Vaillancourt et al., 2005). The gene encoding SyrB2 is part of the syringomycin synthetase gene cluster of
A proposed mechanism for SyrB2 is shown in Scheme 1 (Borowski et al., 2010). In this mechanism, the α-KG binds to the FeII center with its carboxylic and keto groups, while a water molecule occupies the position
Synthetic model studies for SyrB2 have been carried out using sterically hindered α-ketocarboxylate 2,6-di(mesityl)benzoylformate (MesBF) with the FeII complexes LFeCl2 (L =
3.1. Cysteine dioxygenase (PDB: 2B5H)
Cysteine Dioxygenase (CDO) initiates the catabolism of cysteine to pyruvate and sulfate, which is essential for the generation of adequate inorganic sulfate and allows pyruvate to enter central pathways of metabolism. Mammalian CDO was assigned to the cupin superfamily. The crystal structure of recombinant rat CDO was determined at 1.5 Å resolution. The active site coordination of CDO comprises a tetrahedrally coordinated FeII center involving three histidine nitrogens and a water molecule (Simmons et al., 2006). A structure at 1.75 Å was also reported containing a NiII coordinated in distorted octahedral geometry with three histidine nitrogens and three water molecules (Mc Coy et al., 2006). These two eukaryotic structures revealed the presence of a rare cysteinyl-tyrosine cross-link cofactor (Cys93 and Tyr157) while a structure from a prokaryote (
Several mechanisms have been proposed for CDO. A mechanism was postulated after the crystal structure of human CDO was available (Scheme 2) (Ye et al., 2007). This proposal starts with FeII coordinated by His86, His88, and His140 and a water (McCoy et al., 2006). The hydroxyl group of Tyr157 participates in hydrogen bonded to the coordinated water. Upon addition of substrate, the water molecule is displaced by the thiol group of cysteine with the amino group additionally bonded. This binding geometry allows the carboxyl group of cysteine to engage in the hydrogen bonding network formed by the second coordination sphere (i.e. Tyr157, Tyr58, and His155). The dioxygen then binds in an ‘end-on’ fashion. Homolytic scission of the O−O bond occurs in concert with abstraction of a hydrogen atom from the Tyr157, forming a tyrosyl radical. The electron in the O−O bond is used to form a bond with the iron center resulting in a oxoferryl species, FeIV=O. The radical on the phenoxyl group then abstracts a hydrogen atom from cysteine’s thiol. The ferryl species attacks the lone pair on cysteine’s sulfur, forming a single S−O bond. This intermediate then undergoes reductive elimination to form an S=O bond and FeII. The sulfinic acid group is deprotonated and finally, CSA is released from the active site. It is likely that the thiol group would become deprotonated once bonded to the metal since its pKa would drop significantly.
Another mechanism that does not employ radical coupling to produce a thioperoxide is shown in Scheme 3 (Joseph & Maroney, 2007). This mechanism proposes that cysteine initially binds to the iron center by the amine and thiolate groups. In the next step, the
FeII-thiolate complexes [(
3.2. Gentisate 1,2-dioxygenase (PDB: 3BU7)
Gentisate 1,2-dioxygenase catalyze dioxygen incorporation into various organic compounds and plays a key role in the complex degradation pathway of mono- and polycyclic aromatic and hetero-aromatic compounds (Equation 2). The crystal structure of gentisate 1,2-dioxygenase from
The proposed catalytic mechanism for the N domain of GDOsp, is shown in Scheme 4 (Chen et al., 2008). First, gentisate binds to the active site and displaces one water molecules to chelate with the FeII ion by its deprotonated carboxylate at C1 and phenolic hydroxyl at C2. The Hydroxyl at C5 participates in a hydrogen bond with the Asp175 carboxylic side chain, which, additionally is stabilized by the side chain of Gln108. The chelation and H-bond network position the gentisate substrate at the active site. The substrate dioxygen then coordinates to the FeII ion forming a Fe–superoxide complex as illustrated in the mechanisms of other extradiol dioxygenases (Kovaleva & Lipscomb, 2007). This complex is stabilized by the imidazole side chain of conserved His162 through electron transfer and hydrogen-bond rearrangement. The attack of the FeIII-bound superoxide at C1 affords an alkylperoxo intermediate. A subsequent Criegee rearrangement of this intermediate results in O−O bond scission and insertion of the first oxygen atom into the aromatic ring to generate an anhydride intermediate. The subsequent transfer of the hydroxyl group from FeII ion to C2 and resonance rearrangement would give the product maleylpyruvate acid.
3.3. Acetylacetone-cleaving enzyme (PDB: 3BAL)
The crystal structure of Dke1 from
Bxe_A2876 is an FeII-dependent oxygenase from
4.1. Quercetin 2,3-dioxygenase (PDB: 1JUH)
Quercetinase is produced by various filamentous fungi when grown on rutin as the sole carbon and energy source. The reaction involves dioxygenation of quercetin to yield the corresponding depside (phenolic ester 2-protocatechuoylphloroglucinol carboxylic acid) and carbon monoxide (Equation 4). Crystal structures available for both the eukaryotic copper containing (
The dioxygenation mechanism (Scheme 6) that has been proposed from structural studies involves binding of quercetin to the metal copper(II) center followed by reversible formation of a flavonol radical (centered on C2). Reaction of this species with dioxygen leads to an endoperoxide (C2−C4) that decomposes generating the depside and CO (Steiner et al., 2002). In this reaction sequence, dioxygen would access the copper center from the enzyme surface through a predicted channel from molecular simulations (van den Bosch et al., 2004). In addition to these major structural data, biomimetic studies (Grubel et al., 2010; Malkhasian et al., 2007; Pap et al., 2010) and computational investigations (Fiorucci, 2004, 2006, 2007; Siegbahn, 2004) have recently increased our knowledge on these enzymes. However, despite this progress, questions relating to the function of both the prokaryotic and the eukaryotic enzymes, and in particular about the use of different redox metals for the activation of either the substrate or dioxygen, remain to be answered. Recently a quercetinase with a preference for NiII and CoII was discovered (Merkens et al., 2008).
4.2. Pirin (PDB: 1J1L)
Pirin is a newly identified nuclear protein that interacts with the oncoprotein B-cell lymphoma 3-encoded (Bcl-3) and nuclear factor I (NFI) (Pang et al., 2004). The crystal structure of human Pirin at 2.1 Å resolution shows it to be a member of the functionally diverse cupin superfamily. The enzymatic role for Pirin is most likely in biological redox reactions involving oxygen, and provides compelling evidence that Pirin requires the participation of the metal ion for its interaction with Bcl-3 to co-regulate the NF-κB transcription pathway and the interaction with NFI in DNA replication. Substitution of Fe by heavy metals thus provides a unique pathway for these metals to directly influence gene transcription. The determined structure suggests a new role for iron in biology and that Pirin may be involved in novel gene regulation mechanisms. The enzyme is widespread in mammals (including humans), plants, fungi and prokaryotes (Wendler et al., 1997). There are crystal structures for human pirin (PDB code 1J1L) and a pirin homologue from
4.3. Oxalate decarboxylase (PDB: 1UW8)
Oxalate is produced by plants and microbes by the hydrolysis of oxaloacetate or by the oxidation of glyoxylate or ascorbate (Franceschi & Nakata, 2005). Oxalate secreted by fungi promotes the degradation of lignin (Shimada et al., 1997). Accumulation of oxalate in leafy plants such as spinach and
Next, oxygen binds the MnII to generate a MnIII-superoxide species. The reversible electron transfer from coordinated oxalate to MnIII superoxide complex is accompanied by a proton transfer from the protonated substrate to a nearby residue. Based on the OxDC crystal structure, the Glu333 could function as the general base in the deprotonation reaction due to its close proximity to the bound substrate. Decarboxylation and C−C bond cleavage in a second step, facilitated by the partial positive charge on the carbon that will become formate, then results in formation of a formate radical (Mathusamy et al., 2006). The formate radical then acquires a proton, possibly from residue Glu162, and an electron from the enzyme bound manganese ion to yield the product formate bound MnIII superoxide. Loss of formate and dioxygen lead to the resting state of OxDC containing MnII in an octahedral environment.
4.4. Oxalate oxidase (PDB: 1FI2)
Oxalate oxidase (OxOx), also known as germin, is expressed by plants such as wheat and barley and catalyzes the manganese-dependent oxidative decarboxylation of oxalate to carbon dioxide and hydrogen peroxide (Equation 6), and protects plants from the toxic effects of oxalate (Dunwell et al., 2004).
The structure of OxOx (Woo et al., 2000) contains a manganese ion bound to the side chains of conserved glutamate and histidine residues in a site that is located toward the narrow end of the barrel-like domain. Studies by Whittaker and coworkers (Whittaker et al., 2007) have suggested that the catalytically active form of OxOx is likely the MnIII form and not the MnII form as was previously thought. In this mechanism (Scheme 8), the active, resting MnIII enzyme binds oxalate (step 1) as the monoanion to form an enzyme-substrate complex. Protein side chain residues (Asn75 and Asn85) stabilize the oxalate group via hydrogen bonding interactions. Oxalate has been shown to anaerobically reduce the MnIII form of the enzyme to the MnII form (step 2) (Whittaker & Whittaker, 2002). The reduction of the MnIII to MnII is closely associated with generation of an oxalyl free radical (seen in Scheme 8 as potentially binding in a chelating manner). The oxalyl radical is very unstable and is known to undergo rapid C−C bond fission nonenzynmatically in aqueous solution (
An alternative mechanism for OxOx that has also been proposed (Burrell et al., 2007) (Scheme 9) begins with MnII becoming oxidized to MnIII-superoxo after binding to oxalate. Internal redox results in MnII-superoxo bonded to one electron oxidized oxalate. CO2 is lost resulting in a MnII-superoxo bound carbanion. The next step invokes a percarbonate intermediate which decomposes to release H2O2 and CO2 regenerating the resting state. There has been recent interest in using synthetic compounds to model the active site of OxOx (Fuller et al., 2006; Fuller et al., 2005; Makowska-Grzyska et al., 2003; Scarpellini et al., 2008).
4.5. Acireductone dioxygenase (PDB: 1VR3)
Acireductone Dioxygenase (ARD) is an enzyme involved in methionine salvage pathway (Scheme 10), which regulates aspects of the cell cycle. The addition of polyamines to cells accelerates their DNA replication and division, whereas inhibition of polyamine biosynthesis arrests DNA replication and prevents continuation of the cycle. Methylthioadenosine (MTA) is a inhibitor for the biosynthesis of polyamine. The MTA concentration in biological systems is therefore tightly regulated. This control is effected via the methionine salvage pathway, where MTA is recycled through a series of reactions that convert its 5-methylthio-D-ribose group to methionine. This pathway has been found in many organisms including
Depending on the metal bound at the active site, ARD catalyzes two different reactions. ARD with ferrous iron bound promotes the cleavage of 1,2-dihydroxy-5-methylthiopent-1-en-3-one to a precursor of methionine and formate. The NiII-bound variant catalyzes an off-pathway reaction, leading to products methylthiopropionate, CO and formate thus preventing the recycling to methionine (Dai, 1999, 2001). The proposed catalytic pathway for ARD in its FeII and NiII forms (Pochapsky et al., 2002) involves initial bidentate coordination of the deprotonated substrate to the metal center (Scheme 11, A). Dioxygen reduction by the metal-substrate complex involving either inner or outer-sphere mechanism (B), facilitated by the reactive substrate. Following formation of the peroxide intermediate (C), the reaction branches into two paths depending on the metal involved. The FeII-dependent pathway I begins (D) with nucleophilic attack on the adjacent carbonyl carbon to yield a four-membered ring (E), which then decomposes into formate acid and α-oxo-acid products (F).
For pathway II, the reaction of the NiII enzyme (D’) is suggested to involve intramolecular rotation of the metal-bound substrate across its C2−C3 bond such that the C3 carbonyl group is now coordinating (E’) and the peroxide attack is directed towards the C3 carbonyl carbon (F’), with opening of the five-membered dioxolane ring to yield formate, carbon monoxide, and the corresponding carboxylate products (G’). From EXAFS studies (Al-Mjeni et al., 2002), substrate binding is believed to cause dissociation of one water and one histidine ligand. Efforts to model the NiII form of the enzyme have most notably been carried out by Berreau and coworkers (Berreau et al., 2011; Grubel et al., 2010; Rudzka et al., 2010; Szajna-Fuller, 2007a, 2007b; Szajna, 2004, 2005).
5.1. Bacilysin biosynthesis protein BacB (PDB: 3H7J)
Bacilysin is a non-ribosomally synthesized dipeptide antibiotic that is active against a wide range of bacteria and some fungi. Synthesis of bacilysin (L-alanine-[2,3-epoxycyclohexano-4]-L-alanine) is achieved by proteins in the
6.1. RemF protein
RemF is a polyketide cyclase involved in the biosynthesis of the aromatic pentacyclic metabolite resistomycin (Scheme 13) in
6.2. Carotenoid oxygenase
Carotenoid oxygenases (CarOs) are involved in the biosynthesis of retinal and structurally related pigments and have been reported in animals, plants, and more recently cyanobacteria (Ruch et al., 2005). They are described as FeII-dependent enzymes and catalyze the 15–15′ bond cleavage of a carotene precursor, as shown in Scheme 14. CarOs are
very different from the other enzymes discussed with regard to both structure and catalytic mechanism. The structure of CarO from the cyanobacterium
Proteins possessing the cupin DSBH fold are present in all realms of life and can participate in a diverse set of enzymatic reactions. In many cases, these enzymes are assisted by various first row transition metal cofactors. The number of metalloproteins discovered within this class appears to be increasing at a rapid rate. High throughput structural methods and utilization of expression systems has assisted in the increased discovery of cupin enzymes. The catalytically active metal binding sites consist of a variation of Histidine, Glutamine, Aspartate/Glutamate, and/or Tyrosine ligands. This chapter was focused on proteins which had been structurally elucidated and with new potentials for investigations regarding their mechanisms. The mechanisms employed by these enzymes are diverse and their detailed investigation can be probed using synthetic model systems. Since synthetic models can be systematically altered and subjected to conditions not be suitable to biological investigation, there is much promise in slowing down catalytic reactions using low temperature methods and potentially isolating intermediates which can then be investigated by various physical methods and in some cases, by crystallography. Furthermore, physical studies on model complexes along with computational investigations can potentially provide information regarding correlations between electronic and structural parameters.
Financial support from Oakland University and NSF Grant No. CHE-0748607 is gratefully acknowledged. The National Science Foundation (NSF) award (CHE-0821487) is also acknowledged.