Toxoflavin lyase requires a novel 1-His-2-carboxylate facial triad

High-resolution crystal structures are reported for apo, holo, and substrate-bound forms of a toxoflavin-degrading metalloenzyme (TflA). In addition, the degradation reaction is shown to be dependent on oxygen, Mn(II), and dithiothreitol in vitro. Despite its low sequence identity with proteins of known structure, TflA is structurally homologous to proteins of the vicinal oxygen chelate superfamily. Like other metalloenzymes in this superfamily, the TflA fold contains four modules that associate to form a metal binding site; however, the fold displays a rare rearrangement of the structural modules indicative of domain permutation. Moreover, unlike the 2-His-1-carboxylate facial triad commonly utilized by vicinal oxygen chelate dioxygenases and other dioxygen-activating non-heme Fe(II) enzymes, the metal center in TflA consists of a 1-His-2-carboxylate facial triad. The substrate-bound complex shows square-pyramidal geometry in which one position is occupied by O5 of toxoflavin. The open coordination site is predicted to be the dioxygen binding site. TflA appears to stabilize the reduced form of toxoflavin through second-sphere interactions. This anionic species is predicted to be the electron source responsible for reductive activation of oxygen to produce a peroxytoxoflavin intermediate.     

Addition of an external electron donor to in vitro assays of cysteine dioxygenase precludes the need for exogenous iron

Cysteine dioxygenase (CDO) utilizes a 3-His facial triad for coordination of its metal center. Recombinant CDO present in cellular lysate exists primarily in the ferrous form and exhibits significant catalytic activity. Removal of CDO from the reducing cellular environment during purification results in the loss of bound iron and oxidation of greater than 99% of the remaining metal centers. The as-isolated recombinant enzyme has comparable activity as the background level of L-cysteine oxidation confirming that CDO is inactive under the aerobic conditions required for catalysis. Including exogenous ferrous iron in assays resulted in non-enzymatic product formation; however, addition of an external reductant in assays of the purified protein resulted in the recovery of CDO activity. EPR spectroscopy of CDO in the presence of a reductant confirms that the recovered activity is consistent with reduction of iron to the ferrous form. The as-isolated enzyme in the presence of L-cysteine was nearly unreactive with the dioxygen analog, but had increased affinity when pre-incubated with an external reductant. These studies shed light on the discrepancies among reported kinetic parameters for CDO and also juxtapose the stability of the 3-His and 2-His/1-carboxylate ferrous enzymes in the presence of dioxygen.     

Substrate Binding Mechanism of a Type I Extradiol Dioxygenase

A meta-cleavage pathway for the aerobic degradation of aromatic hydrocarbons is catalyzed by extradiol dioxygenases via a two-step mechanism: catechol substrate binding and dioxygen incorporation. The binding of substrate triggers the release of water, thereby opening a coordination site for molecular oxygen. The crystal structures of AkbC, a type I extradiol dioxygenase, and the enzyme substrate (3-methylcatechol) complex revealed the substrate binding process of extradiol dioxygenase. AkbC is composed of an N-domain and an active C-domain, which contains iron coordinated by a 2-His-1-carboxylate facial triad motif. The C-domain includes a β-hairpin structure and a C-terminal tail. In substrate-bound AkbC, 3-methylcatechol interacts with the iron via a single hydroxyl group, which represents an intermediate stage in the substrate binding process. Structure-based mutagenesis revealed that the C-terminal tail and β-hairpin form part of the substrate binding pocket that is responsible for substrate specificity by blocking substrate entry. Once a substrate enters the active site, these structural elements also play a role in the correct positioning of the substrate. Based on the results presented here, a putative substrate binding mechanism is proposed.     

In vivo self-hydroxylation of an iron-substituted manganese-dependent extradiol cleaving catechol dioxygenase

The homoprotocatechuate 2,3-dioxygenase from Arthrobacter globiformis (MndD) catalyzes the oxidative ring cleavage reaction of its catechol substrate in an extradiol fashion. Although this reactivity is more typically associated with non-heme iron enzymes, MndD exhibits an unusual specificity for manganese(II). MndD is structurally very similar to the iron(II)-dependent homoprotocatechuate 2,3-dioxygenase from Brevibacterium fuscum (HPCD), and we have previously shown that both MndD and HPCD are equally active towards substrate turnover with either iron(II) or manganese(II) (Emerson et al. in Proc. Natl. Acad. Sci. USA 105:7347–7352, 2008). However, expression of MndD in Escherichia coli under aerobic conditions in the presence of excess iron results in the isolation of inactive blue-green iron-substituted MndD. Spectroscopic studies indicate that this form of iron-substituted MndD contains an iron(III) center with a bound catecholate, which is presumably generated by in vivo self-hydroxylation of a second-sphere tyrosine residue, as found for other self-hydroxylated non-heme iron oxygenases. The absence of this modification in either the native manganese-containing MndD or iron-containing HPCD suggests that the metal center of iron-substituted MndD is able to bind and activate O₂ in the absence of its substrate, employing a high-valence oxoiron oxidant to carry out the observed self-hydroxylation chemistry. These results demonstrate that the active site metal in MndD can support two dramatically different O₂ activation pathways, further highlighting the catalytic flexibility of enzymes containing a 2-His-1-carboxylate facial triad metal binding motif.     

Structural studies on the reaction of isopenicillin N synthase with the truncated substrate analogues delta-(L-alpha-aminoadipoyl)-L-cysteinyl-glycine and delta-(L-alpha-aminoadipoyl)-L-cysteinyl-D-alanine

Isopenicillin N synthase (IPNS), a non-heme iron(II)-dependent oxidase, catalyzes conversion of the tripeptide delta-(l-alpha-aminoadipoyl)-l-cysteinyl-d-valine (ACV) to bicyclic isopenicillin N (IPN), concomitant with the reduction of dioxygen to two molecules of water. Incubation of the "truncated"substrate analogues delta-(l-alpha-aminoadipoyl)-l-cysteinyl-glycine (ACG) and delta-(l-alpha-aminoadipoyl)-l-cysteinyl-d-alanine (ACA) with IPNS has previously been shown to afford acyclic products, in which the substrate cysteinyl residue has undergone a two-electron oxidation. We report X-ray crystal structures for the anaerobic IPNS/Fe(II)/ACG and IPNS/Fe(II)/ACA complexes, both in the absence and presence of the dioxygen analogue nitric oxide. The overall protein structures are very similar to those of the corresponding IPNS/Fe(II)/ACV complexes; however, significant differences are apparent in the vicinity of the active site iron. The structure of the IPNS/Fe(II)/ACG complex reveals that the C-terminal carboxylate of this substrate is oriented toward the active site iron atom, apparently hydrogen-bonded to an additional water ligand at the metal; this is a different binding mode to that observed in the IPNS/Fe(II)/ACV complex. ACA binds to the metal in a manner that is intermediate between those observed for ACV and ACG. The addition of NO to these complexes initiates conformational changes such that both the IPNS/Fe(II)/ACG/NO and IPNS/Fe(II)/ACA/NO structures closely resemble the IPNS/Fe(II)/ACV/NO complex. These results further demonstrate the feasibility of metal-centered rearrangements in catalysis by non-heme iron enzymes and provide insight into the delicate balance between hydrophilic-hydrophobic interactions and steric effects in the IPNS active site.     

Versatility of biological non-heme Fe(II) centers in oxygen activation reactions

Oxidase and oxygenase enzymes allow the use of relatively unreactive O2 in biochemical reactions. Many of the mechanistic strategies used in nature for this key reaction are represented within the 2-histidine-1-carboxylate facial triad family of non-heme Fe(II)-containing enzymes. The open face of the metal coordination sphere opposite the three endogenous ligands participates directly in the reaction chemistry. Here, data from several studies are presented showing that reductive O2 activation within this family is initiated by substrate (and in some cases cosubstrate or cofactor) binding, which then allows coordination of O2 to the metal. From this starting point, the O2 activation process and the reactions with substrates diverge broadly. The reactive species formed in these reactions have been proposed to encompass four oxidation states of iron and all forms of reduced O2 as well as several of the reactive oxygen species that derive from O-O bond cleavage.     

Cytochrome P450: substrate and prosthetic-group free radicals generated during the enzymatic cycle

During the enzymatic cycle of the cytochromes P450, dioxygen binds to the ferrous haemprotein when the resting ferric haemprotein has undergone a one-electron oxidation after substrate binding. A further one-electron reduction generates an intermediate that is isoelectronic with a peroxide dianion coordinated to a ferric iron. Heterolytic cleavage of the omicron--omicron bond generates water and a species which is formally an oxene (oxygen atom) coordinated by iron(III). However, on the basis of model reactions and by analogy to the catalases and peroxidases, this active oxidizing intermediate is formulated as an oxo-FeIV porphyrin pi-cation radical. The radical is stabilized by delocalization on the porphyrin macrocycle and the high oxidation state is achieved by oxidizing both the metal and the porphyrin ring of the haemprotein. Hydrogen atom abstraction from a saturated hydrocarbon substrate generates a substrate free radical, constrained by the protein binding site, and the equivalent of a hydroxyl radical bound to iron(III). Coupling of the 'hydroxy' and substrate radicals generates hydroxylated product and resting protein. For olefins an initial electron transfer to oxidized haemprotein gives a substrate cation radical. Further reaction of this radical can give the epoxide, the principal product; an aldehyde or ketone by rearrangement; or an alkylated haemprotein resulting in suicide inhibition.     

Dioxygen activation by copper, heme and non-heme iron enzymes: comparison of electronic structures and reactivities

Enzymes containing heme, non-heme iron and copper active sites play important roles in the activation of dioxygen for substrate oxidation. One key reaction step is CH bond cleavage through H-atom abstraction. On the basis of the ligand environment and the redox properties of the metal, these enzymes employ different methods of dioxygen activation. Heme enzymes are able to stabilize the very reactive iron(IV)-oxo porphyrin-radical intermediate. This is generally not accessible for non-heme iron systems, which can instead use low-spin ferric-hydroperoxo and iron(IV)-oxo species as reactive oxidants. Copper enzymes employ still a different strategy and achieve H-atom abstraction potentially through a superoxo intermediate. This review compares and contrasts the electronic structures and reactivities of these various oxygen intermediates.     

Characterization of metal ligand mutants of phenylalanine hydroxylase: Insights into the plasticity of a 2-histidine-1-carboxylate triad

The iron atom in the nonheme iron monooxygenase phenylalanine hydroxylase is bound on one face by His285, His290, and Glu330. This arrangement of metal ligands is conserved in the other aromatic amino acid hydroxylases, tyrosine hydroxylase and tryptophan hydroxylase. A similar 2-His-1-carboxylate facial triad of two histidines and an acidic residue are the ligands to the iron in other nonheme iron enzymes, including the α-ketoglutarate-dependent hydroxylases and the extradiol dioxygenases. Previous studies of the effects of conservative mutations of the iron ligands in tyrosine hydroxylase established that there is some plasticity in the nature of the ligands and that the three ligands differ in their sensitivity to mutagenesis. To determine the generality of this finding for enzymes containing a 2-His-1-carboxylate facial triad, the His285, His290, and Glu330 in rat phenylalanine hydroxylase were mutated to glutamine, glutamate, and histidine. All of the mutant proteins had low but measurable activities for tyrosine formation. In general, mutation of Glu330 had the greatest effect on activity and mutation of His290 the least. All of the mutations resulted in an excess of tetrahydropterin oxidized relative to tyrosine formation, with mutation of His285 having the greatest effect on the coupling of the two partial reactions. The H285Q enzyme had the highest activity as tetrahydropterin oxidase at 20% the wild-type value. All of the mutations greatly decreased the affinity for iron, with mutation of Glu330 the most deleterious. The results complement previous results with tyrosine hydroxylase in establishing the plasticity of the individual iron ligands in this enzyme family.     

Oxalate decarboxylase and oxalate oxidase activities can be interchanged with a specificity switch of up to 282,000 by mutating an active site lid

Oxalate decarboxylases and oxalate oxidases are members of the cupin superfamily of proteins that have many common features: a manganese ion with a common ligand set, the substrate oxalate, and dioxygen (as either a unique cofactor or a substrate). We have hypothesized that these enzymes share common catalytic steps that diverge when a carboxylate radical intermediate becomes protonated. The Bacillus subtilis decarboxylase has two manganese binding sites, and we proposed that Glu162 on a flexible lid is the site 1 general acid. We now demonstrate that a decarboxylase can be converted into an oxidase by mutating amino acids of the lid that include Glu162 with specificity switches of 282,000 (SEN161-3DAS), 275,000 (SENS161-4DSSN), and 225,000 (SENS161-4DASN). The structure of the SENS161-4DSSN mutant showed that site 2 was not affected. The requirement for substitutions other than of Glu162 was, at least in part, due to the need to decrease the Km for dioxygen for the oxidase reaction. Reversion of decarboxylase activity could be achieved by reintroducing Glu162 to the SENS161-4DASN mutant to give a relative specificity switch of 25,600. This provides compelling evidence for the crucial role of Glu162 in the decarboxylase reaction consistent with it being the general acid, for the role of the lid in controlling the Km for dioxygen, and for site 1 being the sole catalytically active site. We also report the trapping of carboxylate radicals produced during turnover of the mutant with the highest oxidase activity. Such radicals were also observed with the wild-type decarboxylase.     

Crystallographic evidence for dioxygen interactions with iron proteins

The interaction of dioxygen with iron plays a key role in many important biological processes, such as dioxygen transport in the bloodstream and the reduction of dioxygen by iron in respiration. However, the catalytic mechanisms employed, for example in ligand oxidation, are not fully understood at the current time despite intensive biochemical, spectroscopic and structural studies. This review outlines the structural evidence obtained by X-ray crystallographic methods for the nature of the interactions between dioxygen and the metal in iron-containing proteins. Proteins involved in iron transport or electron transfer are not included.     

The structure of 3-methylaspartase from Clostridium tetanomorphum functions via the common enolase chemical step.

Methylaspartate ammonia-lyase (3-methylaspartase, MAL; EC ) catalyzes the reversible anti elimination of ammonia from L-threo-(2S,3S)-3-methylaspartic acid to give mesaconic acid. This reaction lies on the main catabolic pathway for glutamate in Clostridium tetanomorphum. MAL requires monovalent and divalent cation cofactors for full catalytic activity. The enzyme has attracted interest because of its potential use as a biocatalyst. The structure of C. tetanomorphum MAL has been solved to 1.9-A resolution by the single-wavelength anomalous diffraction method. A divalent metal ion complex of the protein has also been determined. MAL is a homodimer with each monomer consisting of two domains. One is an alpha/beta-barrel, and the other smaller domain is mainly beta-strands. The smaller domain partially occludes the C terminus of the barrel and forms a large cleft. The structure identifies MAL as belonging to the enolase superfamily of enzymes. The metal ion site is located in a large cleft between the domains. Potential active site residues have been identified based on a combination of their proximity to a metal ion site, molecular modeling, and sequence homology. In common with all members of the enolase superfamily, the carboxylic acid of the substrate is co-ordinated by the metal ions, and a proton adjacent to a carboxylic acid group of the substrate is abstracted by a base. In MAL, it appears that Lys(331) removes the alpha-proton of methylaspartic acid. This motif is the defining mechanistic characteristic of the enolase superfamily of which all have a common fold. The degree of structural conservation is remarkable given only four residues are absolutely conserved.     

Substrate binding site of naphthalene 1,2-dioxygenase: functional implications of indole binding

The three-dimensional structure of the aromatic hydroxylating enzyme naphthalene dioxygenase (NDO) from Pseudomonas sp. NCIB 9816-4 was recently determined. The refinement of the structure together with cyclic averaging showed that in the active site of the enzyme there is electron density for a flat aromatic compound. This compound appears to be an indole adduct, which in Escherichia coli is derived from tryptophan present in the rich culture medium. An indole-dioxygen adduct has been built which fits the electron density convincingly. Support for this interpretation was obtained from crystals of the enzyme purified from cells grown in the absence of tryptophan which had an empty substrate pocket. These types of crystals were soaked in indole solutions and the position of indole in this complex was similar to the corresponding part in the modelled indole-oxygen adduct. This suggests that a peroxide bound to iron end-on attacks the substrate and forms this intermediate. The substrate position has implications for the substrate specificity of the enzyme. Docking studies with indole, naphthalene and biphenyl inside the substrate pocket of NDO suggest the presence of subpockets where the one close to the active site iron is reserved for the binding of the aromatic ring which is hydroxylated upon catalysis. The plausible location for the binding of dioxygen is between this pocket and the catalytic iron. This is in accordance with the enantiospecificity of the products.     

Aprataxin forms a discrete branch in the HIT (histidine triad) superfamily of proteins with both DNA/RNA binding and nucleotide hydrolase activities

Ataxia with oculomotor apraxia type 1 (AOA1) is an early onset autosomal recessive spinocerebellar ataxia with a defect in the protein Aprataxin, implicated in the response of cells to DNA damage. We describe here the expression of a recombinant form of Aprataxin and show that it has dual DNA binding and nucleotide hydrolase activities. This protein binds to double-stranded DNA with high affinity but is also capable of binding double-stranded RNA and single-strand DNA, with increased affinity for hairpin structures. No increased binding was observed with a variety of DNA structures mimicking intermediates in DNA repair. The DNA binding observed here was not dependent on zinc, and the addition of exogenous zinc abolished DNA binding. We also demonstrate that Aprataxin hydrolyzes with similar efficiency the model histidine triad nucleotide-binding protein substrate, AMPNH2, and the Fragile histidine triad protein substrate, Ap4A. These activities were significantly reduced in the presence of duplex DNA and to a lesser extent in the presence of single-strand DNA, and removal of the N-terminal Forkhead associated domain did not alter activity. Finally, comparison of sequence relationships between the histidine triad superfamily members shows that Aprataxin forms a distinct branch in this superfamily. In addition to its capacity for nucleotide binding and hydrolysis, the observation that it also binds DNA and RNA adds a new dimension to this superfamily of proteins and provides further support for a role for Aprataxin in the cellular response to DNA damage.     

Incorporation of oxygen into the succinate co-product of iron(II) and 2-oxoglutarate dependent oxygenases from bacteria, plants and humans

The ferrous iron and 2-oxoglutarate (2OG) dependent oxygenases catalyse two electron oxidation reactions by coupling the oxidation of substrate to the oxidative decarboxylation of 2OG, giving succinate and carbon dioxide coproducts. The evidence available on the level of incorporation of one atom from dioxygen into succinate is inconclusive. Here, we demonstrate that five members of the 2OG oxygenase family, AlkB from Escherichia coli, anthocyanidin synthase and flavonol synthase from Arabidopsis thaliana, and prolyl hydroxylase domain enzyme 2 and factor inhibiting hypoxia-inducible factor-1 from Homo sapiens all incorporate a single oxygen atom, almost exclusively derived from dioxygen, into the succinate co-product.     

Transition metal-catalyzed nonoxidative decarboxylation reactions

Decarboxylases typically utilize an organic cofactor or a transition metal coupled with dioxygen to activate their substrates. The recent characterization of alpha-amino-beta-carboxymuconate-epsilon-semialdehyde decarboxylase (ACMSD) has revealed that this enzyme adopts a TIM-barrel (beta/alpha)(8) fold and employs a mononuclear transition metal center to decarboxylate the substrate in an oxidant-independent fashion. Thus, ACMSD represents a type of decarboxylation reaction that has been so far uncharacterized in biological systems. Several close homologues of ACMSD were analyzed, including isoorotate decarboxylase (IDCase), 5-carboxyvanillic acid decarboxylase (5-CVD), gamma-resorcylate decarboxylase (gamma-RSD), and 4-oxalomesaconate hydratase (OMAH). These enzymes are involved in the catabolism of tryptophan and vanillate, the biodegradation of hydroxylbenzoates, and the thymidine salvage pathways in certain organisms. They possess the signature sequence motifs of the amidohydrolase superfamily and likely share the same structural and mechanistic characteristics as that of ACMSD. Analysis of the sequence conservation and evolutionary relationship of ACMSD-related proteins suggests an emerging ACMSD protein family that includes ACMSD and ACMSD-like decarboxylases and hydratases with diverse substrate specificities, many of which are poorly understood in regard to their functions and mechanisms. Progress in the biochemical and structural characterization of ACMSD not only sheds light on the active site of this protein family but also promises the elucidation of the detailed catalytic mechanism of these novel transition metal-dependent nonoxidative decarboxylation reactions.     

X-ray absorption spectroscopy structural investigation of early intermediates in the mechanism of DNA repair by human ABH2

Human ABH2 repairs DNA lesions by using an Fe(II)- and αKG-dependent oxidative demethylation mechanism. The structure of the active site features the facial triad of protein ligands consisting of the side chains of two histidine residues and one aspartate residue that is common to many non-heme Fe(II) oxygenases. X-ray absorption spectroscopy (XAS) of metallated (Fe and Ni) samples of ABH2 was used to investigate the mechanism of ABH2 and its inhibition by Ni(II) ions. The data are consistent with a sequential mechanism that features a five-coordinate metal center in the presence and absence of the α-ketoglutarate cofactor. This aspect is not altered in the Ni(II)-substituted enzyme, and both metals are shown to bind the cofactor. When the substrate is bound to the native Fe(II) complex with α-ketoglutarate bound, a five-coordinate Fe(II) center is retained that features an open coordination position for O(2) binding. However, in the case of the Ni(II)-substituted enzyme, the complex that forms in the presence of the cofactor and substrate is six-coordinate and, therefore, features no open coordination site for oxygen activation at the metal.     

Life as aerobes: are there simple rules for activation of dioxygen by enzymes?

Numerous biological systems involve reaction with dioxygen in the absence of readily accessible spectroscopic signals. We have begun to develop a set of "generic" strategies that will allow us to probe the mechanisms of dioxygen activation. In particular, we wish to understand the nature of the dioxygen binding step, the degree to which electron transfer to dioxygen is rate limiting, whether reactive species accumulate during turnover and, finally, whether proton and electron transfer to dioxygen occur as coupled processes. Our strategy will be introduced for an enzyme system that uses only an organic cofactor in dioxygen activation (glucose oxidase). Two key features emerge from studies of glucose oxidase: (1) that formation of the superoxide anion is a major rate-limiting step and (2) that electrostatic stabilization of the superoxide anion plays a key role in catalysis. Similar themes emerge when our protocols are applied to enzymes containing both an active site metal center and an organic cofactor. Finally, enzymes that rely solely on metal centers for substrate functionalization will be discussed. In no instance, thus far, has evidence been found for a direct coupling of proton to electron transfer in the reductive activation of dioxygen.     

The Structure of an alternative form of Paracoccus pantotrophus cytochrome cd(1) nitrite reductase

Cytochrome cd(1) nitrite reductase is a bifunctional enzyme, which can catalyze the 1-electron reduction of nitrite to nitric oxide and the 4-electron reduction of dioxygen to water. Here we describe the structure of reduced nitrite reductase, crystallized under anaerobic conditions. The structure reveals substantial domain rearrangements with the c domain rotated by 60 degrees and shifted by approximately 20 A compared with previously known structures from crystals grown under oxidizing conditions. This alternative conformation gives rise to different electron transfer routes between the c and d(1) domains and points to the involvement of elements of very large structural changes in the function in this enzyme. In the present structure, the c heme has a His-69/Met-106 ligation, and this ligation does not change upon oxidation in the crystal. The d(1) heme is penta-coordinated, and the d(1) iron is displaced from the heme plane by 0.5 A toward the proximal ligand, His-200. After oxidation, the iron moves into the d(1) heme plane. A surprising finding is that although reduced nitrite reductase can be readily oxidized by dioxygen in the new crystal, it cannot turnover with its other substrate, nitrite. The results suggest that the rearrangement of the domains affects catalysis and substrate selectivity.     

Structural and spectroscopic studies shed light on the mechanism of oxalate oxidase

Oxalate oxidase (EC 1.2.3.4) catalyzes the conversion of oxalate and dioxygen to hydrogen peroxide and carbon dioxide. In this study, glycolate was used as a structural analogue of oxalate to investigate substrate binding in the crystalline enzyme. The observed monodentate binding of glycolate to the active site manganese ion of oxalate oxidase is consistent with a mechanism involving C-C bond cleavage driven by superoxide anion attack on a monodentate coordinated substrate. In this mechanism, the metal serves two functions: to organize the substrates (oxalate and dioxygen) and to transiently reduce dioxygen. The observed structure further implies important roles for specific active site residues (two asparagines and one glutamine) in correctly orientating the substrates and reaction intermediates for catalysis. Combined spectroscopic, biochemical, and structural analyses of mutants confirms the importance of the asparagine residues in organizing a functional active site complex.