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Structural and mechanistic studies on 2-oxoglutarate-dependent and related Christopher J Schofield and Zhihong Zhang

Mononuclear nonheme-Fe(II)-dependent oxygenases comprise The 2OG-dependent oxygenases have emerged as the an extended family of oxidising enzymes, of which the largest known family of nonheme oxidising enzymes [7,8] 2-oxoglutarate-dependent oxygenases and related enzymes are (Figure 1a). Their occurrence is ubiquitous, having been the largest known subgroup. Recent crystallographic and identified in many organisms ranging from prokaryotes to mechanistic studies have helped to define the overall fold of eukaryotes. Recent evidence also indicates that a 2OG- the 2-oxoglutarate-dependent enzymes and have led to the dependent , prolyl 4-hydroxylase, is expressed by identification of coordination chemistry closely related to that of the P. bursaria Chlorella virus-1 [9]. Oxidative reactions catal- other nonheme-Fe(II)-dependent oxygenases, suggesting ysed by 2OG-dependent are steps in the related mechanisms for dioxygen activation that involve - biosynthesis of a variety of metabolites, including materials mediated electron transfer. of medicinal or agrochemical importance, such as plant ‘hor- mones’ (e.g. gibberellins) and antibiotics (e.g. cephalosporins Address and the β-lactamase inhibitor clavulanic acid). The Oxford Centre for Molecular Sciences and the Department of Chemistry, The Dyson Perrins Laboratory, South Parks Road, Oxford In mammals, the best-characterised 2OG-dependent oxy- OX1 3QY, UK genase is prolyl-4-hydroxylase, which catalyses the Current Opinion in Structural Biology 1999, 9:722–731 post-translational hydroxylation of proline residues in col- lagen. Mammalian prolyl-4-hydroxylase is an α β 0959-440X/99/$ — see front matter © 1999 Elsevier Science Ltd. 2 2 All rights reserved. tetramer, in which the oxygenase activity is associated with the α subunit and the β subunit has an identical sequence Abbreviations to that of protein disulfide [10,11]. Two mem- 2OG 2-oxoglutarate 3,4-PCD protocatachuate-3,4- bers of the 2OG-dependent oxygenase family, 4-HPPD 4-hydroxyphenylpyruvate dioxygenase deacetoxycephalosporin C synthase (DAOCS) and pro- ACCO 1-amino-1-cyclopropanecarboxylic acid oxidase line-4-hydroxylase, are used currently in genetically ACV L-δ-(α-aminoadipoyl)-L-cysteinyl-D-valine engineered fermentation processes [12,13]. BphC 2,3-dihydroxybiphenyl 1,2 dioxygenase CAS clavaminate synthase DAOCS deacetoxycephalosporin C synthase All studied 2OG-dependent oxygenases have an absolute IPNS isopenicillin N synthase requirement for Fe(II) and catalyse a variety of two-elec- NDO napthalene dioxygenase tron oxidations, including hydroxylation, desaturation and PDB Protein Data Bank oxidative ring closure reactions [7,8]. In almost all cases, SLO-1 soybean TfdA (2,4-dichlorophenyoxy)acetate dioxygenase the oxidation of the ‘prime’ substrate is coupled to the con- version of 2OG into succinate and CO2. One of the oxygens of the dioxygen molecule is incorporated into suc- Introduction cinate. In the case of desaturation reactions, the other The stereoselective oxidation of an unactivated alkane car- dioxygen-derived oxygen is presumably converted to bon–hydrogen bond is probably the most difficult common water. In hydroxylation reactions, the partial incorporation functional group interconversion in chemistry. In nature, of oxygen from dioxygen into the alcohol product occurs such reactions are most often carried out by metal-depen- with significant levels of exchange of oxygen from water dent oxygenases or oxidases. The best characterised of these being observed [14,15]. enzymes are the cytochrome P450 , for which detailed structural and mechanistic information is Two enzymes, isopenicillin N synthase (IPNS) and available (see [1,2]). It is now clear that oxygenases/oxidases 1-amino-1-cyclopropanecarboxylic acid oxidase (ACCO), that use no other than iron constitute a ‘superfamily’ have evolved from the same structural platform as the of redox enzymes. This extended family includes both 2OG-dependent oxygenases, but do not use 2OG as a diiron-using enzymes, for example, methane monooxyge- cosubstrate (Figure 1b,c). IPNS catalyses the four-electron nase and ribonucloetide reductase, and monoiron-using oxidation of a tripeptide to give the penicillin nucleus and enzymes. The latter enzymes include those dependent on has been extensively studied [16,17]. ACCO catalyses the Fe(III), for example, lipoxygenase and the intradiol cleaving ultimate step in the biosynthesis of the plant signalling catechol dioxygenases, and those dependent on Fe(II), for molecule ethylene from ACC, using ascorbate as a cosub- example, the extradiol strate and CO2 as an activator [7,8]. cleaving catechol dioxygneases, tyrosine/phenylalanine The first crystal structure to be reported for a member of hydroxylases, benzene/napthalene dioxygenases and the the 2OG-dependent oxygenase and related fam- 2-oxoglutarate (2OG)-dependent oxygenases [3–6]. ily was that of IPNS [18]. Structures of two more typical 2-oxoglutarate-dependent oxygenases and related enzymes Schofield and Zhang 723

Figure 1

'2OG oxygenase', Fe(II) (a) RH ROH

H2O, O2 CO2, H2O O + + CO H HOOC 2 HO2C CO2H 2OG Succinate

(b) SH RCOHN RCOHN IPNS, Fe(II) S H N N O O CO2H CO2H O2 2 H2O Isopenicillin N L-δ-(α-aminoadipoyl) -L-cysteinyl-D-valine (ACV) R = L-δ-(α-aminoadipoyl)

(c) NH2 ACCO, Fe(II) Ascorbate + + HCN + CO2 + Dehydroascorbate CO2H

ACC O2 2 H2O

(d) RCOHN S DAOCS, Fe(II) RCOHN S

N N O O CO2H CO H Penicillin N O2 CO2 + H2O 2 R = D -δ-(α-aminoadipoyl) + + Deacetoxycephalosporin C (DAOC) 2OG Succinate

(e) H H NH OH NH CAS (1) PAH OH N N N O NH NH2 O NH NH2 O NH2 CO2H CO2H CO2H Proclavaminic acid CAS (2)

O OH O NH2 CAS (3) O H NH2 N N N H O O O CO2H CO2H CO2H Clavulanic acid Dihydroclavaminic acid Clavaminic acid

Current Opinion in Structural Biology

Reactions catalysed by 2OG-dependent oxygenases and related ACCO reaction. (d) The DAOCS reaction. (e) The trifunctional role of enzymes. (a) General scheme for hydroxylation by a 2OG dioxygenase. CAS in clavulanic acid biosynthesis. Names of the oxygenase enzymes A water molecule is shown before and after reaction to emphasise the are in red. PAH, proclavaminate amidinohydrolase. exchange of oxygen with dioxygen. (b) The IPNS reaction. (c) The

oxygenases, DAOCS [19••] and clavaminate synthase step in cephalosporin biosynthesis. CAS catalyses three (CAS) ([20]; Z Zhang et al., unpublished data), have been reactions during clavulanic acid biosynthesis, encom- recently determined (Figure 1d,e). DAOCS catalyses the passing hydroxylation, oxidative ring closure processes ring expansion of penicillin N to DAOC, the committal and desaturation, making it an excellent case study for 724 Proteins

Figure 2

Views derived from the PDB-deposited coordinates for the crystal (2OG and ACV) are shown for clarity. Note the conserved ‘jelly-roll’ structures of (a) DAOCS–Fe–2OG [19••], (b) IPNS–Fe–ACV [18] core (in red) in (a) DAOCS and (b) IPNS, but that the overall fold of and (c) a single subunit of homo-octameric BphC complexed to iron BphC is completely different [34], despite the closely related iron [34]. Colours used in the ball-and-stick models are red for oxygen, blue coordination chemistry. In (c), the nonjelly roll β-strand core for nitrogen, yellow for carbon, cyan for sulfur and pink for iron. Helices encompassing the is in red, other β strands are in blue. and loops are in green. Only certain iron ligands and substrates

investigating the structural factors directing the chemos- 2OG-dependent oxygenases, including mammalian electivity and regioselectivity of 2OG-dependent enzymes (e.g. the prolyl hydroxylases [10,11]), will contain a oxygenases [14,21,22]. This article reviews recent stud- similar β-barrel core. The recently reported structure of ies on 2OG-dependent oxygenases, making a brief 4-hydroxyphenylpyruvate dioxygenase (4-HPPD), which comparison with other mononuclear nonheme-Fe- oxidises 4-HPP to homogentisate [23••], however, reveals dependent oxidising enzymes. Note that comparisons that nature has evolved more than one platform for non- between mononuclear and dinuclear iron enzymes are heme oxygenases to catalyse the oxidative decarboxylation beyond the scope of this review, but the distinction is of 2-oxoacids (see below). somewhat arbitrary. Attention is also drawn to other recent reviews on the chemistry of nonheme oxygenases In the crystal structure of IPNS complexed to Mn(II) [sub- [3–6], including the 2OG-dependent oxygenases [7,8]. stituting for Fe(II)], the active site metal was ligated by two water molecules and the sidechains of Gln330, Three-dimensional topology and active site Asp216, His214 and His270 [18]. The presence of two his- coordination chemistry of 2-oxoglutarate- tidines and a carboxylate ligand was anticipated by dependent oxygenases and related enzymes spectroscopic and sequence analysis studies [3]. Ligation The structure of IPNS revealed a β-strand core folded into by Gln330, the penultimate residue in the C terminus, was a distorted jelly-roll motif [18] (Figure 2). Sequence com- unexpected and this residue is not essential for catalysis parisons suggest that many 2OG-dependent oxygenases will [24,25]. Crystal structures of DAOCS–Fe(II) [19••] and have a similar fold to IPNS, a prediction substantiated by CAS–Fe(II) complexes reveal that Fe(II) is coordinated by the crystal structure of DAOCS. Sequence analyses of CAS the two histidine residues of an analogous HXD/E…H and other 2OG-dependent oxygenases [22] revealed little motif, but CAS is unusual in that it uses a glutamate, rather overall similarity with the DAOCS/IPNS subfamily, leading than an aspartate, residue as it carboxylate ligand. There is to the proposal that convergent evolution to a common no evidence for a fourth protein iron ligand in either mechanism and active site chemistry occurred within the DAOCS or CAS. wider family of 2OG-dependent and related oxygenases. The presence of a jelly-roll topology in the CAS structure, as The HXD/E…H iron-binding motif is highly conserved in in IPNS and DAOCS, in which certain 2OG-binding and 2OG-dependent and related oxygenases [4]. Mutagenesis, iron-binding residues are located on analogous strands of the chemical modification and/or spectroscopic studies are all core, suggests the occurrence of divergent evolution within consistent with the presence of this motif in other 2OG- the family of 2OG-dependent oxygenases. Sequence analy- dependent oxygenases, including prolyl-4-hydroxylase [11], ses might have been misled by inserts between β strands of proline hydroxylase [26], (2,4-dichlorophenyoxy)acetate the jelly-roll motif in CAS. It seems possible that all of the dioxygenase (TfdA) [27], CAS [28,29••] and ACCO [30••]. 2-oxoglutarate-dependent oxygenases and related enzymes Schofield and Zhang 725

Figure 3

(a) DAOCS

O O O O O O – O O – O – O O O + O2 H183 H183 H183 O O O X . O Fe O Fe Fe O D185 D185 D185 O (H2O) H243 H243 H243 Penicillin N Penicillin N Penicillin N

O O O O C and/or O O O O – O – O – O O O O His His His ? O ? O Fe O Fe Fe Asp O Asp Ð CO O Asp 2 His Penicillin N His Penicillin N His Penicillin N

Products O O - (OH ) O 2 RCOHN S . His O Fe N Asp O O I CO2H His II Penicillin N

Gln330 + ACV, Gln330 O NHCOR (b) IPNS H O + 2 H214 ÐH2O, ÐH X HO C NH Fe 2 H S .O D216 H214 H2O H279 H O + O2 Fe H2O D216 H279 Ð isopenicillin N, + Gln330, + H+ ÐH 2O

O NHCOR O NHCOR N HO2C N S HO C 2 H214 S HO O H214 Fe H Fe H2O D216 H2O D216 H279 H279

Current Opinion in Structural Biology

Partial mechanisms for (a) DAOCS ([19••,58] and references therein) discussion. I shows the putative radical intermediate in DAOCS and (b) IPNS ([18,52] and references therein). Dioxygen and catalysis. II shows a possible intermediate in the exchange of oxygen dioxygen-derived species are in red. 2OG and derived species are in from water to dioxygen (there is evidence that exchange can also occur green. Iron is in orange. (a) Oxygen is shown bound as a peroxide after reaction of the ferryl [15]). The position from which the ferryl radical, but a superoxide/peroxide is also possible. See text for reacts is uncertain. 726 Proteins

Furthermore, as pointed out by Hegg and Que [4], a ‘facial (C109, C112 and C114) and by the mainchain nitrogen 2-His-1-carboxylate’ triad of iron-ligating residues is present atoms of Ser113 and Cys114. The thiol sidechains of in all enzymes containing mononuclear nonheme Fe sites for Cys112 and Cys114 are reported to be oxidised to sulfinic which structures have been reported. These include Fe(II)- and sulfenic acids, assisting NO binding. It seems possible dependent enzymes, such as IPNS [18], DAOCS [19••], CAS that oxidation of the sidechains of Cys112 and Cys114 [20,28,29••], LigAB protocatachuate 4,5-dioxygenase [31••], occurs by reaction with dioxygen in an Fe-mediated catechol-2,3-dioxygenase [32], 2,3-dihydroxybiphenyl 1,2 process [53••,54•]. dioxygenase (BphC) [33,34], napthalene dioxygenase (NDO) [35••], phenylalanine hydroxylase [36–38], tyrosine hydroxy- Binding of substrates and mechanistic proposals lase [39,40] and 4-HPPD [23••], and Fe(III)-dependent for 2-oxoglutarate-dependent oxygenases enzymes, such as protocatachuate-3,4-dioxygenase (3,4- Kinetic studies of 2OG-dependent oxygenases indicate an PCD) [41–44], soybean lipoxygenase (SLO-1) [45,46] and ordered sequential mechanism, with the binding of 2OG rabbit 15-lipoxygenase [47]. Also note that the conserved triad followed by that of the prime substrate, then dioxygen has evolved on structural platforms with different overall [10,55]. Product release then occurs in the order bicarbon- topologies, for example, those of 2OG-dependent oxygenas- ate (or CO2), succinate and prime product, with the order of es (and IPNS and ACCO), those of the class I/II extradiol release of the last two dependent on their relative concen- dioxygenases and HPPD, those of the class III extradiol tration. The evidence for any reaction steps subsequent to dioxygenases [28] and those of the tyrosine/phenylalanine the formation of an enzyme–Fe–2OG complex is limited, hydroxylases. The secondary structure elements in which the although it is widely believed that an Fe(IV)=O species triad residues are located can be either β strands, α helices or effects oxidation of the prime substrate [56,57]. loops, but they seem generally to be located in relatively Crystallographic and spectroscopic analyses of 2OG-depen- ‘rigid’ parts of the structure, consistent with the ligation of dent oxygenases are starting to define the structures of the these residues to the metal during the catalytic cycle. intermediates involved in the reaction pathway.

The presence of such an iron-binding motif is not limited The crystal structure of a complex of DAOCS bound to to iron-dependent enzymes; it also occurs in Mn(II)- Fe(II) and 2OG demonstrates that the 2OG cofactor is lig- dependent extradiol oxygenases and in Zn(II)-dependent ated to the metal in a bidentate fashion via its 1-carboxylate enzymes such as thermolysin. The latter contain and 2-oxo groups [19••,20]. A water molecule, trans to an HEXXH…E motif, in which the Zn(II) ligands are pro- His279, remains ligated to the metal. The terminal carboxy- vided by the sidechains of the two histidines and the late of 2OG is bound to a conserved RXS motif in DAOCS. C-terminal glutamate of the motif [48]. A link between the Spectroscopic studies of complexes of CAS with Fe(II) and iron and zinc enzymes has also been provided by the dis- 2OG imply a similar iron coordination arrangement in solu- covery that peptide deformylase from Escherichia coli, tion [28,29••]. Related studies of Cu(I) complexes with originally thought to be a zinc-dependent hydroxylase, TfdA implied monodentate ligation of the 2OG [24]; how- instead uses Fe(II) [49,50]. The iron is coordinated by two ever, it seems possible that this reflects the substitution of a histidines and a cysteine residue, and undergoes inactiva- catalytically inactive metal in these studies. Spectroscopic tion under aerobic conditions, implying that an studies demonstrate that, on binding the monocyclic β-lac- iron-mediated reaction with dioxygen has occurred. Fe(II) tam substrate, the CAS–Fe(II)–2OG complex changes from is also present in the structure of galactose-1-phosphate six-coordinate octahedral to five-coordinate square pyrami- uridylyltransferase [51], in which it is ligated in a tetrahe- dal geometry [28,29••]. Crystallographic analyses of two dral fashion by two histidines and two cysteine residues; CAS–Fe(II)–2OG–substrate complexes also revealed that however, the iron is believed to play a structural, rather the occupancy of the iron-ligated water is significantly than a catalytic, role. reduced on substrate binding. These analyses all suggest that dioxygen binding is triggered by the binding of the In the case of Fe(II) enzymes, there is the ‘problem’ of the prime substrate to the enzyme–Fe–2OG complex, as reaction with dioxygen leading to the generation of reactive implied from kinetic studies. Possible mechanisms for the oxidising species. This may explain (in part) why cysteine formation of the ferryl species involve electron transfer from residues are normally not used as ligands in Fe(II) non- the iron to the dioxygen, simultaneously permitting the for- heme enzymes. Indeed, the oxidation of an iron-linked mation of an iron-bound superoxide/peroxide species and thiol is elegantly exploited in a biosynthetic sense by IPNS the activation of the keto group of 2OG for nucleophilic [52]. In a remarkable example of post-translational modifi- attack to form a peroxo species that can collapse to give the cation, it seems that the autocatalytic oxidation of cysteine reactive ferryl species and CO2/succinate or an iron-ligated thiols occurs in the case of nitrile hydratase, an industrially anhydride (Figure 3). used Fe(III)-dependent enzyme involved in the hydrolysis of acrylonitrile. As isolated from Rhodoccocus sp. N-771, the The position of dioxygen binding to the iron in 2OG- enzyme is in an inactive form as a result of nitrosylation of dependent oxygenases is not yet unequivocally the active site iron and is activated by photolysis. Its active established. It is reasonable to propose that dioxygen can site iron is ligated by the sidechains of three cysteines bind into the position occupied by the water molecule. So 2-oxoglutarate-dependent oxygenases and related enzymes Schofield and Zhang 727

Figure 4

Comparison of the DAOCS coordination chemistry with other mononuclear iron (a) DAOCS (b) ACCO (c) 4-HPPD oxygenases and thermolysin. Proposed metal O . O coordination chemistry for O O (a) DAOCS–Fe–2OG–O , based upon the - O HO O 2 O O H H crystal structure of DAOCS–Fe(II)–2OG H183 N His E322 •• O O [19 ], (b) ACCO based upon spectroscopic Fe . O Fe •• . O Fe studies [30 ], (c) 4-HPPD based upon the Asp185 O His O H161 O O crystal structure of 4-HPPD–Fe–acetate H243 Asp H240 [35••], (d) the extradiol dioxygenase catechol (peniclllin N) 2,3-dioxygenase based on the enzyme–Fe(II)–acetone crystal structure (d) Catechol 2,3-dioxygenase (e) Thermolysin ([32]; see also studies on other extradiol R dioxygenases [31••,33,34]) and 1 (e) thermolysin [48]. See also other R2 reviews [4,5]. The proposed (but unproven) position of dioxygen binding in (a–d) is OH(H) indicated. O H153 Zn . O H142 O Fe E166 O H214 H146 E265 Current Opinion in Structural Biology

far, it has not been possible to obtain a structure of pathway can be biased towards processes involving radical DAOCS complexed with its prime (penicillin N) sub- intermediates [5,16]. Note that the natural reaction for strate. Modelling studies indicate that it can be bound in DAOCS is itself an exception, with the ring expansion the DAOCS active site in such a way as to project its pro- process probably proceeding via a 3β-methyl radical on S methyl group towards the iron centre [58]; however, if penicillin N generated by a ferryl-mediated hydrogen the proposed intermediate ferryl species is generated in abstraction process. Biasing the reaction pathway of 2OG- the position opposite His183 of DAOCS, it is directed dependent oxygenases to deliberately generate radicals away from the substrate and it is possible that the ferryl may lead to inhibition. Indeed, 5-oxaproline-containing reacts from the position opposite His243. This might be peptides have recently been shown to cause mechanism- achieved by binding dioxygen to a different form of the based inhibition of prolyl-4-hydroxylase, most probably via complex compared with that observed crystallographically a radical process [59]. or via rearrangement of a pentacoordinate ferryl species subsequent to the release of CO2. If such a speculation is Mechanism of isopenicillin N synthase correct, it may provide a partial explanation for the obser- Mechanistic studies on IPNS are more advanced than those vation that, during hydroxylation processes catalysed by on the 2OG-dependent oxygenases. On binding L-δ-(α- 2OG-dependent oxygenases, incorporation of oxygen aminoadipoyl)-L-cysteinyl-D-valine (ACV) to the IPNS atoms from both dioxygen and water is observed, as water active site, one of the two water molecules (that trans to may bind to the pentacoordinate species. Asp216) ligated to the iron is displaced and the amide sidechain of Gln330 is displaced by the thiol of ACV [5,52]. Following studies of prolyl-4-hydroxylase, the oxygen The ACV valine carboxylate is in position to form electrosta- exchange process has been studied in DAOCS and CAS, tic interactions with the same RXS motif that is responsible with the level of incorporation of oxygen from labelled for binding the terminal carboxylate of 2OG in DAOCS. The dioxygen or water being substrate dependent [11,12]. ACV valine βC–H, which must be cleaved during penicillin Together with kinetic isotope effects, results using an formation, was observed to be directed away from the iron unnatural substrate (exoemthylene cephalosporin C) with centre, implying that rotation of the valine Cα–Cβ bond DAOCS have been taken to imply that the exchange occurs during catalysis. Reaction of the IPNS–Fe(II)–ACV process can occur either prior to or subsequent to the reac- crystals with the dioxygen analogue NO led to the binding of tion of the ferryl species. NO to the iron trans to Asp216, implying that dioxygen binds similarly. The oxygen of the NO was observed to be equidis- During hydroxylation processes, the reaction appears to be tant between the NH of the cysteinyl valine peptide bond highly stereoselective, favouring a mechanism involving and the cysteinyl pro-S β hydrogen, both of which are the direct insertion of a ferryl-derived dioxygen into a C–H removed during IPN formation. It was proposed that, bond, rather than involving radical or Fe–C bonded inter- excepting the thiol hydrogen, iron–dioxygen-derived species mediates. When substrate analogues are used, including are responsible for removal of all the requisite hydrogens cyclopropyl radical traps, it seems that the reaction from ACV during formation of the penicillin nucleus. 728 Proteins

ACC oxidase In contrast, the Fe(II)-using extradiol dioxygenases appear to ACCO contains characteristic sequence motifs of 2OG- employa mechanism that is more closely related to that of dependent oxygenases, including the RXS motif, which the 2OG-dependent oxygenases. Thus, in both cases, cur- (from the structural work on DAOCS) binds to the terminal rent proposals involve activation of dioxygen by binding carboxylate of 2OG. It is unique in having a requirement directly to the iron, forming superoxide/peroxide intermedi- ates. Evidence has been provided for lactone and for ascorbate as a cosubstrate and CO2 as an activator, char- acteristics that make it interesting to study from a semiquinone intermediates [62–64] and a Crigee rearrange- mechanistic viewpoint. Mutagenesis and spectroscopic ment has been proposed as a key step — an analogous studies have implied that ACCO also employs an HXD…H rearrangement is a possibility in 2OG-dependent catalysis. motif to bind its active site iron [60,61]. Mechanistic pro- posals have included schemes involving the generation of 4-HPPD an Fe(IV)=O intermediate by the initial reaction of ascor- A compelling mechanistic link between the 2OG-depen- bate with dioxygen. Recent spectroscopic studies on dent and catechol dioxygenases is provided by 4-HPPD. ACCO have revealed that ACCO–Fe(II)–ACC–NO com- This enzyme catalyses the conversion of the 2-oxoacid plexes are readily formed, but analogous complexes with 4-hydroxyphenyl pyruvate to homogentisate in a process ascorbate substituting for ACC cannot be formed. The involving oxidative rearrangement and decarboxylation. It results demonstrate that ACC is bound to iron in a biden- also catalyses the oxidation of other amino-acid-derived tate manner via both its carboxylate and amino groups. 2-oxoacids, including the conversion of α-ketoisocaproate Consequently, a new outline mechanism for ACCO has to β-hydroxyisovalerate [65]. Thus, it has parallels to both been proposed: initial binding of ACC to ACCO–Fe(II) 2OG-dependent oxygenases and IPNS catalysis in that lowers the redox potential of the metal centre, activating it they catalyse an oxidative decarboxylation process and the for dioxygen binding and superoxide formation, in analogy four-electron oxidation of a single substrate, respectively; with the mechanism for 2OG-dependent oxygenases. however, the crystal structure of 4-HPPD clearly places it Abstraction of a hydrogen atom from the ACC amino group in the structural family of extradiol dioxygenases (class III) •• can lead to fragmentation to form ethylene, CO2 and HCN. [23 ]. The active site iron was once again bound by the The role of the ascorbate in this mechanism is to reduce the 2-His-1-carboxylate (glutamate) motif (Figure 4). On the peroxide intermediate to prevent formation of reactive oxi- basis of the crystal structure, the proposed binding model dising species. Indeed, ACCO from tomato is susceptible to for the enzyme substrate was strikingly similar to that such damage, undergoing partial fragmentation. Alternative observed for the 2OG-dependent oxygenases. Dioxygen mechanistic scenarios, such as the intermediacy of a was proposed to bind opposite Glu322 [23••]. Fe(IV)=O species, are also possible and the mechanism of CO2 activation remains obscure. Lipoxygenases catalyse the two-electron oxidation of fatty acids containing a 1,4-diene to give hydroperoxides. They Relationships of the 2-oxoglutarate-dependent contain a single Fe(III) ligated by three histidines and the oxygenases with other nonheme oxygenases carboxylate of the C-terminal residue Ile839. The Catechol dioxygenases sidechain of asparagine is part of a hydrogen-bonding net- Both from mechanistic and structural viewpoints, the cate- work around the iron, but probably does not directly chol dioxygenases are probably the best characterised of interact with it. As isolated, lipoxygenases contain Fe(II) the mononuclear iron oxygenases. They catalyse steps in and are activated by conversion to high-spin Fe(III) the bacterial metabolism of aromatic compounds, [45–47]. Their mechanism may involve the generation of a catalysing the ring fission of catechol, protocatachuate and free radical of the 1,4-diene substrate, but the process by gentisate. On the basis of the position of aromatic ring which this generated and whether or not an Fe–dioxygen cleavage and the requirement for Fe(III) or Mn(II)/Fe(II), complex is involved in both the hydrogen abstraction and they can be classified into two groups. The intradiol dioxy- peroxide formation steps are uncertain [45–47]. genases cleave the C–C bond between the hydroxyl groups and require Fe(III), whereas the extradiol enzymes Phenylalanine hydroxylase and cleave the C–C bond adjacent to the hydroxyl groups and Two subgroups of mononuclear-Fe-using oxygenases require Mn(II) or Fe(II). utilise additional cofactors. Phenylalanine hydroxylase and tyrosine hydroxylase are closely related Fe(II)- and pterin- There is an important mechanistic difference between the dependent oxygenases of medicinal importance [36–40]. Fe(III)-dependent intradiol dioxygenases and the other Structures have been reported for both tyrosine and oxygenases described here. Very detailed spectroscopic phenylalanine hydroxylases. The active site iron is coordi- and structural studies of 3,4-PCD indicate that the iron nated by a 2-His-1-carboxylate motif and by the pterin retains high spin during catalysis and thus does not bind cofactor. It seems unlikely that the aromatic substrates dioxygen directly. Instead, it has been proposed that bind- coordinate the iron, but it is possible that metal ligation by ing of 3,4-PCD to iron activates the substrate for reaction the phenolic products is a mechanism for product inhibi- with dioxygen [43,44]. tion (see [66] for a review). 2-oxoglutarate-dependent oxygenases and related enzymes Schofield and Zhang 729

Napthalene dioxygenase 4. Hegg EL, Que L: The 2-His-1-carboxylate facial triad — an emerging structural motif in mononuclear non-heme iron(II) The interesting crystal structure of napthalene dioxyge- enzymes. Eur J Biochem 1997, 250:625-629. nase, one of a family of enzymes catalysing the •• 5. Que L, Ho RYN: Dioxygen activation by enzymes with cis-hydroxylation of aromatic rings [35 ], has been recent- mononuclear non-heme iron active sites. Chem Rev 1996, ly reported. All members of the subfamily contain a Rieske 96:2607-2624. [2Fe–S] cluster. The cluster transfers electrons to the non- 6. Feig AL, Lippard SJ: Reactions of non-heme iron (II) centres with heme Fe(II), which catalyses addition of dioxygen to the dioxygen in biology and medicine. Chem Rev 1994, 94:759-805. substrate. The crystal structure reveals that the Rieske 7. Prescott AG, John P: Dioxygenases: molecular structure and role cluster and Fe(II) centre are connected via hydrogen in plant metabolism. Annu Rev Plant Physiol Plant Mol Biol 1996, 47:245-271. bonds between a single residue (Asp205), suggesting a route for electron transfer. The Fe(II) is coordinated by a 8. Prescott AG: A dilemma of dioxygenases (or where biochemistry and molecular-biology fail to meet). J Exp Bot 1993, 44:849-861. water molecule, the sidechains of His208 and His213 and, 9. Eriksson M, Myllyharju J, Tu HM, Hellman M, Kivirikko KI: Evidence for in a bidentate manner, by the carboxylate of Asp362. 4-hydroxyproline in viral proteins — characterization of a viral prolyl 4-hydroxylase and its peptide substrates. J Biol Chem 1999, 274:22131-22134. Conclusions 10. Kivirikko KI, Pihlajaniemi T: Collagen hydroxylases and the protein The factors that govern the selectivity of different proteins disulfide isomerase subunit of prolyl 4-hydroxylases. for binding different metals in vivo are not yet clearly Adv Enzymol Relat Areas Mol Biol 1998, 2:325-400. established. 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