722 Structural and mechanistic studies on 2-oxoglutarate-dependent oxygenases and related enzymes 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 oxygenase, 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 dioxygenases are steps in the related mechanisms for dioxygen activation that involve iron- 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 isomerase [10,11]. Two mem- 2OG 2-oxoglutarate 3,4-PCD protocatachuate-3,4-dioxygenase 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 lipoxygenase 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 monooxygenases, 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 cofactor 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 enzyme 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 active site 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.