Available online at www.sciencedirect.com ScienceDirect Catalytic strategies of the non-heme iron dependent oxygenases and their roles in plant biology Mark D White and Emily Flashman Non-heme iron-dependent oxygenases catalyse the these enzymes play in plant biology. Targeted manipula- incorporation of O2 into a wide range of biological molecules tion of these enzymes could have beneficial effects for and use diverse strategies to activate their substrates. Recent plant growth and/or stress tolerance, addressing the 21st kinetic studies, including in crystallo, have provided century issue of food security. We therefore highlight experimental support for some of the intermediates used by those of potential agricultural (and/or health) interest. different subclasses of this enzyme family. Plant non-heme iron-dependent oxygenases have diverse and important Resolving intermediates in non-heme iron biological roles, including in growth signalling, stress dependent oxygenase catalysis responses and secondary metabolism. Recently identified To activate O2, non-heme iron-dependent oxygenases use roles include in strigolactone biosynthesis, O-demethylation in a redox active iron, coordinated octahedrally at their active morphine biosynthesis and regulating the stability of hypoxia- site with water, usually in a vicinal facial triad arrangement responsive transcription factors. We discuss current structural [1]. During turnover, H2O is sequentially displaced by and mechanistic understanding of plant non-heme iron substrate/co-substrate and O2, which is reductively acti- oxygenases, and how their chemical/genetic manipulation vated enabling a specific substrate modification. Enzyme could have agricultural benefit, for example, for improved yield, subfamilies are proposed to employ different reactive stress tolerance or herbicide development. iron-oxo species to activate their substrate. These species are short-lived intermediates that are difficult to study, Address but methodological advances over the past 15 years have Chemistry Research Laboratory, 12 Mansfield Road, Oxford OX1 3TA, UK enabled an accumulation of indirect and direct evidence for their formation. For example, a combination of freeze Corresponding author: Flashman, Emily trapping techniques and Mo¨ssbauer spectroscopy allowed (emily.fl[email protected]) the characterisation of a transient Fe(IV)-oxo species in 2-oxoglutarate (2OG)-dependent and pterin-dependent Current Opinion in Chemical Biology 2016, 31:126–135 oxygenases (e.g. TauD and Phenylalanine hydroxylase, This review comes from a themed issue on Bioinorganic chemistry respectively, Figure 1a) [2–4]. This high valent intermediate has not been identified in all non-heme Edited by R David Britt and Emma Raven iron-dependent oxygenases (formation of an Fe(IV)-oxo intermediate usually requires co-oxidation of another species, e.g. 2OG) and others are proposed to modify http://dx.doi.org/10.1016/j.cbpa.2016.02.017 their substrates via lower valent alternatives. Here we summarise current evidence for activating intermediates 1367-5931/# 2016 The Authors. Published by Elsevier Ltd. This is an across other members of this enzyme family. open access article under the CC BY license (http://creativecommons. org/licenses/by/4.0/). Catechol dioxygenases Catechol (intradiol and extradiol) dioxygenases catalyse Introduction the oxidative cleavage of catechol substrates as part of Non-heme iron-dependent oxygenases are a widespread bacterial aromatic degradation pathways [5]. Extradiol family of functionally diverse enzymes that facilitate the dioxygenases cleave the C–C bond adjacent to a hydroxyl incorporation of molecular oxygen into a range of biomo- group to form ring-opened products where O atoms are lecules, reactions that are often inaccessible through syn- incorporated into aldehyde and carboxylate functional thetic approaches. Motivated by biomimetic catalyst groups (Figure 1b). Fe(II) is ligated by the common design, extensive research has been conducted to under- His/His/Glu motif [1] with substrate hydroxyl groups stand their chemistry, providing detailed mechanistic coordinating directly to the metal prior to O2 binding. insights into a number of associated enzyme subfamilies. Mechanistic hypotheses based on studies with homopro- Unique or unifying catalytic strategies employed by these tocatechuate dioxygenase (HPCD) propose no overall enzymes continue to be identified, as do novel and impor- change in the oxidation state of the Fe(II) during turnover tant biological roles. In this Review, we briefly highlight of the wild type enzyme, with Fe(II) acting as a conduit recent advances in the mechanistic understanding of non- for electron transfer between O2 and substrate to generate heme (mono-) iron dependent oxygenases before focus- Fe(II)-superoxo and semiquinone radical intermediates sing on the (ever-emerging) range of important roles that (Figure 1b) which recombine to form an alkylperoxo Current Opinion in Chemical Biology 2016, 31:126–135 www.sciencedirect.com Non-heme iron dependent oxygenases in plant biology White and Flashman 127 Figure 1 (a) His 99 20G, O Succinate , CO 2 TauD 2 Asp101 – O SO His O 3 NH2 H N + S – His255 O His Asp 2 HO O FeIV 20G Taurine Taurine Aminoacetaldehyde O – O SO3 Sulfite OOC – H2N (b) His155 O2 – HPCD COO HO – O COO • – O O – HO O O His OH O II HPCA Fe His214 Homoprotocatechuate α-Hydroxy-δ-carboxymethyl Glu O His 267 cis-muconic Semialdehyde – Glu COO (c) O Tyr 2 447 – 3,4 PCD O HO COO – Tyr – O 480 – O HO O COO PCA HO COO O – His462 Tyr Tyr Protocatechuate β-Carboxymuconate FeIII His460 His O O His (d) NAD(P)H, O2 NDO NAD(P) Asn Nap 201 OH HO Asp 362 O His208 HO Asp His213 Naphthalene 1,2-Dihydronaphthalene FeIII His Asp His (e) O Cys 2 CDO – His – 88 COO – COO O + COO + S – His SH H N 140 H3N 3 O H2N His 86 L-Cysteine His O L-Cysteine Sulfinic Acid FeIII – • O His S His (f) Glu O 176 2 HEPD HEP – – O O3P – • O P OH OH O 3 + – His129 H O O 2-Hydroxyethylphosphonate Glu O Hydroxymethylphosphonate His FeIII 182 P Formic Acid His O OH His O Current Opinion in Chemical Biology Non-heme iron-dependent oxygenase-catalysed reactions, active site structures and reaction intermediates. Left panels: The active site structures of non-heme iron-dependent oxygenases, showing the coordination of amino acid residues (light blue cylinders), substrate (green cylinders) and co-substrate (coral cylinders) to the iron (maroon sphere). A purple mesh shows the electron density of the metal cofactor and water molecules are represented as red balls. Right panels: The corresponding non-heme iron-dependent oxygenase reaction schemes and iron- oxo intermediates, following the route of molecular oxygen (red). Unresolved/currently proposed reaction species are coloured blue. For references, see text. (a) Taurine Dioxygenase (TauD) catalyses oxidation of taurine to aminoacetaldehyde and sulphite via a high valent Fe(IV)-oxo species (PDB ID: 1GQW). (b) Homoprotocatechuate dioxygenase (HPCD) catalyses the oxidative cleavage of homoprotocatechuate (HPCA) to a- hydroxy-d-carboxymethyl-cis-muconic-semialdehyde using a Fe(II)-superoxo/semiquinone intermediate (PDB ID: 4GHG). (c) Protocatechuate 3,4- Dioxygenase (3,4-PCD) catalyses the oxidative cleavage of protocatechuate (PCA) to b-carboxymuconate via a Fe(III)-alkylperoxo intermediate. (PDB ID: 3PCA). (d) Naphthalene Dioxygenase (NDO) catalyses the dihydroxylation of naphthalene (Nap) via a Fe(III)-(hydro)peroxo intermediate (PDB ID: 1O7G). (e) Cysteine dioxygenases (CDO) catalyse the oxidation of cysteine (Cys) to cysteine sulfinic acid via a (putative) Fe(III)-superoxo intermediate (PDB ID: 2IC1). (f) 2-Hydroxyethylphosphonate dioxygenase (HEPD) catalyse the oxidation of 2-hydroxyethylphosphonate (HEP) to hydroxymethylphosphonate and formic acid via a (putative) Fe(III)-superoxo species (PDB ID: 3GBF). www.sciencedirect.com Current Opinion in Chemical Biology 2016, 31:126–135 128 Bioinorganic chemistry intermediate [6]. These species have been observed in proposed to occur either via coupled O–O cleavage and crystallo upon reaction of an anaerobic HPCDÁFe(II)Á4- substrate oxidation or via rearrangement of the hydro- NC complex with O2, where 4-NC (4-nitrocatechol) is a peroxo intermediate to cleave O–O and yield a high non-physiological substrate used to slow the reaction valent Fe(V)oxo-hydroxo species which subsequently (note care should be taken in interpreting the radical oxidises the substrate (discussed in detail in [10]). Inter- nature or oxidation status of intermediates observed estingly, a recent study correlating rates of Rieske cluster crystallographically without additional analysis) [7]. oxidation and product formation in benzoate 1, 2-dioxy- The use of HPCD variants and substituted active site genase implicates another possible mechanism, whereby metals has allowed spectroscopic detection of catalytical- substrate activation is achieved by a Fe(III)-superoxo ly competent Fe/Co/Mn(III)-superoxo intermediates up- species prior to electron transfer from the Rieske cluster on slower rates of catalysis (reviewed in [6]). As only [12]. It is possible that different oxidative approaches are Fe(II) species have been observed in the native reaction, utilised by different members of this diverse enzyme sub- the Fe(II) state may indeed be maintained or it may be that class. an Fe(III)-superoxo is so transient that it remains undetect- able with current technical capabilities. Recently, in crystallo Cysteine dioxygenases studies of homogentisate 1,2-dioxygenase