Unleashing the Synthetic Power of Plant Oxygenases:From Mechanism to Application1[OPEN]
Andrew J. Mitchell,a and Jing-Ke Wenga,b,2,3
aWhitehead Institute for Biomedical Research, Cambridge, Massachusetts 02142bDepartment of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139
Plant-specialized metabolites account for arguablythe largest and most diverse pool of natural productsaccessible to humans. Conferring the plants’ selectivetraits, such as UV defense, pathogen resistance, andenhanced nutrient uptake, these chemicals are crucial toa species’ viability. During biosynthesis of these com-pounds, a vast array of specialized enzymes catalyzediverse chemical modifications, with oxidation beingone of the most predominant (Smanski et al., 2016;Dong et al., 2018). Considering these observations, it is notsurprising that within plant genomes, two families of ox-ygenases are the most abundant: the cytochrome P450monooxygenases (P450s) and the iron/2-oxoglutarate-dependent oxygenases (Fe/2OGs). These and otheroxygenases represent the synthetic workhorses of plant-specialized metabolism and also play key roles inprimary metabolism, cellular regulation, and fitness.Furthermore, the challenging and selective chemistrythey catalyze cannot currently be matched by syn-thetic chemists. Since many plant natural productsserve as valuable pharmaceuticals and commoditychemicals, plant oxygenases represent a promisingtoolset for synthetic biologists to manipulate planttraits or develop biocatalysts (Harvey et al., 2015).Here, we review families of plant oxygenases and theirchemistry and suggest potential applications.
CYTOCHROME P450S
P450s (or CYPs) are hemoproteins that catalyzeNADPH- and O2-dependent hydroxylation, demeth-ylation, epoxidation, cyclization, and desaturation re-actions, among others. P450s form a vast superfamily of
proteins, the genes for which are found in bacteria, in-sects, fish, mammals, plants, and fungi. P450 nomen-clature is based on amino acid sequence similarity,assigning proteins with greater than 40% sequenceidentity into the same family and proteins with greaterthan 55% sequence identity into the same subfamily(Nelson, 2006). Genomes of higher plants contain alarge number of P450s. For example, the Arabidopsis(Arabidopsis thaliana) genome encodes 246 full-lengthP450s,accounting for approximately 1% of the Arabidopsis genecomplement, and the Jatropha curcas (Barbados nut)genome is estimated to encode greater than 400 P450s(Schuler, 2015). Plant P450s have been shown to par-ticipate in a variety of pathways, including the bio-synthesis of phenylpropanoids, alkaloids, terpenoids,lipids, cyanogenic glycosides, and glucosinolates, aswell as plant growth regulators such as auxin, gib-berellins, jasmonic acid, and brassinosteroids.All eukaryotic P450s are strictly membrane bound
and dependent upon partner flavin-dependent reduc-tase proteins that provide reducing equivalents. Somealso recruit cytochrome b5s as additional electron
AADVANCES
• Plant oxygenase activity occurs widely in natural
product biosynthesis with regio- and stereo-
selectivity that current synthetic standards
cannot match.
• Next-gen sequencing technology has greatly
facilitated the discovery of novel oxygenases
belonging to several enzyme families that can
be harnessed for desirable enzymatic
transformations.
• Directed evolution and rational design of
bacterial oxygenases to diversify chemical
scaffolds and catalyze novel reactions highlight
the untapped potential of their plant
counterparts.
• Oxygenases can be used as a means to
manipulate many facets of plant physiology.
1This work was supported by grants from the Edward N. and DellaL. Thome Memorial Foundation, the Family Larsson-Rosenquist Foun-dation, and the National Science Foundation (grant no. 1709616).J.-K.W. was supported by the Arnold and Mabel Beckman Foun-dation, Pew Charitable Trusts (grant no. 27345), and the KinshipFoundation (grant no. 15-SSP-162).
2Author for contact: [email protected] author.A.J.M. and J.-K.W. wrote the article.[OPEN] Articles can be viewed without a subscription.www.plantphysiol.org/cgi/doi/10.1104/pp.18.01223
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donors. These transmembrane domains make it diffi-cult to obtain recombinant protein; therefore, it ischallenging to obtain structural data for eukaryoticP450s. To date, a handful of human P450s and one plantmember have been successfully crystallized by replac-ing the N-terminal signal peptide sequence to increaseprotein solubility. These structures are largely helical,with the heme buried in a solvent-exposed central cleftand an axial Cys residue additionally coordinating theiron cofactor (Fig. 1A). Oxygen activation generates thecharacteristic compound I intermediate (Fig. 1B) thatcatalyzes hydrogen atom transfer (HAT) from thesubstrate. While the mechanism of hydroxylation islargely understood (Fig. 1B; Box 1), control of the re-action outcome by nonhydroxylating members is acontinuing field of study.
Many characterized plant P450s catalyze hydroxyl-ation (Schuler, 2015) of sp3 or sp2 C-H bonds, but theirarsenal of catalysis is continually expanding as newplant natural product chemistry is discovered. P450hydroxylases typically display strong preference to-ward a selective C-H bond of their substrate, but manyhave been shown to exhibit a general promiscuity,catalyzing a small percentage of altered regiospecificoutcomes and/or activity upon related chemicalscaffolds. For example, CYP76C subfamily P450s fromArabidopsis can install multiple hydroxyls intomonoterpenol substrates (Fig. 1C; Höfer et al., 2014).Other P450s have dual functionality, such as Avenasativa (oat) CYP51H10 that hydroxylates C16 andepoxidates the C12-C13 bond of b-amyrin (Fig. 1C;Geisler et al., 2013) or ent-kaurene oxidase (CYP701A)that catalyzes three consecutive oxidation reactions toproduce the GA precursor ent-kaurenoic acid (Fig. 1C;Helliwell et al., 2001; Mafu et al., 2016). In the latter re-action, the second turnover generates a dihydroxylatedcarbon center that dehydrates to an aldehyde prior to athird oxidation. In a similar manner, P450-dependentdemethylation likely proceeds through hydroxylation,followed by decomposition to formaldehyde and thedemethylated product (Kellner et al., 2015a). Anotherspontaneous rearrangement following hydroxylase ac-tivity by a P450 is thought to occur in (+)-menthofuranproduction byMentha spicata (mint) CYP71A32 (Fig. 1D),wherein the newly installed hydroxyl group acts as anucleophile (Bertea et al., 2001). In hydroxylation-derived reactions such as the latter, it is likely that ad-ditional protein residues aid in rearrangement.
Other P450s favor radical rearrangement of theirsubstrate immediately following HAT but prior to hy-droxylation. The ability to slow -OH rebound in thesesystems is not well understood but gives rise to uniqueP450 catalysis, for example, the ring contraction of ent-7-OH-kaurenoic acid (Fig. 1D) by CYP88A (Helliwellet al., 2001) and the oxidative ring opening of loganinto secologanin (Fig. 1D) by CYP72A1 or secologaninsynthase (Irmler et al., 2000). In both systems, it is likelythat radical rearrangement post HAT leads to -OH re-bound at a different C atom than initially targeted thatis ultimately dehydrated to give the final product
(Hakamatsuka and Hashim, 1991; Yamamoto et al.,2000). Whereas these P450s delay hydroxylation,others completely suppress it by utilizing compound IIfor a second HAT, generating two substrate radicalsthat undergo recombination to form a new bond(Fig. 1B, inset). This strategy is utilized by P450s forolefin installation and the intra/intermolecular cycli-zation of C-C or C-O bonds, the latter being used ex-tensively during plant benzylisoquinoline alkaloidbiosynthesis (Kellner et al., 2015a, 2015b; Kilgore et al.,2016). The intramolecular C-C coupling of (S)-reticulineand (R)-reticuline is catalyzed by Coptis japonicaCYP80G2 and Papaver somniferum CYP719B1, respec-tively (Fig. 1E; Kellner et al., 2015b). In both systems,HAT from hydroxyl groups on opposite rings leads toC-C bond formation via radical rearrangement(Mizutani and Sato, 2011). Subsequent enolization thenrearomatizes, a step possibly facilitated by active-siteresidues. Both of these P450s must bind the phenolicrings such that the hydroxyls are primed for HAT, butthe altered chirality likely brings different C atomswithin close proximity for radical coupling. A morerecently identified P450, CYP96T1 from daffodil(Narcissus pseudonarcissus), catalyzes a similar C-Ccoupling of 49-O-methylnorbelladine to noroxomaritidinebut produces an enantiomericmixture (Fig. 1E), thought toarise from flexibility of the C-C coupled product prior tospontaneous N-C ring closure (Kilgore et al., 2016). Thecontinued research on properties of P450s that enabletargeting of anO-H bond and/or utilization of compoundII for a second HAT step will yield valuable informationfor the rational design of atypical P450 reactivity.
Much of the P450’s catalytic capability extends fromits ability to generate high-valent iron species that allowit to target otherwise unreactive C-H bonds. However,the ferric-peroxo species (Fig. 1B) is also thought to actas a nucleophile during the Baeyer-Villiger oxidationcatalyzed by CYP85A2 in brassinosteroid biosynthesis(Fig. 1F; Nomura et al., 2005). In this system, the peroxointermediate likely attacks the steroid backbone ke-tone and undergoes a Criegee-like rearrangement toproduce the expanded ester-containing ring of thebrassinosteroids. The examples described above rep-resent only a subset of known plant P450 reactions, alist that is likely to expand in the future.
Plant P450s have become a focal point of syntheticbiology research due to their abundance and the chal-lenging oxidative chemistry they catalyze. Total syn-thetic routes to many clinically used plant metabolitesare crippled by efficiency, selectivity, and cost, often atsteps that plant oxygenases catalyze with ease (King-Smith et al., 2018). Chemo-enzymatic approaches usingplant P450s expressed in Escherichia coli and yeast hostshave overcome some of these bottlenecks, enablingmore efficient production of highly complex com-pounds. Among others, semisynthesis of the antima-larial drug artemisinin is a prominent example (Paddonand Keasling, 2014). Nevertheless, expression of plantP450s in nonplant hosts can be challenging, as theyrequire coexpression of partner reductases, need to be
Figure 1. A, 3D structure of the plant P450 allene oxide synthase and zoomed-in view of the active site (PDB 3DSI). The hemescaffold is shown as green sticks, the ferric ion as an orange sphere, and a hydroxylated substrate mimic as yellow sticks.An active-site Asn residue helps tether the substrate above the heme. B, The mechanism of hydroxylation by the P450s invokingthe potent compound I oxidant. A detailed discussion can be found in Box 1. The faded inset depicts an alternative non-hydroxylating pathway utilizing compound II for a second HAT step. C to F, Select plant P450 reactions discussed in the text thatcatalyze promiscuous hydroxylations (C), rearrangement coupled with hydroxylation (D), phenolic C-C bond coupling (E), and a
localized to the endoplasmic reticulummembrane, andmay produce numerous undesired products (O’Reillyet al., 2011; O’Connor, 2015). Additionally, some P450shave been shown to preferentially operate synergisti-cally with other P450s (Quinlan et al., 2012) or in mul-tienzyme metabolon complexes (Jørgensen et al., 2005;Ralston and Yu, 2006; Bassard et al., 2017), which mustbe considered when designing synthetic systems in-volving P450s. These challenges can be alleviatedthrough the use of a heterologous plant host for whichnew strategies have been devised to achieve improvedactivity. One promising approach is to syntheticallytarget P450s to the thylakoid membranes of the chlo-roplasts in planta (Nielsen et al., 2013). This not onlyinsulates the engineered pathway from other metabolicpathways of the hosts but also obviates the dependencyupon a partner reductase, as photosynthesis can pro-vide the reducing equivalents required for catalysis(Lassen et al., 2014). This strategy, first tested in 2013,has since been used to reconstitute the entire dhurrinbiosynthetic pathway via CYP79A1 (Gnanasekaranet al., 2016) and was recently adapted to produceartemisinic acid, the precursor to artemisinin, in to-bacco (Nicotiana tabacum) leaves (Fuentes et al., 2016).
The P450 scaffold itself also holds great potential forengineering novel catalysts, as demonstrated by pio-neering work of the Arnold lab and others. Throughhigh-throughput directed evolution approaches, bac-terial P450-BM3 and others have been evolved exten-sively for a wide range of oxidations on a diverse set ofsubstrates (Jung et al., 2011; Renata et al., 2015; Arnold,2018). P450-BM3 mutants are being used in the chemo-enzymatic synthesis of plant metabolites such asnigaelladine A (Loskot et al., 2017), a norditerpenoidalkaloid fromNigella glandulifera (Chen et al., 2014). TheP450-BM3 system was targeted because it is naturallyfused to its partner reductase, a trait not observed forany known plant P450. However, successful fusion ofplant P450s to partner reductases has enabled an in-crease in their catalytic efficiency (Didierjean et al.,2002; Schückel et al., 2012; Sadeghi and Gilardi, 2013).Directed evolution of P450s has also yielded novel ac-tivities and even some previously unobserved for anynatural catalyst, such as carbene/nitrene insertions togenerate cyclopropyl/butyl groups, aziridination, andsulfimidation, greatly expanding the utility of this en-zyme scaffold (Coelho et al., 2013; Wang et al., 2014;Brandenberg et al., 2017; Chen et al., 2018a). Such ex-haustive evolution of any plant P450 has yet to be ex-plored, owing to their difficulty in heterologousexpression, but their native promiscuity could yieldimproved access to a plethora of plant secondary me-tabolites. For example, researchers observed that onlythree amino acid variations were needed to efficientlyalter P450 activity in carnosic acid biosynthesis (Scheler
et al., 2016). Significant advancement in P450 structureand mechanistic control are necessary to expand suchefforts in the future.
FE/2OGS
The second largest oxygenase family in plants is theFe/2OGs, represented by ;130 genes in Arabidopsis(Hagel and Facchini, 2018). The Fe/2OGs participate inmany of the same plant pathways as P450s and alsofunction in histone modification and DNA repair, rolesconserved in all domains of life. These oxygenases uti-lize a nonheme ferrous cofactor and a 2OG cosubstratefor O2 activation to functionalize unreactive C-H bonds,concomitant with the production of CO2 and succinatecoproducts. Plant Fe/2OGs mainly catalyze hydroxyl-ation reactions, and other reactivities include C/N/Odemethylation, olefin installation, C-/O,C bond cou-pling, and methoxy migration (Hagel and Facchini,2018). Furthermore, bacterial Fe/2OGs catalyze C-Ncyclization, epoxidation, halogenation, azidation, epi-merization, and endoperoxdiation (Vaillancourt et al.,2005; Steffan et al., 2009; Chang et al., 2014, 2016;Matthews et al., 2014; Dunham et al., 2018). The widerange of catalytic functions, their cytosolic nature, andthe absence of a required partner reductase make Fe/2OGs a valuable source of novel biocatalysts.
The mechanism and structure/function relationshipsof Fe/2OGs have been studied extensively and arelargely understood for the common hydroxylationoutcome (Fig. 2, A and B; Box 2; Bollinger et al., 2015).Structurally, they are defined by a conserved cupin ordouble-stranded b-helix, and the ferrous cofactor iscoordinated by a conserved 2His-1Glu/Asp (HxD/ExnH) facial triad with the 2OG cosubstrate coordi-nated via its C1 carboxylate and C2 carbonyl (Fig. 2A).The substrate binds in the general vicinity such that itstarget carbon is positioned closest to the iron center. Noplant Fe/2OG has been characterized biophysically ingreat depth, and only four x-ray crystal structures havebeen obtained (Wilmouth et al., 2002; Sun et al., 2015;Kluza et al., 2018). Besides a histone demethylase, thethree other plant Fe/2OG structures are involved inplant specializedmetabolism and share a highly similarsecondary structure (;2.5 Å root-mean-square devia-tion), despite disparity in substrates and reactivity.This conservation may extend to most specialized plantFe/2OGs, suggesting these scaffolds could be moretractable for engineering purposes than their bacterialcounterparts.
The specialization of plant Fe/2OGs is prominentlydisplayed by those involved in flavonoid biosynthesis(Fig. 2C). The flavonoid precursor naringenin acts asthe substrate for both flavanone-3-hydroxylase (F3H)
Figure 1. (Continued.)Baeyer-Villiger-like oxidation (F). Possible independent reaction outcomes catalyzed by CYP76s are depicted by different colors.Dashed arrows represent spontaneous reactions.
and flavone synthase I; the former hydroxylates C3,whereas the latter desaturates along the C2-C3 bond toyield apigenin. The F3H reaction product dihy-drokaempferol serves as the substrate for flavonolsynthase (FLS) that also desaturates the C2-C3 bondto yield kaempferol. Alternatively, the ketone ofdihydrokaempferol can be reduced by the NADPH-dependent reductase DFR to give various leucoantho-cyanidins that are desaturated once again along theC2-C3 bond by a fourth Fe/2OG, anthocyanidin syn-thase (Saito et al., 1999). This desaturation triggersspontaneous dehydration of C4 to produce theoxinium-containing anthocyanidins leading to the vi-brant pigments of many flowering plants. These fourFe/2OGs are an excellent group of related enzymesfrom which structural features dictating substrate pro-miscuity and specificity can be learned. Structures ofanthocyanidin synthase bound to narigenin or dihy-drokaempferol (Wilmouth et al., 2002; Welford et al.,2005) suggest that these Fe/2OGs, assuming a roughlysimilar bindingmode, initially target C3.While this is inline with hydroxylation by F3H, desaturation via initialtargeting of C3 would not correlate to standing andrecently supported mechanistic proposals of either anadjacent-heteroatom-assisted or dual HAT pathway(Dunham et al., 2018), the latter of which would requirethe C2-H bond to be directed toward the iron center.
Further analysis of these Fe/2OGs may reveal a thirddesaturation strategy. It is worth noting that an otherknown plant Fe/2OG catalyzing desaturation, glucor-aphasatin synthase1 from Raphanus raphanistrum subsp.sativus (radish; Fig. 2C), installs an alkene adjacent to anS atom of its glucoerucin substrate, consistent with theproposed heteroatom-assisted mechanism (Kakizakiet al., 2017; Dunham et al., 2018).Other plant Fe/2OGs have repurposed their hy-
droxylase activity to trigger rearrangement similar toP450 reactions. For theN-Lys demethylases involved inhistone regulation, these rearrangements occur spon-taneously, as the charged amino group is an inherentlygood leaving group (Tsukada et al., 2006; Chen et al.,2011). In other cases, such as codeine/thebaine-6 O-demethylase and protopine O-dealkylase (Fig. 2D;Farrow and Facchini, 2013), assistance from surround-ing active-site residues for proton donation is likely.These Fe/2OGs are the first members shown to catalyzeO-demethylation, and the dealkylation activity of pro-topine O-dealkylase was previously observed only forP450s (Farrow and Facchini, 2013). Other rearrange-ments are catalyzed by the Fe/2OGs Phex30848 (or 2-ODD) and BX13 that catalyze the C-C bond formationof (2)-yatein (Lau and Sattely, 2015) and an oddmethoxy migration in benzoxazinoid biosynthesis(Handrick et al., 2016), respectively (Fig. 2E). The exact
BBOX 1. Catalytic mechanism of hydroxylation by P450s
In their resting state, P450s possess a heme-
bound ferric ion coordinated by an axial Cys
residue opposite a water molecule. Binding of the
primary substrate triggers displacement of water
and reduction of iron to the ferrous state via the
partner reductase. Reaction with oxygen and a
second reducing equivalent leads to a ferric-
peroxy/hydroperoxy species that undergoes
proton-assisted O-O bond scission to generate
Compound I, an Fe(IV)-oxo-porphyrin π radical
(Meunier et al., 2004; Rittle and Green, 2010).
Compound I targets the closest C-H bond of the
substrate for HAT to produce a substrate-centered
radical and Fe(IV)-OH species (Compound II).
Subsequent attack of the -OH by the C• produces
the hydroxylated product and regenerates the
resting ferric cofactor. This “rebound” of the -OH
group can be delayed, allowing for radical
rearrangement of the substrate prior to
hydroxylation (Meunier et al., 2004).
Other noncanonical outcomes proceed through a
second HAT step, catalyzed by Compound II and
followed by radical coupling, direct attack of an
alkene by Compound I, or attack by the ferric-
peroxo species upon electrophilic carbons (Krest
et al., 2013). It is likely that additional features of
the active site promote these noncanonical
reactivities either through substrate positioning,
catalyzing additional acid/base chemistry, or
tuning the electronic properties of the iron-oxo
species (Green et al., 2004; Behan et al., 2006;
Rittle and Green, 2010; Yosca et al., 2013). For
example, the identity and environment of the
coordinating axial residue can change the
oxidative potential of the system (Yosca et al.,
2013). Exhaustive biophysical characterization of
bacterial P450s and related heme oxidases has
helped researchers understand these versatile
catalysts.
Box 1 Catalyticmechanism of hydroxylation by P450s.Green et al. (2004);Meunier et al. (2004); Behan et al. (2006); Rittle andGreen (2010); Krestet al. (2013); Yosca et al. (2013).
mechanisms remain unclear but could invoke rear-rangement of thecarbon radical post HAT instead of an-OH-modified product. Other Fe/2OGs can catalyzeadditional oxidations on their initially hydroxylatedproducts. Identified as the main contributor to the fa-mous dwarf phenotype characterized by reduced levels
of the growth hormone GA in an Oryza sativa (rice)mutant (Sasaki et al., 2002), GA 20-oxidase catalyzesthree consecutive oxidations of the C20 methyl of GA53to produce GA20 (Fig. 2F). GA 20-oxidase converts thismethyl group to a hydroxyl, formal, and carboxylgroup consecutively, of which the final reaction triggers
Figure 2. A, Overall double-stranded b-helix (DSBH) fold and active site of the plant Fe/2OG anthocyanidin synthase bound tosubstrate analog naringenin (PDB 2BRT). Naringenin is shown as yellow sticks, the 2OG cosubstrate as green sticks, the ferrouscofactor as an orange sphere, a water molecule as a red sphere, and the iron-binding facial triad residues and 2OG-stabilizingresidues as gray sticks. B, The standing mechanistic proposal for Fe/2OG-dependent hydroxylation. A detailed discussion can befound in Box 2. The faded inset depicts possible routes to coordination relocation of the key ferryl intermediate to possiblysuppress the canonical hydroxylation reactivity. C to F, Select plant Fe/2OGs described in the text that participate in flavonoidbiosynthesis and catalyze olefin installation (C), catalyze demethylation or dealkylation (D), catalyze unique rearrangements (E),and catalyze iterative turnovers upon the same substrate (F). Dashed arrows represent spontaneous reactions. ANS, Anthocya-nidin synthase; CODM, codeineO-demethylase; FLS, flavonol synthase; FSI, flavone synthase I; GA20ox, GA 20-oxidase; GRS1,glucoraphasatin synthase1; H6H, hyoscyamine-6b-hydroxylase; PODA, protopine O-dealkylase; T6ODM, thebaine-6O-demethylase; DIMBOA, 2-(2,4-dihydroxy-7-methoxy-1,4-benzoxazin-3-one)-b-D-glucopyranose; TRIBOA, 2-(2,4,7- trihy-droxy-8-methoxy-1,4-benzoxazin-3-one)-b-D-glucopyranose; 2-ODD, Phex30848.
rearrangement, installing the characteristic carboxylbridge. Another dual-function Fe/2OG is the epoxide-forming hyoscyamine-6b-hydroxylase in Solanaceaeplants that first hydroxylates the C6 position prior toC-O ring closure with C7 in two independent turnovers(Ushimaru et al., 2018; Fig. 2F). Hyoscyamine-6b-hydroxylase differs from other epoxidizing Fe/2OGsthat directly target alkenes in a single turnover (Changet al., 2016) and is more similar to other C-O-cyclizingmembers such as clavaminate synthase (Zhou et al.,2001) and the loline oxidase LolO (Pan et al., 2018).Additional studies are required to fully understand theintricacies of the reaction outcome in this enzymefamily.Although not as heavily studied or utilized as bio-
catalysts comparedwith P450s, Fe/2OGs could serve asvaluable synthetic biology tools for engineering pur-poses. Their involvement in numerous plant hormoneand defense pathways makes them good targets foraltering certain plant traits, as demonstrated by thegeneration of pathogen resistance Solanum tuberosum(potato) through CRISPR targeting of the DOWNYMILDEW RESISTANCE6 gene, an Fe/2OG involvedin plant defense signaling (Zeilmaker et al., 2015).
Specialized plant Fe/2OGs in particular could be use-ful due to the wide array of substrate chemical scaf-folds they accommodate and a potentially conservedsecondary structure. For example, a recent reviewproposed a plausible chemo-enzymatic synthesis fornatural and modified vinblastine/vincristine alkaloidsthrough the use of desacetoxyvindoline-4-hydroxylasefrom Catharanthus roseus (Madagascar periwinkle;King-Smith et al., 2018). Such approaches using bacte-rial Fe/2OGs for natural product synthesis have al-ready been demonstrated (Zhang et al., 2018; Zwickand Renata, 2018). Moreover, Fe/2OGs could be engi-neered to produce novel halogenated natural pro-ducts in planta, a strategy exemplified by engineeringchlorinated monoterpene indole alkaloid productionin Madagascar periwinkle using bacterial FAD-dependent halogenases (Runguphan et al., 2010). Itwas recently demonstrated that a bacterial Fe/2OGamino acid hydroxylase could be modified to catalyzehalogenation of its native substrate, albeit at reducedefficiency (Mitchell et al., 2017a). Theoretically, plantFe/2OG hydroxylases could be engineered to producenovel halogenated compounds as well. Researchershave also recently developed a promising chemical
BBOX 2. Catalytic mechanism of hydroxylation by Fe/2OGs
An original proposal for hydroxylation by prolyl-4-
hydroxylase (P4H) in 1984 has largely stood up to
rigorous characterization (Majamaa et al., 1984).
The ferrous cofactor is coordinated by a conserved
HisXAsp/GluXnHis protein motif and 2OG in a
bidentate mode. The remaining coordination site
is occupied by a water molecule in the absence of
primary substrate, protecting the enzyme from
unproductive oxidation (Proshlyakov et al., 2017).
Binding of the substrate in the active site displaces
the water molecule and triggers reaction with O2,
generating a ferric superoxide that attacks the C2
carbonyl of the coordinated 2OG cosubstrate. This
complex rapidly decomposes to succinate, CO2,
and a high-valent ferryl (Fe(IV)-oxo) intermediate,
originally characterized by Bollinger and Krebs
(Price et al., 2003a; Price et al., 2003b). The bicyclic
and peroxysuccinate species are computationally
predicted, but a recent in crystallo study
surprisingly captured the latter, possibly due to
movement of an active-site Arg residue prior to O-
O bond scission (Ye et al., 2012; Mitchell et al.,
2017b). The ferryl species catalyzes HAT from the
closest C bond, and the resulting C• attacks the
redirection of the C•
-
ferric-hydroxyl, giving rise to the hydroxylated
product and regenerating the ferrous cofactor.
Exchange of succinate for another molecule of
2OG then allows for multiple turnovers. Alternate
reaction outcomes are believed to diverge post
HAT by the ferryl intermediate via redirection of
the substrate radical. Characterization of bacterial
Fe/2OGs catalyzing halogenation, epimerization,
and desaturation have helped reveal some of these
underlying mechanistic controls (Blasiak et al.,
2006; Chang et al., 2014; Mitchell et al., 2016;
Martinez et al., 2017; Dunham et al., 2018). Another
notion gaining support for avoidance of a
hydroxylation outcome is the relocation of the
ferryl intermediate to a more distal coordination
site, sacrificing HAT efficiency but favoring
(Mitchell et al., 2016; Srnec et
al., 2016; Martinie et al., 2017; Mitchell et al.,
2017b). This coordination isomerization could
occur after ferryl formation or could result from an
altered 2OG binding mode that has been observed
in many Fe/2OG crystal structures. Further
characterization will be needed to engineer
reaction outcomes into Fe/2OG scaffolds.
Box 2 Catalytic mechanism of hydroxylation by Fe/2OGs. Majamaa et al. (1984); Price et al. (2003a, 2003b); Blasiak et al. (2006); Ye et al. (2012);Chang et al. (2014); Mitchell et al. (2016, 2017b); Srnec et al. (2016); Martinie et al. (2017); Martinez et al. (2017); Proshlyakov et al. (2017);Dunham et al. (2018).
handle upon Fe/2OG activity in vivo through the use ofamodified or bumpedN-oxalylglycine (NOG) chemicalinhibitor (Sudhamalla et al., 2018). NOG is a structuralanalog of 2OG that nonspecifically inhibits Fe/2OGactivity. By expanding the active site of an Fe/2OGprotein involved in nucleotide repair via rational de-sign, it was demonstrated that NOG harboring smallaliphatic groups at the C4 position could selectivelyinhibit only the engineered enzyme while wild-typeactivity was unaffected. This approach could be ap-plied to transgenic plants to selectively regulate artifi-cially engineered Fe/2OG-gated pathways.
FLAVIN MONOOXYGENASES
Flavin-dependent monooxygenases (FMOs) utilizean organic FAD cofactor and O2 for oxidation. Theirreactions are also dependent on reducing equivalentsfrom a NADPH cosubstrate, and, in contrast to metal-loenzymes capable of accessing high-valent oxo species
to target unreactive C-H bonds, FMO activity is re-stricted to the targeting of nucleophilic or electrophilicsubstrates. Currently, only three sets of plant FMOs areknown, the FMO1s, glucosinolate-S-oxidases (GS-Oxs),and YUCCAs, all with highly specific biochemicalfunctions (Schlaich, 2007). This is in stark contrast tohomologous human FMOs that lack specificity andplay a general role in xenobiotic metabolism (Lattardet al., 2004). Bacterial FMOs are vastly more diversifiedthan both aforementioned sets, with known memberscapable of halogenation, oxidative rearrangements, andmore (Huijbers et al., 2014). The term FMO can be ap-plied across many classes of FAD/O2-utilizing en-zymes, most of which have not been identified in plantgenomes. Plant FMOs are defined by a double Ross-mann fold that simultaneously binds FAD andNADPHvia conserved motifs in the N/C termini (Fig. 3A;Huijbers et al., 2014). Reduction of the FAD cofactor byNADPH allows for the activation of O2 to produce thecommon hydroperoxyflavin or C4a(OOH)FAD inter-mediate for catalysis, observed in all characterized
Figure 3. A, The double Rossmann fold and active site of a bacterial cyclohexanone monooxygenase bound to its caprolactoneproduct (PDB 4RG3). No plant FMOhas been crystallized, but this bacterial Baeyer-Villigermonooxygenase (BVMO) is predictedto share an overall secondary structure. The FAD cofactor is shown as green sticks, the NADPH cosubstrate as cyan sticks, theproduct as yellow sticks, and the C4a atom that reacts with O2 is labeled. B, The proposed mechanisms for plant FMO activityutilizing the C4a(OOH)FAD intermediate as an electrophile (blue path) or nucleophile (red path). C to E, Select plant FMOreactions discussed in the text that catalyze hetero-atom oxidation (C), oxidative decarboxylation (D), and a C-C coupling re-action catalyzed by the plant FAD-dependent oxidase BBE (E).
FMOs (Fig. 3B). Structures of bacterial FMOs have alsosuggested a second role of the bound NADP+ in stabi-lizing the reactive intermediate, which needs to be in-vestigated further (Alfieri et al., 2008).Among the three classes of plant FMOs, two catalyze
heteroatom oxidation and the other catalyzes oxidativedecarboxylation. Arabidopsis FMO1 was first identi-fied as an enzyme linked to plant systemic acquiredresistance (Mishina and Zeier, 2006), but its functionwas only resolved recently. Two groups indepen-dently discovered that FMO1 is responsible for theN-hydroxylation of pipecolic acid (Fig. 3C), producingthe mobile signal molecule of the systemic acquiredresistance pathway (Chen et al., 2018b; Hartmann et al.,2018). This reaction likely proceeds via nucleophilicattack upon the C4a(OOH)FAD intermediate and isshared by the GS-Oxs (Hansen et al., 2007; Kong et al.,2016). GS-Oxs catalyze the S-oxidation of thiol-glucosinolates to their respective sulfinyls, with ho-mologs participating in alliin biosynthesis in Alliumsativum (garlic; Fig. 3C; Yoshimoto et al., 2015). The GS-Oxs are highly selective toward glycosylated substrates(Hansen et al., 2007), further highlighting the distinc-tion compared with the promiscuous human FMOs.The YUCCA family FMOs catalyze the oxidative de-carboxylation of indole pyruvate to indole-3-acetic acid(IAA; Fig. 3D), the primary plant growth hormone(Mashiguchi et al., 2011). YUCCAs were initially pro-posed to catalyze the N-hydroxylation reaction earlierin the IAA pathway (Zhao et al., 2001), but follow-upstudies clarified their bona fide activity (Mashiguchiet al., 2011; Stepanova et al., 2011). This reaction pro-ceeds via nucleophilic attack by the C4a(OOH)FAD onthe a-keto group and subsequent decarboxylation withO-O bond cleavage to produce IAA (Dai et al., 2013).Detection of a short-lived C4a(OOH)FAD species isconsistent with its nucleophilic role compared withother FMOs (Dai et al., 2013). The YUCCAs are chem-ically similar to the BVMOs found in bacteria that oxi-dize linear or cyclic ketones to new ester compoundsvia rearrangement of a Criegee intermediate betweenthe substrate and peroxy-flavin (Mascotti et al., 2015).No BVMO has been identified in plants, but thesebacterial enzymes share conserved sequence and sec-ondary structure features with plant and human FMOs,suggesting that plant FMOs could potentially be engi-neered to BVMO-like enzymes (Huijbers et al., 2014). Infact, a human FMO was recently shown to catalyze aBVMO-like reaction (Fiorentini et al., 2017). There arealso Baeyer-Villiger-like oxidations of plant metaboliteswith unknown biosynthetic routes, such as the degra-dation of (+)-camphor in Salvia officinalis (sage; Croteauet al., 1984). The BVMOs have been used in numerouschemo-enzymatic applications, and their selectivity hasalso been successfully engineered (van Beek et al., 2012;Li et al., 2018a; Woo et al., 2018).Other notable classes of FAD-dependent oxidases
found in plants include the berberine-bridging enzymes(BBEs) and FAD-epoxidases. BBE, or (s)-reticuline oxi-dase from Eschscholzia californica (California poppy),
catalyzes a C-C coupling reaction (Fig. 3E), producingH2O2 as a by-product, and utilizes a bicovalently teth-ered FAD cofactor (Daniel et al., 2017). Other membersparticipate in similar cyclizations during cannabinoidmetabolism (Daniel et al., 2017). FAD epoxidases spe-cifically target alkenes of carotenoids or the triterpenoidprecursor squalene and have a different source of re-ducing equivalents from other FMOs (Büch et al.,1995; Han et al., 2010). It is also worth mentioning thebacterial flavo-halogenases that generate a reactivehypohalous ion to chlorinate and brominate electron-rich substrates (van Pée and Patallo, 2006; Payne et al.,2016). Improvement of the aforementioned in plantahalogenation of indole alkaloids (Runguphan et al.,2010) could lead to promising new therapeutics, as itis estimated that one-third of the drugs in clinical trialsare halogenated (Fraley and Sherman, 2018), and thepresence of halogens can significantly enhance drugpotency (Imai et al., 2008; Gillis et al., 2015).
CAROTENOID CLEAVAGE DIOXYGENASES
First identified in Zea mays (maize; Schwartz et al.,1997), the carotenoid cleavage dioxygenases (CCDs)are a specialized class of non-heme-iron-dependentenzymes that cleave the electron-rich alkene chainof various carotenoids and apocarotenoids. Whileb-carotenes are important antioxidants and pigmentsthemselves, CCD activity produces the precursors forthe important signaling hormones strigolactone andabscisic acid, along with many notable fragrant com-pounds. The identification of plant CCDs has sincerevealed mammalian homologs involved in retinalproduction and bacterial homologs capable of metab-olizing noncarotenoid substrates. In plants, CCD1,CCD4, CCD7, and CCD8 and the neoxanthin cleavageenzymes are universally conserved (Auldridge et al.,2006; Harrison and Bugg, 2014), the latter of whichspecifically target epoxidized carotenoids during thebiosynthesis of abscisic acid, a key regulator of droughttolerance and seed dormancy (Fig. 4C). A number ofspecialized plant CCDs have also been identified, suchas CCD2 from Crocus sativus, which cleaves zeaxanthinduring corcin biosynthesis (Frusciante et al., 2014), andlycopene cleavage dioxygenase from Bixa orellana,which is capable of cleaving the b-carotenoid precursor(Satpathy et al., 2010).The cleavage activity of CCDs is catalyzed by a single
ferrous cofactor and O2 molecule, drawing all four re-ducing equivalents from the primary substrate. Struc-tural characterization and sequence comparison ofCCDs revealed a conserved seven-bladed b-propellerarchitecture housing a 4-His coordinationmotif (Fig. 4A;Messing et al., 2010; Sui et al., 2013), also present in thebacterial lignostilbene dioxygenases that catalyze sim-ilar oxidative cleavage reactions (Bugg et al., 2011).Three second-sphere Glu residues that form hydro-gen bonds with the His residues are also conserved (Suiet al., 2013; Priya and Siva, 2015). The exact mechanism
for CCD-catalyzed cleavage remains in question, butthe current proposal invokes the action of a Fe(III)-superoxide species (Fig. 4B; Box 3). Limited biochemicaland structural studies of the plant CCDs have beenreported to date.
The specialization of plant CCDs is exemplifiedby members CCD7 and CCD8 that act sequentiallyto produce carlactone, the direct precursor tostrigolactone, a more recently identified plant hormonethat controls root and shoot branching. Similar to CCD1and CCD4 (Harrison and Bugg, 2014), CCD7 catalyzesthe selective 9/10 cleavage of b-carotene; however, itspecifically recognizes 9-cis-b-carotene (Fig. 4C) anddisplays no activity upon the more abundant trans-conformer (Bruno et al., 2014). Its 9-cis-b-apo-109-car-otenal product then serves as the substrate for CCD8,the most chemically unique plant CCD, catalyzing se-quential dioxygenations to produce carlactone (Fig. 4C;Harrison et al., 2015). The first cleavage reaction ofCCD8 is likely akin to others, but the second results in aunique rearrangement to produce the enol ether ofcarlactone. CCD8 seems to not release its initial cleav-age product, an observation consistent with the recentproposal involving a covalent enzyme-product com-plex (Harrison et al., 2015). Alternatively, this could beachieved via direct coordination to the iron cofactor.
Pre-steady-state kinetic analysis of CCD8 under sub-molar O2 concentrations and anaerobic crystallo-graphic experiments would help decipher thismechanism. For other CCDs, substrate positioningseems to be themajor factor dictating reaction outcome.Engineering of CCD activity could be used for theproduction of high-value rose (Rosa spp.) ketones, suchas ionones and damascenones, used in fragrances andoils, but it will require improved structural and mech-anistic knowledge to efficiently alter reactivity.
EXTRADIOL-CLEAVING DIOXYGENASES
Similar in reactivity to the CCDs, the extradiol-cleaving dioxygenases (ECDs) are iron-dependent en-zymes that catalyze oxidative cleavage of C-C bonds.However, ECDs selectively target catechols to cleavethe adjacent phenolic bonds, hence the name extradiol(Lipscomb, 2008). These enzymes contain a single fer-rous cofactor coordinated via two His and one Aspresidues, with their diol substrates occupying two othercoordination sites (Lipscomb, 2008). In plants, there area number of annotated ECDs, but few members havebeen functionally characterized. DOPA 4,5-dioxygen-ase (DODA) is responsible for the production of
Figure 4. A, The seven-bladed b-propeller structure and active site of iron-bound Vp14, a plant CCD from maize. The four iron-coordinatingHis and the conserved second-sphereGlu residues are shown as gray sticks, the ferrous cofactor as an orange sphere,and a water molecule as a red sphere. An end-on coordinating O2 is modeled and shown as a red stick, but the accuracy of thismodel is suspect due to low resolution of the structure (;3.1 A). Structures of bacterial CCDs (sub-2 A resolution) contain clearwater molecules modeled at said location. B, Mechanistic proposals for CCD dioxygenation invoking either a dioxetane (bluepath) or Criegee-like (red path) rearrangement. These two possibilities are explained further in Box 3. C, Schematic of conservedCCD reactions catalyzed in higher plants. Hollow arrows represent multiple and/or non-CCD enzyme reactions. DWARF27 is a2Fe-2S-cluster-dependent carotenoid isomerase. NCED, Neoxanthin cleavage enzyme.
betalamic acid, the betalain pigment precursor, viacleavage of L-DOPA (Fig. 5A; Christinet et al., 2004;Chung et al., 2015), LigB from Arabidopsis cleavescaffealdehyde (Fig. 5A;Weng et al., 2012), and a third isproposed to be involved in stizolob(in)ic acid biosyn-thesis (Saito and Komamine, 1976). The 4,5 cleavagereaction of DODA produces 4,5-seco-DOPA thatspontaneously rearranges to betalamic acid with highenantiomeric selectivity in both in planta and in vitroassays, suggesting that DODA may also play an addi-tional role (Christinet et al., 2004). Bacterial ECDs cat-alyze a wide range of catechol cleavage reactions, someofwhich have been expressed inMedicago sativa (alfalfa)for bioremediation (Wang et al., 2015). It is possible thatsome of the uncharacterized plant ECDs have addi-tional biosynthetic roles.
RIESKE-TYPE OXYGENASES
Rieske-type oxygenases (RSOs) represent a smallfamily of non-heme-iron-dependent oxygenases inplants. These enzymes contain separate ferrous- and2Fe-2S-cluster-binding domains, the latter of whichtransfers electrons to the iron center via a bridging Aspresidue during the catalytic mechanism for oxygen ac-tivation (Ferraro et al., 2005; Barry and Challis, 2013).Crystallized RSOs from bacteria revealed functional
trimers with these domains juxtaposed. All higherplants possess a set of RSOs involved in chlorophylldegradation (Gray et al., 2004), and the only otherknown plant RSO is choline monooxygenase(Rathinasabapathi et al., 1997), which produces theosmoprotectant Gly-betaine (Fig. 5B; Hibino et al.,2002). RSOs are more abundant in bacterial genomescatalyzing known monohydroxylations and dihydrox-ylations, demethylations, N oxidation, and even C-Cbond coupling (Barry and Challis, 2013), highlightingthe synthetic potential of the RSO scaffold. A recentstudy identified a novel RSO from Ocimum basilicum(basil) trichomes that functions as a flavone-8-hydrox-ylase with strong selectivity toward salvigenin (Fig. 5B;Berim et al., 2014). Additional uncharacterized RSOhomologs found in plant genomes may be proven tooperate in specialized metabolism as well.
FATTY ACID OXYGENASES
The oxygenation of fatty acids is utilized by plantsfor the production of signaling and defense com-pounds. All higher plants possess two classesof oxygenases that target polysaturated or unsatu-rated fatty acids. The lipoxygenases (LOXs) aremononuclear nonheme enzymes that catalyze theinsertion of molecular oxygen into unsaturated
BBOX 3. Mechanistic proposals for C-C cleavage by CCDs
An actual dioxygenation event for CCD activity
remained in question upon their initial
characterization, as independent studies reported
contradicting results during isotopically labeled O2
tracing experiments. This discrepancy was
resolved in a more recent study of related bacterial
CCDs where 100% incorporation of both O2 atoms
was clearly observed, with researchers accounting
for back exchange of the aldehyde products. It is
believed that, like most other mononuclear ferrous
oxygenases, activation of O2 proceeds through a
ferric-superoxide species. The next likely step is
radical addition into the nearest substrate alkene
from which two plausible mechanisms have been
proposed. Recombination of the distal O atom
could generate a dioxetane species (blue arrows)
that would rapidly decompose to the cleaved
products. This pathway is fully consistent with a
dioxygenation event and is the most energetically
favored route based on in silico simulation
(Borowski et al., 2008).
An alternate pathway (red arrows) invokes electron
transfer to the ferric ion to create a (+)-carotene-
peroxo adduct, the cation being stabilized via the
adjacent conjugated chain. This cation could be
quenched by an active-site water molecule or
nucleophilic residue that can trigger O-O bond
scission via a Criegee-like rearrangement.
Subsequent attack by the resulting iron-bound
hydroxyl and rearrangement would generate the
cleaved products with incorporation of both O2
atoms. This pathway would invoke acid/base
chemistry in which the conserved active-site Glu
residues could play a role. A recent study on CCD8
implicated the role of an active-site Cys residue in
a Criegee-like mechanism (Harrison et al., 2015), an
intriguing and perhaps more favorable proposal to
ensure selective breakdown as the final proposed
dihydroxy-ether intermediate could plausibly
proceed via loss of either hydroxyl group.
Additional structural and biophysical
characterization is needed to further discern the
mechanism.
Box 3 Mechanistic proposals for C-C cleavage by CCDs. Borowski et al. (2008); Harrison et al. (2015).
bonds to form hydroperoxo units (Fig. 5C; Baysaland Demirdöven, 2007). These compounds are thencleaved during production of various plant volatilesand oxylipins, such as the hormone jasmonic acid(Vick and Zimmerman, 1984; Fuller et al., 2001;Baysal and Demirdöven, 2007). LOXs are abundantin both plants and animals, utilizing three His resi-dues and their C-terminal carboxyl group to coor-dinate an iron cofactor. This assembly differs fromthe di-iron cofactor used by the fatty acid hydroxy-lases (FaHs; Broun et al., 1998). Di-iron enzymesconstitute an abundant superfamily that operatesin critical biological processes in bacteria, such asribonucleotide reduction and methane oxygenation,and catalyzes many monooxygenation or dioxyge-nation reactions such as N-hydroxylation in chlor-amphenicol biosynthesis (Jasniewski and Que, 2018;Komor et al., 2018). FaHs have only been found inselect plant species but are closely related to thefatty acid desaturases (FaDs) present in nearly allhigher plants (Fig. 5C; Broadwater et al., 2002). Se-quence comparison of FaDs and FaHs revealed onlyseven class-specific variations (Broun et al., 1998).Subsequent mutagenesis studies showed that thedesaturation:hydroxylation activity partition ofArabidopsis FAD2 and several FaHs could be ma-nipulated with as few as a single amino acid change(Broun et al., 1998; Broadwater et al., 2002). Manip-ulation of plant LOXs and FaD/FaHs could be usedto gain access to a range of high-value fatty acid
derivatives and to manipulate plant growth anddevelopment.
CYS DIOXYGENASES
Cys dioxygenases (CDOs) are mononuclear non-heme-iron-dependent enzymes that couple the reduc-tion of O2 to the oxidation of a Cys thiol to sulfinic acid.Bacterial and animal CDOs target free Cys in taurinebiosynthesis (Dominy et al., 2006; Stipanuk et al., 2009),but plant CDOs (PCOs) target N-terminal cysteinylresidues of select plant peptides (Weits et al., 2014). Asthese sequences vary significantly, it remains to be de-termined whether PCOs utilize a similar active-site ar-chitecture to their mammalian and bacterial homologs.Briefly, CDOs utilize a cupin fold with a three-His ironmotif to which the L-Cys substrate coordinates via itsthiol and amino groups (McCoy et al., 2006; Simmonset al., 2006). Mammalian CDOs also contain a con-served Cys(S)-Tyr(meta-C) cross-link in the active site(McCoy et al., 2006; Li et al., 2018b), believed to helpstabilize reactive Fe-oxo species (Ye et al., 2007). PCOs,first identified in Arabidopsis, are linked to the regu-lation of hypoxic response and were recently shown tospecifically oxidize the N termini of class VII EthyleneResponse Factors (ERF-VIIs; Fig. 5D; Weits et al., 2014;White et al., 2017). During periods of low cellular oxy-gen, such as flooding, the ERF-VIIs trigger up-regulation of anaerobic genes, enabling the plant to
Figure 5. The representative reactions catalyzed by plant ECDs (A), RSOs (B), the LOXs and FaD/FaHs (C), PCOs (D), and ACCO(E). The role of DODA during the spontaneous formation of betalamic acid remains in question. Molecular oxygen atoms areshown in blue for tracing purposes.Dashed arrows represent spontaneous rearrangements, and hollowarrows representmultisteppathways. CMO, Choline monooxygenase.
enter a generally anaerobic state (Bailey-Serres et al.,2012). Under normal O2 levels, the ERF-VIIs are oxi-dized by PCOs, which initiates their degradation viatheN-end rule pathway, suggestingthat the PCOs act asthe primaryO2 sensor in vivo (Licausi et al., 2011;Whiteet al., 2017). Flashman and coworkers recently providedsupport for this notion through the kinetic characteri-zation of all five Arabidopsis PCOs, which displayedapparent Km values toward O2 within the physiologi-cal range (White et al., 2018). These findings and thein vivo role of PCOs are analogous to the regulation ofthe hypoxia-inducible transcription factors in animalsby a set of Fe/2OGs, the hypoxia-inducible transcrip-tion factor hydroxylases, or PHDs, which display aneven stronger O2 dependence (Ehrismann et al., 2007;Tarhonskaya et al., 2014). Arabidopsis PCOs were alsoshown to possess unique substrate preferences, variedenzymatic rates, and O2 dependence, suggesting non-redundant roles in vivo (White et al., 2018). Suppres-sion of PCO activity could be used to increase thestability and abundance of ERF-VIIs, leading to im-proved flood tolerance and crop recovery (Licausi et al.,2010; Gibbs et al., 2011).
AMINOCYCLOPROPANECARBOXYLATE OXIDASE
Found inall higherplants, aminocyclopropanecarboxylateoxidase (ACCO) catalyzes the final step of ethylenebiosynthesis via the Yang cycle (Fig. 5E; McKeon et al.,1995). Produced in nearly all plant tissues, ethylene is amajor plant hormone controlling fruit ripening, senes-cence, and other aspects of plant growth (Iqbal et al.,2017). Manipulation of ethylene levels, both exoge-nously and within the plant, is routinely exercised tohasten ripening and extend shelf life. Tissue-specificregulation of ACCO activity is a powerful strategy tomodulate many aspects of plant physiology (Tonuttiet al., 1997; Ben-Amor et al., 1999; Lemus et al., 2007).ACCO utilizes a ferrous cofactor and O2 to produce oneequivalent of ethylene, CO2, and cyanide from ACC(Rocklin et al., 2004). This reaction is also dependentupon ascorbate, presumably providing reducingequivalents, and an apparent requirement for CO2 orbicarbonate that remains to be understood. ACCO isstructurally related to the Fe/2OGs, but its mechanism
is not 2OG dependent (Hagel and Facchini, 2018). ACCbinds in a pocket analogous to that of 2OG and coor-dinates in a bidentate manner via its amino and car-boxyl groups. HAT from C2 by an Fe(IV)-oxo species isbelieved to initiate breakdown to ethylene and cyanoformate,the latter of which is shunted away from the active sitebefore it decomposes (Murphy et al., 2014). Interestingly,bacteria also use an Fe/2OG-like enzyme for ethyleneproduction, knownas the ethylene-forming enzyme,whichdirectly oxidizes the iron-bound 2OGmolecule to ethyleneand twomolecules of CO2 (Martinez and Hausinger, 2016;Martinez et al., 2017; Zhang et al., 2017). Ethylene-formingenzymes have garnered much interest as a sustainablesource for bioethylene production, as their 2OG substrate ischeap and it does not produce a toxic by-product like cy-anide compared with ACCO.
CONCLUSION
Although the enzyme families discussed here do notinclude all plant oxygenases, those chosen provide arepresentative picture of the diverse chemistry andcatalytic strategies utilized by oxygenases in plantmetabolism. While many of the reaction outcomes arechemically redundant (i.e. hydroxylation), each systemhas its own advantages regarding their adaptability,selectivity, and/or accessibility as potential syntheticbiocatalysts. P450s have garneredmuch of the attentiondue to their abundance, plasticity, and established en-gineering efforts. However, the influx of plant genomicdata has helped illuminate the activity and role of nu-merous other, less studied oxygenases in plant metab-olism.Many of these oxygenase families have proven tobe useful biocatalysts in bacterial systems, highlightingtheir synthetic potential. Similar endeavors using plantcatalysts have presented many difficulties, as manyplant enzymes are unstable in vitro, harbor suboptimalactivity, and/or require additional partnering en-zymes. Establishment of in vivo optimization tech-niques, such as directed evolution, in plant cells mayultimately circumvent this issue and allow the rapidgeneration of novel plant catalysts. A continued effortto enhance basic structural and biochemical knowledgeof each oxygenase family should be emphasized to laythe foundation for future rational design of enzymeactivity. If such strategies can be routinely accessed, thepresence of oxygenases throughout plant genomes willgive synthetic biologists access to nearly all facets ofplant biochemistry and physiology.Received October 2, 2018; accepted January 14, 2019; published January 22,2019.
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