A Dioxobilin-Type Fluorescent Chlorophyll Catabolite as a Transient Early Intermediate of the Dioxobilin-Branch of Chlorophyll Breakdown in Arabidopsis thaliana

Chlorophyll breakdown in higher plants occurs by the so called “PaO/phyllobilin” path. It generates two major types of phyllobilins, the characteristic 1-formyl-19-oxobilins and the more recently discovered 1,19-dioxobilins. The hypothetical branching point at which the original 1-formyl-19-oxobilins are transformed into 1,19-dioxobilins is still elusive. Here, we clarify this hypothetical crucial transition on the basis of the identification of the first natural 1,19-dioxobilin-type fluorescent chlorophyll catabolite (DFCC). This transient chlorophyll breakdown intermediate was isolated from leaf extracts of Arabidopsis thaliana at an early stage of senescence. The fleetingly existent DFCC was then shown to represent the direct precursor of the major nonfluorescent 1,19-dioxobilin that accumulated in fully senescent leaves.

Aremarkable cytochrome P450 enzyme (CYP89A9) was identified recently in Arabidopsis thaliana (A. thaliana) that catalyzed the in vitro deformylation of the "primary" fluorescent Chl catabolite (pFCC) to the corresponding epimeric "primary" DFCCs (pDFCCs). [6] In weakly acidic solution, such apair of pDFCC epimers isomerized rapidly to apair of DNCCs.T hus,t wo key steps of the "dioxobilin" branch of chlorophyll breakdown appeared to be clarified. [6] Unfortunately,t hese results provided no conclusion with respect to the stereochemical outcome of the hypothetical DFCC-DNCC isomerization, [5b] nor was am ajor natural step of the dioxobilin path clearly identified by the in vitro enzyme reaction. We have now "trapped" at ransiently existent, natural DFCC in an early senescence stage of de-greening leaves of the model plant A. thaliana,a nd have explored its isomerization to aD NCC.T his isomerization occurred rapidly,w as highly stereoselective,a nd cleanly furnished At-DNCC-33, which is the major natural DNCC in senescent leaves of A. thaliana (this DNCC was provisionally named At-NDCC-1 previously). [6] To trap early intermediates of Chl breakdown in wild-type A. thaliana,fresh leaf extracts were analyzed after two days of incubation in the dark, that is,atanearly stage of senescence. Analysis by HPLC revealed av ariety of Chl catabolites (see Figure 2a nd Figure S2 in the Supporting Information), consistent with similar earlier observations. [6,7] Nonfluorescent fractions were observed with absorptions near 315 nm (classified as NCCs), as well as more prominent compounds with weak absorptions near 237 nm and 274 nm, but none near 315 nm, and thus were provisionally classified as NDCCs ("nonfluorescent" DCCs). Strikingly,aminor fluorescent fraction was also detected that showed two characteristic bands near 237 nm and near 360 nm (see Figure 3), which were conspicuously similar to those of DFCCs characterized earlier as the product of the P450-catalyzed deformylation of pFCC. [6] Roughly 130 mgo ft he unknown fluorescent compound 1 with as tandard retention time of 32.8 min was isolated by semipreparative HPLC from A. thaliana leaves,k ept in the dark for two days (212 g) or three days (148 g). Analysis of 1, now named At-DFCC-33, by positive-ion ESI mass spectrometry,r evealed ap seudo-molecular ion [M + H] + at m/z 619.0, consistent with am olecular formula of C 33 H 38 N 4 O 8 . Fragments at m/z 575.1 and 434.2 indicated subsequent loss of CO 2 and of ring A. Thus,t he DFCC 1 was revealed to be an isomer of At-DNCC-33 (2), the main Chl catabolite in senescent leaves of A. thaliana (previously At-NDCC-1). [6,8] Signals of 31 (of its 32) carbon-bound hydrogen atoms were observed and assigned in a600 MHz 1 HNMR spectrum of At-DFCC-33 (in CD 3 OD,273 K). Among them were three methyl group singlets (at high field) and ad oublet at d = 1.15 ppm, assigned to the H 3 C13 1 methyl group on the basis of correlations in 2D NMR spectra. Amultiplet at d = 2.79 was assigned to HC13, and another, at d = 2.74, to the direct neighbor HC12. From analysis of the set of correlations from 1 H, 1 H-ROESY, 1 H, 1 H-COSY, 1 H, 13 C-HSQC,a nd 1 H, 13 C-HMBC spectra, the constitution of rings Ba nd ring Cw as deduced to be the same as in FCCs. [9] Tw oddatd = 4.43 ppm and d = 4.83 ppm indicated hydrogen atoms at positions C4 and C16, as observed earlier in spectra of DNCCs. [5,6] In addition, a2 -hydroxyethyl side chain was identified at position C3. Analysis of the 2D NMR spectra revealed the structure of At-DFCC-33 (1,s ee Figure S6 in the Supporting Information). Thed erived structure is consistent with the observed UV spectrum, in which an absorption band near 360 nm is seen, characteristic of the common B/C chromophore of FCCs and DFCCs.A na dditional absorption band near 320 nm was absent, which is ac haracteristic of the formylpyrrole unit of the type-I phyllobilins,s uch as FCCs and NCCs.T hus,afirst representative of the elusive natural DFCCs could be characterized.
. . characterize the isomerization of the isolated, native At-DFCC-33 (1), and to identify its isomerization product, DFCC 1 was stored in potassium phosphate buffer (100 mm) at pH 5a tr oom temperature.Ahighly stereoselective conversion of the DFCC 1 was observed, and monitored by UV/Vis spectroscopy.U nder these conditions, 1 exhibited ah alf-life of 32 min and isomerized with first order kinetics (k = 0.022 min À1 ). After 220 min, the solution exhibited the typical UV spectrum of aD NCC (see Figure 3B). As ingle product was formed (HPLC), which had the retention time of authentic At-DNCC-33 (2,s ee Figure S8 in the Supporting Information). Ap ositive-ion ESI mass spectrum of the presumed isomerization product was also consistent with the molecular formula C 33 H 38 N 4 O 8 ([M + H] + at m/z 619.0). A CD spectrum of the isomerization product of the DFCC 1 showed the same features as authentic At-DNCC-33 (2), and of other DNCCs isolated from senescent leaves of A. thaliana (see Figure S1 in the Supporting Information). [8] Thenonfluorescent product of the acid-induced isomerization of At-DFCC-33 (1)w as,t hus,i dentified as At-DNCC-33 (2), the main Chl catabolite found in senescent leaves of A. thaliana. [6,8] Thed iscovery of an atural 1,19-dioxobilin-type FCC (DFCC), as well as its selective isomerization to the DNCC 2,r eported here,s upport the crucial role of such fleetingly existent fluorescent intermediates of Chl breakdown in ah igher plant, such as (wild-type) A. thaliana.I nterestingly, in recent work on Chl catabolites of senescent leaves of an A. thaliana mutant, amodified "fluorescent" 1,19-dioxobilintype Chl catabolite (an FDCC) was identified, which carried apuzzling "extra" hydroxymethyl group,afeature also found in some NDCCs from the mutant plant, [11] as well as in wildtype A. thaliana. [8] At-DFCC-33 (1)w as obtained here as asingle stereoisomer,and an epimer of 1 was not identified in the leaf extract. Based on this finding, the hypothetical stereoselective formation of 1 is indicated to take place through enzymatic,o xidative in vivo deformylation of an FCC precursor.T he "puzzling" hydroxymethylations that accompany the in vivo, [8,11] but not the in vitro, [6] deformylation of pFCC,a re not features of the natural pathway to DFCC 1.

Angewandte
Chemie formation of DFCC 1 from 3 2 -OH-pFCC may not occur through hydrolysis of 3 2 -OH-pFCC by MES16 to give 3 2 -OH-O8 4 -demethyl-pFCC (also known as At-FCC-1), [7b] followed by deformylation of At-FCC-1 by CYP89A9 to 1.Instead, two NCCs are found in A. thaliana leaves that are suggested to be downstream products from intact At-FCC-1. [7b] Under standard conditions of extract preparation and isolation, [11] At-DFCC-33 (1)c ould not be isolated in pure form, and solutions of 1 had to be kept cold (0 8 8Co rl ess) to minimize isomerization of 1 to 2.R apid conversion of the DFCC 1 into aD NCC was not unexpected, considering the known isomerization of FCCs to NCCs.S uch isomerizations were observed to be particularly fast in FCCs with af ree 8 2carboxylic acid function, [10c] which is also present in the DFCC 1.A so riginally delineated for "primary" FCCs, [10a,b] the propionic acid function at C12 has been proposed to induce the steroselective isomerization of 1 to 2,w hich, therefore, would be deduced to generate 2 with R configuration of the asymmetric methine group at C10. Indeed, the isomerization of 1 at pH 5produced the natural DNCC 2,whose chiroptical features fall in line with those of most DNCCs identified previously. [5a, 6, 11] Interestingly,this stereochemical outcome is contrary to the stereochemistry of the apparently "aberrant" case of the DNCC from Norway maple, [5b] which, thus,s till requires an alternative explanation.
With the advent of the characterization of At-DFCC-33 (1), an early "bona fide intermediate of the major dioxobilinbranch of Chl breakdown in A. thaliana is now identified. The DFCC 1 is generated as at ransient intermediate near the hypothetical branching point of the PaO/phyllobilin pathway at which the type-II phyllobilins diverge from the first formed 1-formyl-19-oxobilin-type Chl catabolites (or type-I phyllobilins). Identification of 1 corroborates the hypothetical role of DFCCs as natural, short-lived entry points to nonfluorescent type-II phyllobilins,such as the abundant DNCCs.The fleeting existence of At-DFCC-33 (1)a lso made it necessary to isolate 1 from leaves at an early stage of senescence.I n fully senescent, yellow A. thaliana leaves,f luorescent phyllobilins are hardly detectable.However, arange of colorless Chl catabolites (NCCs,D NCCs,a nd NDCCs) were identified as products further downstream of the two breakdown branches. [6][7][8] As deduced for the natural formation of NCCs from the corresponding FCCs, [10a,b] as lightly acidic medium, as provided in the vacuoles,isbeneficial for the rapid isomerization of DFCCs to the corresponding DNCCs.I ndeed, at pH 5 DFCC 1 undergoes rapid stereoselective isomerization to DNCC 2,thus suggesting DFCC 1 is the natural precursor of 2 in A. thaliana.S ince DNCC 2 represents,b yf ar, [6] the major fraction among the phyllobilins in senescent leaves of this plant (see Figure 2and Figure S1 in the Supporting Information), this,i nt urn, gives the transient DFCC 1 an important position in Chl breakdown in such senescent leaves.A ccording to the model of Chl breakdown in higher plants, [3c,d] import of the DFCC 1 into the vacuoles would be required to set the stage for the isomerization to the DNCC 2.
Thec ritical in vivo transition from 1-formyl-19-oxobilintype phyllobilins to 1,19-dioxobilin-type (or type-II) phyllobilins would, thus,m ostly occur by deformylation of 3 2 -OH-pFCC,a ne xcellent in vitro substrate for the FCC-deformylase CYP89A9 ( Figure 5). [6] Thed eformylation product 3 2 -OH-pDFCC (3), from which the DFCC 1 is presumably generated by the cytosolic methyl esterase MES16 has,sofar, remained unidentified in A. thaliana leaf extracts.D eformylation of the original pFCC also occurs on aminor additional path in senescent A. thaliana leaves. [8] It is deduced to give rise to elusive pDFCCs,and to "puzzling" hydroxymethylated iso-DFCCs as precursors of the corresponding group of remarkable NDCCs,w hich were only recently identified. [8,11] Hence,branching of the PaO/phyllobilin path towards type-II phyllobilins occurs in more than one case subsequent to formation of the colorless pFCC.T wo critical branching points from type-I to type-II phyllobilins have now been identified, consistent with the known low (in vitro) selectivity of the deformylase CYP89A9. [6] Hence,the findings reported here allow adeep glimpse into Chl breakdown in A. thaliana, which, while adhering to the PaO/phyllobilin pathway,t akes divergent roads at later stages ( Figure 5).
Breakdown of Chl in senescent A. thaliana leaves shows hallmarks of a" detoxification" process: [3a] it rapidly leads to av ariety of increasingly polar, colorless,a nd nonfluorescent catabolites,a mong which type-II phyllobilins dominate,s uch as the DNCC 2.T heir intriguing 1,19-dioxobilin-type structure is ac onstitutional feature shared with the heme-derived bilins. [12] This common structural property of type-II phyllobilins,and of the heme-derived "bile pigments", is remarkable in light of the diverse important biological roles of hemederived bilins. [12] Hence,inview of their unique chemistry,the ubiquitous phyllobilins [3d,4b] also qualify as candidates for relevant physiological roles in higher plants,a sw ell as, probably,i np lant-eating animals and humans. [13] However, ap hysiological effect of phyllobilins remains remarkably elusive.

Experimental Section
HPLC analysis:207 mg of fresh A. thaliana (wild-type) leaves,kept in the dark for 2days,w ere ground under N 2 ,e xtracted with 41 mL MeOH, and centrifugatedfor 10 min. Theclear supernatant (160 mL) was diluted 1:1with potassium phosphate buffer (pH 7). After further centrifugation at 13 000 rpm for 2min, a2 0mLa liquot was analyzed by HPLC (standard conditions; for details see the Supporting Information).