Isolation and Characterization of 1-Hydroxy-2,6,6-trimethyl-4-oxo-2-cyclohexene-1-acetic Acid, a Metabolite in Bacterial Transformation of Abscisic Acid

We report the discovery of a new abscisic acid (ABA) metabolite, found in the course of a mass spectrometric study of ABA metabolism by the rhizosphere bacterium Rhodococcus sp. P1Y. Analogue of (+)-ABA, enriched in tritium in the cyclohexene moiety, was fed in bacterial cells, and extracts containing radioactive metabolites were purified and analyzed to determine their structure. We obtained mass spectral fragmentation patterns and nuclear magnetic resonance spectra of a new metabolite of ABA identified as 1-hydroxy-2,6,6-trimethyl-4-oxo-2-cyclohexene-1-acetic acid, which we named rhodococcal acid (RA) and characterized using several other techniques. This metabolite is the second bacterial ABA degradation product in addition to dehydrovomifoliol that we described earlier. Taken together, these data reveal an unknown ABA catabolic pathway that begins with side chain disassembly, as opposed to the conversion of the cyclohexene moiety in plants. The role of ABA-utilizing bacteria in interactions with other microorganisms and plants is also discussed.


Introduction
Abscisic acid (ABA) is a versatile isoprenoid signaling molecule produced by a wide range of organisms. It is best studied as an important phytohormone regulating plant growth, development and stress response [1]. The main processes controlled by ABA in plants are the acceleration of abscission, induction of dormancy, inhibition of rooting, and stimulation of stomatal closure [2][3][4]. Significant amounts of ABA and the products of its catabolism are constantly introduced into soil via root exudation, decomposition of abscised shoot tissues and root turnover. It has been shown that ABA transporters located in root sis [22]. Meanwhile, catabolism is also very important for ABA homeostasis. In plants, it is known that ABA can be catabolized to phaseic acid via CYP707A or inactivated by glucose conjugation (ABA-glucose ester) via the enzyme uridine diphosphate-glucosyltransferase (UDP-glucosyltransferase) [44]. However, a complete chemical degradation of ABA still awaits development.
Recently, using a selective ABA-supplemented medium, two bacterial strains were isolated from the rhizosphere of rice (Oryza sativa L.) and assigned to Rhodococcus sp. P1Y and Novosphingobium sp. P6W [45]. Both strains could utilize ABA as a sole carbon source in batch culture and decrease ABA concentrations in tomato roots or leaves upon plant inoculation. We have also shown that Rhodococcus sp. P1Y is able to shorten the acyl moiety of the ABA molecule to form dehydrovomifoliol [46]. This work is devoted to a further study of ABA catabolism in this bacterial species and presents a new bacterial metabolite.

ABA and Its Tritium Labeled Analog
(+)-ABA was purchased from Merck (Sigma-Aldrich, St. Louis, MO, USA). Tritium was introduced into the cyclohexene part of the molecule as described earlier [47]. The radiochemical purity of the final product was 98.5% and the specific radioactivity was 30.5 Ci mmol −1 .

Sample Extraction and Purification
The lyophilized material was extracted with 150 mL of methanol using an ultrasonic bath. The insoluble residue was collected by centrifugation and extracted with 150 mL of methanol again. The re-extraction procedure was performed twice. The combined extract was evaporated on a rotary evaporator at 40 • C. The resulting residue weighing 507.4 mg was dissolved in 150 mL of 0.1 M NaHCO 3 and extracted three times in a separation funnel with 100 mL portions of methylene chloride. The aqueous solution was acidified carefully with acetic acid to pH 4-5 and extracted with ethyl acetate three times with 100 mL portions. The combined ethyl acetate solution was dried over Na 2 SO 4 and evaporated to dryness on a vacuum rotary evaporator Heidolph Hei-VAP Precision (Heidolph Instruments GMBH and CO KG, Schwabach, Germany) at 40 • C. As a result, 461.3 mg of dry yellow residue was obtained.
Preparative chromatographic separation of the residue suspected of containing ABA metabolites was carried out on silica gel (Merck 60) using a Buchi Sepacore MPLC system equipped with a C-630 UV monitor, two C-605 pump modules, an S-620 control unit, and a C-660 fraction collector (Buchi, Flawil, Switzerland). The solvents were (A) n-hexane, (B) EtOAc, and (C) MeOH. The dry residue was dissolved in 50 mL of methanol and loaded onto 5 mL of silica gel, blowing off the methanol in a stream of nitrogen. The resulting material was introduced in hexane into a cartridge, which was connected in series with a Glass Column Buchi 15/460 (81.3 mL) filled with the same silica gel and preconditioned in  Table 1. The eluent flow rate was 15 mL min −1 . Fractions of 45 mL were collected and analyzed by liquid scintillation counting using a QuantaSmart Tri-Carb 2810TR instrument (PerkinElmer, Inc., Waltham, MA, USA). The obtained MPLC fractions were analyzed for their component composition by HPLC using an Acquity class H chromatograph with a PDA detector and a Waters AC-QUITY UPLC BEH C18 column (50 × 2.1 mm, 1.7 µm). Chromatography was performed at 30 • C. The injection volume was 5 µL. The flow rate was 0.3 mL min −1 . The mobile phases were water with 0.1% formic acid (A) and acetonitrile with 0.1% formic acid (B). The gradient was as follows: 0-1 min 0% B, 1-13 min 0% → 90% B; 13-14 min, 90% B; and 14-14.1 min, 90% → 0% B. The detection wavelengths were 265 and 240 nm. Fractions 4-8 contained two compounds with retention times corresponding to ABA and dehydrovomifoliol. Fractions 12-14 contained one unknown compound as the main component with a total content of more than 96%. Fractions 12-14 were combined and evaporated to dryness. Dry residue was dissolved in methanol, recrystallized, and analyzed by HPLC. A total of 155.9 mg (yield 30.7%) of a white crystalline substance was obtained with a chromatographic purity of more than 98%. Identification of the chemical structure of the new ABA metabolite was performed using NMR spectroscopy in combination with mass spectrometry, FTIR spectroscopy, spectrophotometry, and polarimetry.

HPLC-MS Analysis
The substance dissolved in methanol was analyzed by HPLC-MS using a IT-TOF mass spectrometer (Shimadzu Corporation, Kyoto, Japan) equipped with an ESI interface. A Waters BEH-C18 column (50 × 3.0 mm, 3.5 µm, Waters Corporation, Milford, MA, USA) was used. Chromatography was carried out at a temperature +15 • C and a flow rate of 0.3 mL/min. The mobile phases were 0.1% formic acid (A) and acetonitrile +0.1% formic acid (B). The gradient was as follows: 0-1 min 0% B, 1-13 min 0% → 90% B; 13-14 min, 90% B; and 14-14.1 min, 90% → 0% B. The IT-TOF/MS analysis was carried out in full-scan mode, and the mass range was m/z 60−2000 in the negative and positive modes for MS and MS/MS, with a scan rate 2 spectra/sec. The operating parameters of the electrospray ionization sources were as follows: nebulizing gaz (N 2 ) flow rate, 1.5 L/min; drying gas pressure, 100 kPa; CDL temperature, 200 • C; heat block temperature, 200 • C; probe voltage, 3.5 kV for positive mode and -2.5 kV for negative mode; ion accumulation time, 30 ms; collision energy of collision-induced dissociation (CID), 10% for negative mode and Biomolecules 2022, 12, 1508 5 of 20 7% for positive mode; collision gas (Ar), 10% for negative and positive mode; detector voltage, 1.6 kV. All the acquisitions and analyses of data were controlled by LabSolutions LCMSSolution software (release 3.80, Shimadzu Corporation). A standard solution of sodium trifluoroacetic acid (TFA) was used to calibrate the TOF-MS to increase mass accuracy. Parallel UV detection was performed using an SPD-M20A Multiple Wavelength Detector instrument (Shimadzu Corporation).

Spectrophotometry
An ultraviolet/visible (UV/Vis) spectrum was recorded in MeOH on a Beckman Coulter DU 800 spectrometer (Beckman Coulter Inc., Brea, CA, USA). The survey was carried out in a quartz cuvette with an optical path length of 1 cm. Survey parameters: measurement range 200-800 nm, resolution 0.1 nm, scanning speed 120 nm/min. The concentration of the compound was 0.01 mol/L.

Polarimetry
Optical rotation was determined using an automatic polarimeter SAC-i (ATAGO Co., Ltd., Tokyo, Japan) for a monochromatic laser beam with λ = 589 nm. The cell length (optical path) was 50 mm. For measurement, a solution of the substance in water with a concentration of 20 mg/mL was used. The measurement was carried out at room temperature (22.3 • C). The resulting value is the average of 20 measurements.

Melting Point Measurement
The melting point was determined on a PTP-M instrument (Khimlabpribor, Ltd., Klin, Russia) according to the manufacturer's manual.

FTIR Spectroscopic Measurements
For FTIR spectroscopic measurements, a sample of the isolated and purified compound (~1 mg) was dissolved in a minimal volume (~10 µL) of MilliQ water, placed (using a microsyringe) as a thin film on a clean flat ZnSe disk (CVD-ZnSe, "R'AIN Optics", Dzerzhinsk, Russia; ø 1 cm, thickness 0.2 cm) and dried in a drying cabinet at 45 • C (~20 h). Transmission FTIR spectroscopic measurements were performed on a Nicolet 6700 FTIR spectrometer (Thermo Electron Corporation, Beverly, MA, USA; DTGS detector; KBr beam splitter) as reported earlier [46] (with a total of 64 scans (resolution 4 cm −1 ) against the ZnSe disk background; spectra were manipulated using the OMNIC software (version 8.2.0.387) supplied by the manufacturer of the spectrometer). The baseline was corrected using the "automatic baseline correct" function, and the spectra were smoothed using the standard "automatic smooth" function of the software which uses the Savitsky-Golay algorithm (95-point moving second-degree polynomial). The FTIR spectroscopic measurements were repeated three times and were well reproducible.

NMR Spectroscopic Measurements and Experimental Conditions
One-dimensional ( 1 H, 13 C) and two-dimensional (HSQC, HMBC, H2BC) NMR spectra were recorded on a DirectDrive NMR System (Varian, Palo Aho, CA, USA) in D 2 O at 700 and/or 175.8 MHz. The residual signals of H 2 O (for 1 H NMR), as well as of CH 3 OH (for 13 C NMR) which was introduced as a micro-impurity in one of the experiments, were used as internal standards.

Isolation of Metabolite 2
Previously, we reported that the growth of Rhodococcus sp. P1Y on the minimal medium with ABA was accompanied by the degradation of this phytohormone and the accumulation of additional metabolites in the culture liquid. HPLC-MS revealed two putative metabolites, one of which was purified and identified as dehydrovomifoliol [46]. In the present work, we increased the cultivation time after the addition of ABA from 2 Biomolecules 2022, 12, 1508 6 of 20 to 4 h, which increased the accumulation of the second compound ( Figure 1, compound with a retention time (RT) of 4.8 min) in the culture liquid with a simultaneous decrease in the content of dehydrovomifoliol and ABA ( Figure 1, compounds with RT of 5.4 and 6.3 min, respectively). Using the procedures described in Section 2.3 allowed us to purify Compound 2 to an individual form (over 98 percent, Figure 2).
Biomolecules 2022, 12, x 6 of 20 putative metabolites, one of which was purified and identified as dehydrovomifoliol [46]. In the present work, we increased the cultivation time after the addition of ABA from 2 to 4 h, which increased the accumulation of the second compound ( Figure 1, compound with a retention time (RT) of 4.8 min) in the culture liquid with a simultaneous decrease in the content of dehydrovomifoliol and ABA ( Figure 1, compounds with RT of 5.4 and 6.3 min, respectively). Using the procedures described in Section 2.3 allowed us to purify Compound 2 to an individual form (over 98 percent, Figure 2).  Biomolecules 2022, 12, x 6 of 20 putative metabolites, one of which was purified and identified as dehydrovomifoliol [46]. In the present work, we increased the cultivation time after the addition of ABA from 2 to 4 h, which increased the accumulation of the second compound ( Figure 1, compound with a retention time (RT) of 4.8 min) in the culture liquid with a simultaneous decrease in the content of dehydrovomifoliol and ABA ( Figure 1, compounds with RT of 5.4 and 6.3 min, respectively). Using the procedures described in Section 2.3 allowed us to purify Compound 2 to an individual form (over 98 percent, Figure 2). The monoisotopic molecular weight of this compound, according to the results of mass spectrometry (see Section 3.2), was 212.1049 Da. This corresponded to the molecular formula C11H16O4. Bacteria were cultivated using radioactive ABA with a calculated specific radioactivity of 10.57 μCi mmol −1 (10 μCi per 250 mg of ABA with a molecular weight of 264.32 Da). The individual compound had a specific radioactivity of 9.12 μCi mmol −1 (9.55 × 10 4 cpm m −1 ). It should be noted that some discrepancy between the values may be due to the fact that the accumulation of bacterial biomass was carried out in preliminary cultivation using non-radioactive ABA. Probably, a slight overestimation of the specific radioactivity of the starting material can be caused by residual amounts of ABA in the culture medium before the addition of the radioactive preparation. Thus, the initial substrate and the purified compound have a comparable content of the radioactive isotope. This may serve as evidence that the isolated compound is an ABA derivative. Further, this substance was designated "Metabolite 2". The use of a complex of physicochemical methods and instrumental techniques described below made it possible to identify Metabolite 2 as 1-hydroxy-2,6,6-trimethyl-4-oxo-2-cyclohexene-1-acetic acid.

Mass Spectrometric Studies
The high resolution mass spectra of Metabolite 2 obtained in the negative and positive electrospray modes are shown in Figure 3. The interpretation of these spectra gives insight into the structure of the metabolite. The generation of a stable negatively charged molecular ion 211.0960 (calculated 211.0970) under experimental conditions indicates the acidity of the compound. The 167.1062 (calculated 167.1072) ion associated with the elimination of the neutral CO 2 molecule may indicate the presence of a carboxyl group in its molecular structure. The same assumption indirectly confirms the elimination of the CH 3 COOH molecule. The splitting off of a water molecule from a molecular ion obtained in the positive ionization mode suggests the presence of a hydroxyl group in the compound under study.

Mass Spectrometric Studies
The high resolution mass spectra of Metabolite 2 obtained in the negative and positive electrospray modes are shown in Figure 3. The interpretation of these spectra gives insight into the structure of the metabolite. The generation of a stable negatively charged molecular ion 211.  The most probable molecular formula for the 211.0960 ion is C 11 H 15 O 4 , for the 213.1133 ion it is C 11 H 17 O 4 . Along with the structure and known pathways of fragmentation of the initial ABA molecule, this allows us to suggest the most probable structure of the metabolite and the fragmentation scheme of its molecular ions, which are shown in Figure 4.  The most probable molecular formula for the 211.0960 ion is C11H15O4, for the 213.1133 ion it is C11H17O4. Along with the structure and known pathways of fragmentation of the initial ABA molecule, this allows us to suggest the most probable structure of the metabolite and the fragmentation scheme of its molecular ions, which are shown in Figure 4.

NMR Spectroscopy
To confirm the presented hypothetical structure of the Metabolite 2 molecule, we performed one-dimensional ( 1 H, 13 C) and two-dimensional ( 13 C-1 H HSQC, 13 C-1 H HMBC, H2BC) NMR experiments. The results are shown in Figures 5-8, respectively. The original NMR spectral characteristics of Metabolite 2 are presented in Table 2.

NMR Spectroscopy
To confirm the presented hypothetical structure of the Metabolite 2 molecule, we performed one-dimensional ( 1 H, 13 C) and two-dimensional ( 13 C-1 H HSQC, 13 C-1 H HMBC, H2BC) NMR experiments. The results are shown in Figures 5-8, respectively. The original NMR spectral characteristics of Metabolite 2 are presented in Table 2.
The HSQC spectrum made it possible to establish the binding of carbon atoms to the corresponding protons, as well as to determine the nature of atomic groups. Information about the carbon skeleton of the molecule was obtained from the two-dimensional spectra of long-range spin-spin interaction. The proton spin-spin coupling constants were extracted from the 1 H spectrum. The absence of the proton peak of the tertiary hydroxyl group in the 1 H NMR spectrum can be explained by the presence of a stable intramolecular hydrogen bond with the oxygen of the carboxyl group of the side chain (see the FTIR spectroscopic data). The long-range 13 C-1 H interaction constants are schematically shown in Figure 9. The great general similarity of the NMR spectra of Metabolite 2 with those for abscisic acid and dehydrovomifoliol should be emphasized, especially for the cyclic part of the molecule [46], which confirms the common chemical nature of all the three compounds. In fact, all spectra of Metabolite 2 are a reduced version of the spectra of ABA and dehydrovomifoliol, which, apparently, may indicate the origin of this compound from more complex precursors. Summarizing the presented results, we can conclude that, according to the spectral data obtained both in NMR experiments and by mass spectrometry and optical spectrometry, the cyclic part of the molecule in Metabolite 2 is similar to that of original ABA. The structure of the side chain of a molecule can be established by considering its NMR spectra as a whole. The presence of a cross peak in the HMBC spectrum between the carbon atom of the carboxyl group (δ = 183.0) and two protons of the CH 2 group (δ = 2.32, 2.53) indicates the presence of bonding between carbon atoms with δ = 183.0 and δ = 42.4. Similarly, the interaction of a quaternary carbon atom at δ = 79.9 with the above protons indicates the presence of an interaction between carbon atoms with δ = 79.9 and δ = 42.4. Thus, the presented data, in our opinion, are sufficient to establish the structure of the side chain and the position of its attachment to the cyclic part of the molecule.            The HSQC spectrum made it possible to establish the binding of carbon atom corresponding protons, as well as to determine the nature of atomic groups. Info about the carbon skeleton of the molecule was obtained from the two-dimensional of long-range spin-spin interaction. The proton spin-spin coupling constants w tracted from the 1 H spectrum. The absence of the proton peak of the tertiary h group in the 1 H NMR spectrum can be explained by the presence of a stable intram lar hydrogen bond with the oxygen of the carboxyl group of the side chain (see t spectroscopic data). The long-range 13 C-1 H interaction constants are schematically in Figure 9. The great general similarity of the NMR spectra of Metabolite 2 with t abscisic acid and dehydrovomifoliol should be emphasized, especially for the cyc of the molecule [46], which confirms the common chemical nature of all the thr pounds. In fact, all spectra of Metabolite 2 are a reduced version of the spectra of A dehydrovomifoliol, which, apparently, may indicate the origin of this compoun more complex precursors. Summarizing the presented results, we can conclude cording to the spectral data obtained both in NMR experiments and by mass spectr and optical spectrometry, the cyclic part of the molecule in Metabolite 2 is simila of original ABA. The structure of the side chain of a molecule can be established sidering its NMR spectra as a whole. The presence of a cross peak in the HMBC sp between the carbon atom of the carboxyl group (δ = 183.0) and two protons of group (δ = 2.32, 2.53) indicates the presence of bonding between carbon atoms w 183.0 and δ = 42.4. Similarly, the interaction of a quaternary carbon atom at δ = 7 the above protons indicates the presence of an interaction between carbon atoms 79.9 and δ = 42.4. Thus, the presented data, in our opinion, are sufficient to estab structure of the side chain and the position of its attachment to the cyclic part of t ecule. The results obtained allowed us to establish the structure of Metabolite 2 as 1-hydroxy-2,6,6-trimethyl-4-oxo-2-cyclohexene-1-acetic acid ( Figure 10). To the best of our knowledge, this substance has not been previously described. This compound was given the trivial name rhodococcal acid (RA). Additional information about the structure of this compound was obtained by optical spectroscopy.
starts at a carbon atom and ends at a proton. The dotted line denotes weak interactions.
The results obtained allowed us to establish the structure of Metabolite 2 as 1-hydroxy-2,6,6-trimethyl-4-oxo-2-cyclohexene-1-acetic acid (Figure 10). To the best of our knowledge, this substance has not been previously described. This compound was given the trivial name rhodococcal acid (RA). Additional information about the structure of this compound was obtained by optical spectroscopy. The hydroxyl configuration at C1′ is preliminarily based on the studies discussed in Section 3.6.

UV-Visible Absorption Spectrum of Metabolite 2
The UV-visible spectrum of a 0.01 M methanolic solution of the ABA metabolite (Figure 11) shows a single band with an absorption wavelength λmax = 240.6 nm.

UV-Visible Absorption Spectrum of Metabolite 2
The UV-visible spectrum of a 0.01 M methanolic solution of the ABA metabolite ( Figure 11) shows a single band with an absorption wavelength λ max = 240.6 nm. Figure 9. Scheme of long-range spin-spin interactions 13 C-1 H in the Metabolite 2 molecule, constructed basing on the analysis of two-dimensional spectra of HSQC, HMBC, and H2BC. Each arrow starts at a carbon atom and ends at a proton. The dotted line denotes weak interactions.
The results obtained allowed us to establish the structure of Metabolite 2 as 1-hydroxy-2,6,6-trimethyl-4-oxo-2-cyclohexene-1-acetic acid (Figure 10). To the best of our knowledge, this substance has not been previously described. This compound was given the trivial name rhodococcal acid (RA). Additional information about the structure of this compound was obtained by optical spectroscopy. The hydroxyl configuration at C1′ is preliminarily based on the studies discussed in Section 3.6.

UV-Visible Absorption Spectrum of Metabolite 2
The UV-visible spectrum of a 0.01 M methanolic solution of the ABA metabolite (Figure 11) shows a single band with an absorption wavelength λmax = 240.6 nm. The obtained value correlates well with the wavelength calculated according to Woodward's empirical rules [48] for π→π* transitions in conjugated enone systems ( Figure 12): λ max (calculated) = λ 0 + 2 × ∆λalk β = 215 + 2 × 12 = 239 nm. The base value λ 0 = 215 nm was taken to be the absorption maximum for cyclic enones in six-carbon cycles. It should be noted that dehydrovomifoliol, which is also a degradation product of abscisic acid, has a similar spectrum with λ max = 239 nm [46].
The obtained value correlates well with the wavelength calculated according to Woodward's empirical rules [48] for π→π* transitions in conjugated enone systems (Figure 12): λmax (calculated) = λ0 + 2 × Δλalkβ = 215 + 2 × 12 = 239 nm. The base value λ0 = 215 nm was taken to be the absorption maximum for cyclic enones in six-carbon cycles. It should be noted that dehydrovomifoliol, which is also a degradation product of abscisic acid, has a similar spectrum with λmax = 239 nm [46].

FTIR Spectroscopic Characterization
The FTIR spectrum of the isolated and purified title compound (measured as a dry thin film of the pure substance, without a solvent or a matrix) presented in Figure 13 clearly features all the functional groups defined in its structure (see Figure 10). Assignments of the main characteristic bands observed in the FTIR spectrum of the title compound are given in Table 3. Figure 13. A FTIR spectrum of the isolated and purified title compound (measured in the transmission mode). Table 3. Assignments of the main characteristic bands in the FTIR spectrum of the isolated and purified title compound [46,49].

FTIR Spectroscopic Characterization
The FTIR spectrum of the isolated and purified title compound (measured as a dry thin film of the pure substance, without a solvent or a matrix) presented in Figure 13 clearly features all the functional groups defined in its structure (see Figure 10). Assignments of the main characteristic bands observed in the FTIR spectrum of the title compound are given in Table 3.
The obtained value correlates well with the wavelength calculated according to Woodward's empirical rules [48] for π→π* transitions in conjugated enone systems (Figure 12): λmax (calculated) = λ0 + 2 × Δλalkβ = 215 + 2 × 12 = 239 nm. The base value λ0 = 215 nm was taken to be the absorption maximum for cyclic enones in six-carbon cycles. It should be noted that dehydrovomifoliol, which is also a degradation product of abscisic acid, has a similar spectrum with λmax = 239 nm [46].

FTIR Spectroscopic Characterization
The FTIR spectrum of the isolated and purified title compound (measured as a dry thin film of the pure substance, without a solvent or a matrix) presented in Figure 13 clearly features all the functional groups defined in its structure (see Figure 10). Assignments of the main characteristic bands observed in the FTIR spectrum of the title compound are given in Table 3. Figure 13. A FTIR spectrum of the isolated and purified title compound (measured in the transmission mode). Table 3. Assignments of the main characteristic bands in the FTIR spectrum of the isolated and purified title compound [46,49]. The very broad ν(OH) region evidently contains two poorly resolved broadened maxima corresponding both to the tertiary alcoholic hydroxo group (at C1 ; see Figure 10) at~3500-3400 cm −1 (which was observed for dehydrovomifoliol, with a similar structure but lacking the carboxylic group, at 3448 cm −1 [46]) and to the carboxylic OH-group (with a maximum at 3201 cm −1 ). It is natural that in the condensed phase both of these moieties are involved in relatively strong intermolecular H-bonding (see the description of the 1 H NMR data presented above).
The ν(C-H) region comprises a number of bands (see Table 3) typical of methyl and methylene groups; note also the weak ν(=C-H) band (for the group at the C3 position) at 3028 cm −1 and the slight difference in the frequencies of the ν as (CH 2 ) bands for the side chain (position C2) and in the cycle (position C5 ) neighboring the carbonylic group at C4 . The latter, considering its relatively low frequency (1707 cm −1 ) as compared to that in dehydrovomifoliol (1736 cm −1 [46]), is also involved in the intermolecular H-bonding. The positions of the ν(C=C) band (1658 cm −1 ), stretching vibrations of the carboxylic group and of a number of bending modes (see Table 3) are characteristic; the lower-wavenumber region of the spectrum in Figure 13 contains a number of weak bending and stretching bands of the C-C/C-O skeleton which are less specific.
Thus, the main set of characteristic bands listed in Table 3 (see Figure 13) can be useful for the identification of this substance by spectroscopic techniques including FTIR spectroscopy. Table 3. Assignments of the main characteristic bands in the FTIR spectrum of the isolated and purified title compound [46,49].

Polarimetry
An aqueous solution of Metabolite 2 showed the presence of optical rotation: [∝] 20 D = +33.6 • (∆ = 0.6 • ). This clearly indicates the absence of racemization during the biodegradation of the side chain of the ABA molecule. The fact that the isolated substance is not racemic indicates the enzymatic nature of the observed conversion of ABA. Probably, the position of the hydroxyl group at carbon atom 1 of RA coincides with that for ABA, as shown in Figure 10.

Melting Point Measurement
Melting of Metabolite 2 is accompanied by its decomposition. At a temperature of 118-120 • C, a gaseous product is released and the substance passes into a liquid state. Further heating of the substance leads to its evaporation in the temperature range of 220-225 • C. Mass-spectrometric analysis of the compound after the decomposition process by GC-MS allowed it to be identified as 4-oxoisophorone. Thus, the process of decomposition of Metabolite 2 is apparently associated with the elimination of an acetic acid molecule, as shown in Figure 14. of Metabolite 2 is apparently associated with the elimination of an acetic acid molecule, as shown in Figure 14.

Discussion
ABA is well studied as a phytohormone [1,15]. However, the discovery of this compound in a wide range of organisms, including bacteria, algae, fungi, sea sponges and mammals, determined the expansion of the scope of research into the metabolism and functions of ABA [22,37,50]. At the same time, most reports on the chemistry of ABA deal

Discussion
ABA is well studied as a phytohormone [1,15]. However, the discovery of this compound in a wide range of organisms, including bacteria, algae, fungi, sea sponges and mammals, determined the expansion of the scope of research into the metabolism and functions of ABA [22,37,50]. At the same time, most reports on the chemistry of ABA deal with its synthesis [51]. As a result of these studies, it was shown that plants and fungi have evolved different synthesis pathways using carotenoids (either farnesyl diphosphate or farnesyl pyrophosphate) as precursors [52]. Different prokaryotic species probably use independently developed pathways for ABA biosynthesis, carotenoid-dependent and carotenoid-independent [22]. As regards ABA catabolism, it is known that, in addition to the formation of conjugates, mainly with glucose, plants are limited to the modification of the cyclohexene part of the molecule [51,53,54], while no pathways for complete degradation of the carbon skeleton have been found [55]. Another aspect of the problem is that, as discussed in Section 2, the plant moisture monitoring strategies and plant-microbe communication may involve secretion of ABA into the soil via root exudation [6,36]. In addition, ABA is constantly introduced into the soil with dead tissues and falling leaves and accumulates as a result of production by bacteria, fungi, and algae [6]. Despite the significant biological activity of exogenous ABA [2,11,12], plants do not have the ability to control the soil concentration of this phytohormone or otherwise reduce its activity in the environment. It is likely that in the course of evolution, plants delegated this function to the microorganisms associated with them. However, information on the mechanisms of microbial ABA catabolism is very limited [31,37]. It was reported that the introduction of radioactive ABA into non-sterile soil led to the decomposition of this compound to phaseic acid and dehydrophaseic acid [6]. It was also shown that Corynebacterium sp., growing on an ABA-supplemented medium, accumulated a compound with spectral characteristics close to those for dehydrovomifoliol [56]. We have recently isolated this substance from a culture of Rhodococcus sp. P1Y in vitro and characterized it in detail as a metabolite of ABA [46]. In the present study, it was shown that Rhodococcus sp. P1Y carries out further destruction of ABA, shortening the side chain by two more carbon units. Thus, the first steps of ABA microbial catabolism differ from the known transformations of this phytohormone in plants. This is probably due to the fact that it is important for plants to modify the signaling properties of ABA, while for bacteria this compound is a nutrient source of carbon, for which the use of C 2 units of the side chain is preferable. The results obtained allowed us to suggest a catabolic pathway for the utilization of the ABA side chain ( Figure 15). It can be assumed that in each stage of this catabolic pathway, one molecule of acetate is formed, which is used for the metabolism of bacteria. We believe that to date we have only been able to isolate the initial stages of this catabolic pathway. At present, the question on the mechanisms of the presented initial stages of ABA degradation and the enzymes involved in them has yet to be developed. The transcriptome data showed that in Rhodococcus sp. P1Y (data not published) and Novosphingobium sp. P6W [57], the growth of bacteria on a medium with ABA increases the level of fatty acid metabolism, the enzymes of which can be involved in ABA utilization. In this regard, it should be noted that the branch point at the C3 position of ABA may interfere with normal β-oxidation. However, it was previously shown that Rhodococcus ruber [58] and Nocardia cyriacigeorgica [59] can overcome similar branching points of aliphatic and aromatic hydrocarbons, in particular, they can shorten β-methylcinnamic acid by cleaving the C2-unit to form acetophenone. Probably, Rhodococcus sp. P1Y can implement similar mechanisms in relation to ABA during the formation of dehydrovomifoliol. The formation of RA from dehydrovomifoliol can occur as a result of subterminal oxidation with the At present, the question on the mechanisms of the presented initial stages of ABA degradation and the enzymes involved in them has yet to be developed. The transcriptome data showed that in Rhodococcus sp. P1Y (data not published) and Novosphingobium sp. P6W [57], the growth of bacteria on a medium with ABA increases the level of fatty acid metabolism, the enzymes of which can be involved in ABA utilization. In this regard, it should be noted that the branch point at the C3 position of ABA may interfere with normal β-oxidation. However, it was previously shown that Rhodococcus ruber [58] and Nocardia cyriacigeorgica [59] can overcome similar branching points of aliphatic and aromatic hydrocarbons, in particular, they can shorten β-methylcinnamic acid by cleaving the C2-unit to form acetophenone. Probably, Rhodococcus sp. P1Y can implement similar mechanisms in relation to ABA during the formation of dehydrovomifoliol. The formation of RA from dehydrovomifoliol can occur as a result of subterminal oxidation with the participation of Baeyer-Villiger monooxygenase, accompanied by the accumulation of acetate [60]. However, additional studies are required to accurately establish the mechanisms of ABA destruction by soil bacteria. Another limitation of this study is that we were unable to determine the exact stereochemical configuration of the new compound. The configuration shown in Figure 10 is based on indirect information, and an unambiguous conclusion could be drawn from the results of future crystallographic studies.
Regardless of the results of biochemical studies, there are reasons to believe that ABAutilizing bacteria perform an important ecological function of maintaining soil hormonal homeostasis, normalizing the regulation of plant growth, development, and fitness. Since the production of ABA is a virulence factor for many phytopathogens, in particular, phytopathogenic fungi [32], it may be speculated that ABA-degrading bacteria can possess a biocontrol trait, which makes them a promising object for application in agriculture. It is possible that in other ecological niches, such as the digestive tract of phytophages, microbial degradation of ABA may affect interspecies interactions. Thus, a new pathway for ABA biodegradation may become another interesting aspect of the study of the microbiome. The biological activity of the new isolated compound also needs to be studied.

Conclusions
In a culture of the rhizospheric bacterium Rhodococcus sp. P1Y grown in the presence of radioactive ABA, a new metabolite of this phytohormone was found. This metabolite has been identified as a previously unknown compound, 1-hydroxy-2,6,6-trimethyl-4-oxo-2-cyclohexene-1-acetic acid, for which the trivial name rhodococcal acid has been proposed. Basing on the structural formula, rhodococcal acid is a product of a new metabolic pathway of ABA degradation. We have described the beginning of this pathway, which consists in the sequential cleavage of the side chain of the ABA molecule with the formation of dehydrovomifoliol and rhodococcal acid. Further studies can be aimed at searching for and identifying the subsequent products of this pathway, elucidating the chemical reactions and enzymes involved in their catalysis, characterizing the biological properties of the new compound, and the place of ABA-degrading bacteria in interspecies interactions.