Degradation of trifluoroacetophenone by the newly isolated Gordonia sp. strain SH2

RESULTS AND DISCUSSION

Isolation and characterization of strain SH2. Strain SH2 was isolated from a mix of soil samples collected from a plant nursery and a golf course situated in the North of The Netherlands. Enrichment with TFAP as a sole source of carbon and energy resulted in the isolation of a single bacterial culture capable of growing on TFAP. The gram-positive isolate formed orange colonies on LB medium. Sequencing of the PCR amplified 16S rRNA gene yielded a sequence of 1,483 bases that had 98% sequence identity with the 16S rRNA gene of Gordonia bronchialis. It suggests that our newly isolated strain belongs to genus Gordonia.

This strain is designated as Gordonia sp. strain SH2.

The genus Gordonia belongs phylogenetically to the suborder Corynebacterineae within the order Actinomycetales (35). The colors of the Gordonia species cover a broad range, including white, yellow, tannish, orange, red, and pink (39). In recent years, the genus Gordonia has attracted much interest for its potential use in industrial and environmental biotechnology, because of their capacity to degrade substituted and nonsubstituted hydrocarbons, environmental chemicals, other xenobiotics, and natural compounds that are not readily biodegradable (3, 18). Many species of Gordonia have been isolated and characterized that are able to degrade toxic environmental pollutants, such as benzothiophene

and dibenzothiophene by G. desulfuricans and G. amicalis (25), hydrocarbons by G. westfalica (29), G. paraffinivorans and G. alkanivorans (27, 39), and phenol by G. kroppenstedtii (24). Cholesterol degradation has been described for G. cholesterolivorans

(16).

60 80 100 120 140 160 180 0

1 00

50

Abundance (%)

m /z

176 107

79

6269 5 1

6 0 8 0 1 0 0 12 0 14 0 16 0 1 80 1 00

5 0

0

Abundance (%)

m /z 10 5

5 1 77

6 9 1 7 4

6 2

FIG. 2. Biotransformation of TFAP by Gordonia sp. strain SH2. Time series incubations were carried out and samples were taken at the beginning and at time intervals, extracted with ethyl acetate containing dodecane as internal standard (IS), and analyzed by GC and GC/MS.

Degradation of TFAP by whole cells. In order to study the pathway of TFAP, we incubated whole cells of strain SH2 with substrate and analyzed the products formed by GC and GC/MS. Whole cells of the strain SH2 were grown in MMY medium with TFAP, washed with MMY medium and resuspended in 2 ml MMY medium to an OD450 of 2.5. Cells transformed 2 mM TFAP in the presence of 5 mM glucose in MMY medium and produced a low amount of α,α,α-trifluoroacetophenyl alcohol (TFAPA) due to reduction by an alcohol dehydrogenase (ADH) and eluted from the GC at 8.25 min (Fig. 2). TFAPA remained

undegraded and accumulated in the culture medium. The much larger decrease of the concentration of TFAP showed that most TFAP was degraded, with some concomitant formation of trifluorophenyl acetate (TFPA), which may lead to the formation of phenol with the release of trifluoroacetate (TFA). We could not identify trifluoroacetate (TFA) through ion chromatography may be due to the overlapping of TFA with sulfate and phosphate peaks.

A similar result has been reported when the whole cells of fungal species Epicoccum nigrum SSP 1498 and Emericella nidulans CCT 3119 formed two intermediates, an alcohol and an ester, in the first step of the transformation of acetophenone derivatives (2). Our results showed that the strain employed in the biotransformation of TFAP produces enzymes that catalyze bioreduction and Baeyer-Villiger oxidation of aromatic ketones.

Activities in cell-free extract. To detect enzymes that catalyze transformation of TFAP, we used enzyme assays with cell-free extract of cultures induced with TFAP.

Incubation of cell-free extract with TFAP and NADPH resulted in transformation of the substrate. This transformation was not observed without NADPH. Overnight incubation of cell-free extract with 1 mM TFAP and 1 mM NADPH resulted in the formation of α,α,α−trifluoroacetophenyl alcohol. Some phenol was also formed. The formation of phenol most likely involves the combined action of a Baeyer-Villiger monooxygenase and an esterase. Since α,α,α−trifluorophenyl acetate was not observed in the GC-MS chromatograms (Fig. 3A), we assume that the esterase activity was so high that it rapidly transformed the ester into phenol with release of trifluoroacetate. Unfortunately, we could not separate TFA from phosphate and other compounds of the cell-free extract to directly measure it.

The cell-free extract of strain SH2 also transformed phenylacetone with formation of benzyl acetate, which then hydrolyzed to form benzyl alcohol (Fig. 3B) (13). Hence, identification of degradation intermediates and products confirmed the induction of a BVMO in cells of strain SH2 during growth on TFAP.

Substrate specificity of the BVMO. In 100 mM potassium phosphate buffer (pH 7.4) with NADPH, cell-free extract of strain SH2 (from induced cells) transformed a range of acetophenone derivatives into their corresponding phenols and alcohols, indicating the BVMO activity (Table 1). Cell-free extract of the organism failed to transform progesterone, 1-indanone, cyclohexanone, and 3-acetylindole.

DMSO

Time (min)

Abundance

uV (×100,000)

A

DMSO

Time (min)

Abundance

uV (×100,000)

A

Time (min)

Abundance

uV (×100,000)

B

Time (min)

Abundance

uV (×100,000)

B

FIG. 3. Transformation of (A) TFAP and (B) phenylacetone by cell-free extract of Gordonia sp. strain SH2. The reaction mixture contained 1.5 mM substrate, 1.5 mM NADPH and 0.08 mg ml-1 cell-free extract. Intermediates and products were analyzed by GC-MS after overnight incubation at room temperature.

Enantioselective conversion of phenyl sulfides. In addition to converting aliphatic, cyclic and aromatic ketones, cell-free extract of strain SH2 also oxidized phenyl sulfides like benzyl methyl sulfide, methyl phenyl sulfide, ethyl phenyl sulfide and methyl p-tolyl sulfide.

Enantioselective conversions of a variety of sulfoxides by cyclohexanone monooxygenase (CHMO), cyclopentanone monooxygenase (CPMO), hydroxyacetophenone monooxygenase (HAPMO) and phenylacetone monooxygenase (PAMO) has been described (8, 11, 13, 23).

Enantiomerically pure sulfoxides can be used in a wide range of asymmetric reactions for the synthesis of natural products and biologically active ingredients (9, 10).

TABLE 1. List of compounds transformed by the BVMO of strain SH2 containing BVMO.

TABLE 2. Resolution of racemic sulfoxides metabolized by cell-free extract of strain SH2, hydroxyphenylacetone monooxygenase (HAPMO), phenylacetone monooxygenase (PAMO), cyclopentanone monooxygenase (CPMO), styrene monooxygenase (SmoA) and cyclohexanone monooxygenase (CHMO).

BVMO of strain SH2

HAPMO (12)

PAMO (33)

CPMO (23)

SmoA (38)

CHMO (8) Structure e.e. a ( %) e.e. a (%) e.e. a (%) e.e. a (%) e.e. a (%) e.e. a (%)

methyl phenyl sulfide

>99 (S) 99 (S) 41 (R) 100 (S) 75 (R) 99 (R)

ethyl phenyl sulfide

>99 (S) 99 (S) 6 (S) - 92 (R) 47 (R)

benzyl methyl sulfide

>99 (S) 85 (S) 98 (S) - - 54 (R)

methyl p-tolyl sulfide

97 (S) 99 (S) 6 (R) 84 (S) 60 (R) 37 (S)

a The predominantly formed enantiomer is in parenthesis

Using chiral GC for product analysis it was observed that all selected sulfides were converted to the (S)-enantiomer with a high ee (≥ 97%). No formation of (R)-enantiomers was observed (Table 2). HAPMO also has a preference for formation of the (S)-enantiomers (13) with no formation of (R)-enantiomers. In contrast, oxidation of three of the four selected sulfides by SmoA was reported to lead to the formation of (R)-sulfoxides with ee’s exceeding 60% (38). On the other hand, PAMO showed very low enantioselectivity with the same sulfides apart from benzyl methyl sulfide which gave (S)-oxide with a high ee (98%) (33).

Oxidation of methyl phenyl sulfide by CPMO also leads to formation of the (S)-sulfoxide with high enantioselectivity (ee ≥ 99%) (23), while CHMO has a preference for the (R)-enantiomer with a higher (R)-enantiomeric excess (ee = 99%) (8). This finding revealed that for sulfoxidation of methyl phenyl sulfide, the BVMO of strain SH2 and HAPMO are enantiocomplementary with CHMO. In contrast, the BVMO of strain SH2 and HAPMO are enantiocomparable.

Oxidation of sulfides occurs with attack of oxygen to sulfur, resulting in the formation of chiral sulfoxides. Further oxidation may lead to the formation of sulfones, in which two oxygen atoms are covalently attached to sulfur (Fig. 4). The formation of a sulfone appeared to be pH dependent when PAMO was used to oxidize methyl phenyl sulfide to the corresponding (R)-sulfoxide. The enantiomeric excess (ee) values increased 5-fold by raising the pH from 6 to 10 (40). This suggests that the yield of a particular enantiomer can be altered by changing the pH of the reaction mixture.

FIG. 4. General pathway of the BVMO-catalyzed sulfoxidation of organic sulfides to sulfoxides and sulfones.

Conversion of a prochiral bicycloketone. The conversion of a prochiral bicycloketone (bicyclo[3.2.0]hept-2-en-6-one) with the cell-free extract of strain SH2 formed both normal and abnormal lactones with enantiomeric excesses of 44% and 32%, respectively (Fig. 5). It suggests that the BVMO in strain SH2 has no selective preference for either normal or abnormal lactone. Similarly, HAPMO showed low regioselectivity by forming the two lactones (i.e normal and abnormal) in almost equal amounts (22).

FIG. 5. Oxidation scheme of bicyclo[3.2.0]hept-2-en-6-one by cell free-extract of strain SH2 induced with α,α,α−trifluoroacetophenone.

Identification of proteins induced in strain SH2 with TFAP by MALDI-MS/MS.

The degradation pathway of TFAP was explored by identifying the proteins involved in the degradation pathway. For this, we looked for the differential induction of proteins of Gordonia sp. stain SH2 grown with TFAP compared to succinate-induced proteins. Cell-free extracts were analyzed by running two-dimensional gels, followed by mass spectrometry.

From the 2-D gel with TFAP-induced cell-free extract, 55 spots were selected for mass spectrometry. These spots were different from those observed in succinate (Fig. 6). To identify these proteins, spots were excised, destained and digested with trypsin. For most of the spots, we observed good spectra (< 900 Da peptide mass), with peak intensities suitable for MALDI-MS/MS analysis. The fragmentation spectra obtained from the prominent spots were used for protein identification by database searching. The genome of strain SH2 has not been sequenced and is therefore not represented in the Actinobacteria protein database. Since Gordonia sp. belongs to phylum Actinobacteria, the protein database of this phylum was selected to identify the induced proteins. Good matches were obtained with putative phenol hydroxylase of Nocardia farcinica, catechol 1,2-dioxygenase and esterase of Rhodococcus strain RHA1, indicating the presence of homologous genes in these organisms which belong to the family Nocardiaceae.

FIG. 6. Two-dimensional gel electrophoresis Coomassie-stained protein pattern from Gordonia sp.

strain SH2 grown separately with each of succinate and TFAP added in MMY medium. Selected protein spots with increased levels are highlighted by numbered-arrows. They correspond to the proteins listed in Table 3.

A likely BVMO that is involved in the transformation of TFAP was not identified in the samples of the 2-D gel of TFAP-induced proteins. This may be due to the absence of a

homologous BVMO sequence in the database of Actinobacteria. However, other proteins that are directly involved in the degradation pathway of TFAP were identified (Table 3).

Identification of these proteins in strain SH2 when exposed to TFAP indicates the selective induction of the proteins associated with the degradation of TFAP.

In the 2-D gel, proteins of spot 1, 2 and 3 (Fig. 6B) were similar to putative phenol hydroxylase of Nocardia farcinica (Q5YRH7). These three spots correspond to proteins with the same molecular weight (Mr) but different isoelectric point (pI), which may be caused by posttranslational modifications or could be the result of experiment-induced modifications, including urea-mediated carbamylation or deamidation (7, 30). The protein of spot 4 was identified as a homolog of catechol 1,2-dioxygenase of Rhodococcus sp. RHA1 (Q0SDR6) and spot 5 was similar to an esterase of Rhodococcus sp. RHA1 (Q0SB47).

TABLE 3. Proteins with increased levels in Gordonia sp. strain SH2 grown in MMY medium supplemented with 1 mM TFAP as compared to 1 mM succinate.

Spot

4 Catechol 1,2-dioxygenase LWHADDDGYY VELWHA

a Spot number according to Fig. 6B.

b Mascot score represents the probability that the observed match is a random event. Only protein scores with P values of < 0.05 are reported.

Partial purification of proteins induced in cell-free extracts. In order to study the degradation pathway of TFAP, we separated monooxygenase, esterase and catechol 1,2-dioxygenase, which were induced in strain SH2 in the presence of TFAP. Protein fractions obtained from ion-exchange chromatography were tested for BVMO, esterase, phenol hydroxylase, and catechol 1,2-dioxygenase activities. Five prominant bands were observed in the cell-free extract of strain SH2 induced with TFAP (Fig. 7, lane 2, bands A, B C, D, and E) as compared to the succinate induced extract (Fig. 7, lane 3). The highest monooxygenase (BVMO and phenol monooxygenase), esterase and catechol 1,2-dioxygenase activities were found in the concentrated fractions shown in lane 6, lane 4 and lane 5 of Fig. 7, respectively.

Some BVMO activity was also monitored in the fraction of lane 5. This suggests that band E is esterase and D is catechol 1,2-dioxygenase, which was confirmed by 2-D gel electrophoresis and mass spectrometry. The BVMO and phenol monooxygenase can either be band A, B or C and were not separated by Q Sepharose chromatography. Phenol

Degradation pathway of TFAP. The degradation pathway of TFAP was explored by using concentrated protein fractions involved in the important steps of the degradation route.

A concentrated fraction of proteins containing BVMO (Fig. 7, lane 6) was used to monitor the transformation products of TFAP. Decrease in absorbance at 340 nm indicated the utilization of NADPH, which suggests the transformation of trifluoroacetophenone into trifluorophenyl acetate. Formation of trifluoroacetophenyl alcohol (TFPA) was also observed which was formed by the activity of alcohol dehydrogenase. TFPA was not further degraded and remained in the reaction mixture. This suggests that part of the TFAP was reduced by an

FIG. 7. SDS-PAGE of protein fractions.

Lane 1, marker proteins; lane 2, cell-free extract of strain SH2 induced with TFAP;

lane 3, cell-free extract induced with succinate; lane 4, Q fraction of esterase;

lane 5, Q fraction of catechol 1,2-dioxygenase; lane 6, Q fraction of mixed proteins containing BVMO and phenol monooxygenase; band D, catechol 1,2-dioxygenase and band E, esterase.

alcohol dehydrogenase and the rest is oxidized by a BVMO, as mentioned above. The same was observed when derivatives of acetophenone were transformed by cells of the fungi E. nigrum SSP 1498 and E. nidulans CCT 3119 (2). We could not identify trifluorophenyl acetate in the reaction mixture. This may be due to the presence of esterase activity in the concentrated fraction that was used for the TFAP transformation.

Phenol monooxygenase transformed phenol into catechol. Catechol 1,2-dioxygenase (Fig. 7, lane 5, band D) cleaved the aromatic ring to form muconate.

FIG. 8. Proposed pathway for the degradation of α,α,α−trifluoroacetophenone. Abbreviations: ADH, alcohol dehydrogenase and BVMO, Baeyer-Villiger monooxygenase.

CONCLUSION

The newly isolated Gordonia sp. strain SH2 expressed an NADPH-dependent oxygenase that performed a Baeyer-Villiger conversion of acetophenones, and oxidized aromatic sulfides. No enzymes were found that catalyzed conversion of trifluoroacetate, which accumulated as a dead end product. Strain SH2 also possessed esterase, phenol hydroxylase and catechol 1,2-dioxygenase for the cleavage of TFA, hydroxylation of phenol and ring opening of catechol, respectively.

ACKNOWLEDGEMENTS

The authors thank M. I. Arif for identification of strain SH2, R. K. R. Marreddy for helping with running 2-D gel, and J. van Leeuwen for the collection of soil samples from various sites in The Netherlands. S. A. Hasan thanks Higher Education Commission (HEC), Government of Pakistan for financial support.

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