JOURNAL OF BACTERIOLOGY, Oct. 1968, p. 1055-1064Copyright @ 1968 American Society for Microbiology

Vol. 96, No. 4Printed in U.S.A.

Oxidative Degradation of Methyl KetonesII. Chemical Pathway for Degradation of 2-Tridecanone by Pseudomonas

multivorans and Pseudomonas aeruginosa

F. W. FORNEY AND A. J. MARKOVETZ

Department of Microbiology, University of Iowa, Iowa City, Iowa 52240

Received for publication 1 July 1968

A new intermediate was identified in the 2-tridecanone pathway of Pseudomonasmultivorans, formerly designated pseudomonad 4G-9. This intermediate, undecylacetate, was isolated directly from growing cultures of the organism; the structureof the intermediate was determined by infrared spectroscopy and by gas-liquidchromatographic identification of its hydrolytic products. An amended pathway ispresented that accounts for the conversion of 2-tridecanone to provide carbon andenergy for growth. It was shown that all early intermediates in the pathway arisebiologically and sequentially from their precursors. Studies with P. aeruginosashowed that this organism also degrades 2-tridecanone by the pathway characteristicof P. multivorans. Biochemical mechanisms of the pathway are discussed. Discoveryof undecyl acetate confirms our earlier contention that the primary attack on methylketones by bacteria can be by subterminal oxidation.

Aliphatic methyl ketones are a class of carboncompounds of widespread natural occurrence.Information concerning biological degradation ofthese ubiquitous substrates is scant because theanabolic aspect of their metabolism has beenstudied more extensively than the catabolicaspect. It was this fact that first prompted us toinvestigate the microbial catabolism of methylketones. The following report is an extension ofour investigation.

In an earlier publication (5), we reported thatketone-utilizing organisms were easily isolatedfrom soil by elective culturing and that invariablythey were gram-negative, rod-shaped bacteria,presumably aerobic pseudomonads. One ofthese, Pseudomonas strain 4G-9, was partiallycharacterized and was employed to study thedegradation of 2-tridecanone, a representativenaturally occurring methyl ketone.

It was found that dissimilation of 2-tridecanoneleads mainly to the formation of 1-undecanol,2-tridecanol, and undecanoic acid. We inferredfrom the direct accumulation of these metabolitesin culture fluid that they were intermediates on adegradative pathway for the carbon source.Moreover, 1-undecanol, which was physicallyand chemically identified, was the predominantintermediate. A comparison of the structures of1-undecanol and 2-tridecanone clearly indicatedthat the former must be derived from the latterby some reaction which does not involve methylgroup oxidation. On the basis of these findings,

we proposed the following pathway for theutilization of 2-tridecanone by the organism:

2-tridecanone -l 1-undecanol + C2 fragment1 1

undecanoic energy viaacid glyoxalate

I bypass andproducts of tricarbox-(3-oxidation ylic acid cycle

The present study was initiated to provide amore complete chemical characterization of theproposed metabolic pathway for degradation of2-tridecanone, so that some plausible biochemicalexplanation can be offered for the occurrence ofthose intermediates already identified. Evidenceis presented which indicates that the same path-way is present in another member of the genus,P. aeruginosa. Additional data which differ-entiate Pseudomonas 4G-9 are reported in orderto give a meaningful taxonomic identity to theorganism. A preliminary report of the work de-scribed in this paper has been published (F. W.Forney and A. J. Markovetz, Bacteriol. Proc.,p. 51, 1968).

MATERIALS AND METHODS

Materials. The substrates and reagents used werethe purest commercially available. Adsorbosil-CAB,Adsorbosil-4, and RAPSAP, an ethanolic solution ofKOH, were obtained from Applied Science Labora-tories, Inc., State College, Pa. Florisil was purchased

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from Fisher Scientific Co., Chicago, Ill. Materialsused for gas-liquid chromatography (GLC) columnsare well known and are readily available, except asnoted.

Organisms and culture methods. Pseudomonas strain4G-9, which was also used in a previous study (5),and P. aeruginosa strain Sol 20, obtained from J. C.Senez, Marseilles, France, were used in these studies.Media and routine culture methods have been de-scribed elsewhere (5). The biochemical tests employedto characterize Pseudomonas 4G-9 were based onmethods outlined by Stanier, Palleroni, and Doudoroff(15).

For isolation of undecyl acetate, 100 ml of growthmedium containing 0.3% 2-tridecanol was inoculatedfrom a nutrient agar slant and was incubated for 96hr. Each of four 1-liter amounts of growth mediumcontaining 0.2% 2-tridecanol was inoculated with 20ml of this starter culture and was incubated for 30 hr.

"Replacement-culture" cells were grown for 24 hrwith shaking in 1 liter of basal-salts medium contain-ing 50 mmoles of disodium succinate. Cells wereharvested, washed once with sterile basal medium, andthen added to 250 ml of growth medium containing0.2% 1-undecanol. This "replacement" culture wasshaken for 32 hr; at 4- and 18-hr intervals, 0.4%1-undecanol was added.

Chemical methods. Diethyl ether and n-hexane,routinely employed as extracting solvents, were re-distilled before use. Large volumes of culture fluid,freed from cells by centrifugation, were extracted in acontinuous-extraction apparatus for a minimum of72 hr; small volumes of cell-free culture fluid wereextracted twice in a separatory funnel.

"Replacement-culture" fluids were obtained bycentrifugation, acidified to pH 1 with concentratedHCl, and extracted twice with ether in a separatoryfunnel. Ethereal extracts were dried over Na2SO4 andwere decanted away from the drying agent; then thesolvent was removed by flask evaporation under re-duced pressure. The residue was taken up in chloro-form and redried. A portion of the chloroform solu-tion was analyzed for free fatty acids and the re-mainder was used for preparation of fatty acid methylesters (12).

Undecyl acetate was hydrolyzed by the followingprocedure: 0.25 ml of RAPSAP was added to a 15-mlconical centrifuge tube that contained 0.1 ml of thesolution to be hydrolyzed, and this solution was heatedto boiling for 3 min in a hot-water bath. A 1-mlamount of hot distilled water was added to the tube,and the solution was allowed to cool. The mixturewas extracted with two 5-ml portions of hexane whichwere combined and concentrated in a hot-water bathby evaporation under a stream of nitrogen. This solu-tion, which contained hexane-soluble 1-undecanol,was ready for GLC analysis. The water phase remain-ing in the centrifuge tube was concentrated in thesame manner, acidified, and immediately analyzed foracetic acid by GLC.

Undecyl acetate was synthesized by refluxing equi-molar amounts of acetic anhydride and 1-undecanolin dry pyridine.

Purification procedure for biologically derived

undecyl acetate. To recover pure undecyl acetatedirectly from growth culture fluid, we used the follow-ing procedure. The progress of the procedure wasfollowed by monitoring the product from each stepby GLC on the FFAP column.

Step 1. Ether extract from 4 liters of culture fluidwas dried over Na2SO4, and the solvent was removedto yield 9.5 ml of raw extract.

Step 2. A column (2.4 by 110 cm) was packed witha slurry of 300 g of 140/200 mesh Adsorbosil-CABin hexane. Extract from step 1 was applied to thecolumn, and elution was begun with a gradient ob-tained by adding 1.5 liter of ether to 1.0 liter of vigor-ously stirred hexane. Fractions (5 ml) were collectedat a flow rate of 60 ml/hr; those fractions containingundecyl acetate but contaminated with 2-tridecanoneand other nonpolar compounds were pooled into asingle fraction. This fraction had a final volume of 1.4ml after the solvent was removed.

Step 3. To remove 2-tridecanone, the fraction fromstep 2 was washed into a 50-ml Erlenmeyer flaskwith carbonyl-free methanol, and 40% NaHSO3 solu-tion was added in excess to precipitate the bisulfiteaddition compound. This precipitate was removed byfiltration through Whatman no. 42 filter paper on aBuchner funnel and was washed on the funnel withdry hexane to remove residual compounds. The filtratewas transferred to a separatory funnel and was ex-tracted with 300 ml of hexane. The hexane solutionwas washed with 500 ml of distilled water in fiveportions, dried in the funnel with Na2SO4, and de-canted from the drying agent; then the solvent was re-moved. The resulting extract was again treated withNaHSO3 by the procedure outlined. Final volume ofthe fraction after the second treatment was 50 to 75,uliters. Since formation of the ketone addition com-pound is a reversible reaction occurring in a waterphase, more than one treatment usually is necessaryto remove most of the water-insoluble ketone.

Step 4. The fraction from step 3 was dissolved in 2ml of hexane and was spotted on thin-layer chromato-graphic plates (20 by 20 cm) coated with Adsorbosil-4to a thickness of 0.5 nmm. Plates were developed inn-hexane-diethyl ether (95:5, v/v), sprayed lightlywith 0.2% ethanolic 2',7'-dichlorofluorescein, andexamined by ultraviolet light. Silica gel from areas ofthe plates containing esters was removed; compoundswere extracted from the gel with ether. This extract,which still contained 2-tridecanone as an impurity,was again chromatographed on Adsorbosil-4 plates(20 by 20 cm; 0.25 mm thick). These plates were de-veloped in n-hexane-diethyl ether-acetic acid (90: 10: 1,v/v), sprayed, and examined; the areas of silica gelcontaining undecyl acetate were removed and ex-tracted as before. Final volume of the extract aftersolvent removal was 10 Mliters.

Step 5. Final purification of the extract from step4 was accomplished with a column (5 mm by 8 cm) ofFlorisil from which purified undecyl acetate was elutedwith n-hexane-diethyl ether (20:1, v/v). After thesolvent was removed, 6 to 8 ,liters of purified com-pound remained.

Analytical methods. Infrared absorption spectrawere obtained with a model 137B Perkin-Elmer

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spectrophotometer. Samples were analyzed in optical-quality potassium bromide, which was pressed intopellets 5 mm in diameter with a Carle micro-pelletdie assembly (Carle Instruments, Inc., Anaheim,Calif.).GLC analyses were performed with an F & M

model 700 chromatograph equipped for flame ioniza-tion detection. The detector and injection port were

operated at temperatures 50C higher than columntemperatures. Copper columns, 0.25 inch (0.63 cm)in diameter, and helium as the carrier gas were usedfor all analyses. The following columns, flow rates,and column temperatures were employed: (i) 10 ft(304.8 cm), 5% FFAP (Varian Aerograph, WalnutCreek, Calif.) on 100/120 mesh Chromosorb G, 50ml/min, 160 C; (ii) 10 ft (304.8 cm), 20% Reoplex 400on 45/60 mesh Chromosorb W, 60 ml/min, 165 C;(iii) 15 ft (457.2 cm), 20% Versamid 900 on 30/60mesh AW-DMCS Chromosorb W, 100 ml/min, 188 C(designated in Table 3 as column Versamid 900-A);(iv) 20 ft (609.6 cm), 2% Versamid 900 on 100/120mesh Chromosorb G, 75 ml/min, 185 C (designatedin Table 7 as column Versamid 900-B); (v) 6 ft (182.9cm), 150/200 mesh Porapak Q (Waters Assoc., Inc.,Framingham, Mass.), 50 ml/min, 150C; (vi) 8 ft(243.8 cm), 10% Apiezon L on 60/80 mesh Chromo-sorb W, 100 ml/min, 190C; (vii) 10 ft (304.8 cm),5% SE-30 on 100/120 mesh Chromosorb G, 50mil/min, 150 C; (viii) 4 ft (121.9 cm), 80/120 meshPAR-1 (Hewlett-Packard, F & M Scientific Division,Avondale, Pa.), 100 ml/min, 220 C (designated inTable 7 as column PAR-1A); 50 ml/min, 110 C(designated in Table 6 as column PAR-IB); (ix) 8 ft(243.8 cm), 9.1% Lac-2R-446 on 60/80 mesh Diato-port S, 50 ml/min, 140 C.

RESULTS AND DISCUSSIONOrganisms. Identification of Pseudomonas

strain 4G-9 followed the outline of Stanier,Palleroni, and Doudoroff (15). The data reportedin this paper supplement those compiled in ourprevious publication (5). Table 1 itemizes theresults of tests for the general and nutritionalcharacters of diagnostic value for differentiationof species of aerobic pseudomonads. Originally,we reported that strain 4G-9 was monotrichous;however, subsequent electron microscopic studieshave shown that it is lophotrichous. Intracellularpoly-3-hydroxybutyrate was detected both byphase microscopy of wet mounts and by SudanBlack B staining; extracellular hydrolysis of poly-mer was not determined because of lack of puresubstrate. Table 2 presents the positive charac-ters of greatest differential value in the recognitionof P. multivorans; the reactions of strain 4G-9are also listed. Strain 4G-9 conforms to 13 of the14 selected characters which define the idealphenotype of P. multivorans; in addition, thisstrain utilizes the following substrates tested as

sole carbon and energy sources: salicin, malonate,succinate, fumarate, glutarate, pimelate, suberate,

TABLE 1. Reactions of Pseudomonas strain 4G-9compared with reactions of P. multivorans

in tests for the general and nutritionalcharacters of diagnostic value for

differentiation of species ofaerobic pseudomonads

Reactions of ReactionsCharacters Pseudomo- of P.

nas 4G-9 multivoransa

No. of flagella............... >1 >1Poly-,3-hydroxybutyrate as

cellular reserve ..........+b +

PigmentsFluorescent................ -Phenazine....- vPhenazine .................. _v

Methionine requirement...... _Growth at4C.......................41 C....................... v

Extracellular hydrolasesGelatin.................... +Poly-,B-hydroxybutyrate . ndStarch.....................

Oxidase reaction............. + +Arginine dihydrolase.........Cleavage mechanism for di-

phenols.................. nd orthoUtilization, as sole carbon

and energy source, of:D-Fucose, D-glucose, tre-

halose, cellobiose, mal-tose, inositol, mannitol,2-ketogluconate, glyco-late, DL-lactate, pelargo-nate, adipate, m-hydrox-ybenzoate, testosterone,acetamide, arginine, va-line, 6-amino valerate,betaine, putrescine....... + + or v

Starch, maleate, norleu-cine, D-tryptophan....

a Data from Stanier, Palleroni, and Doudoroff(15).

b Symbols: +, positive; -, always negative;v, variable; nd, not determined.

azelate, and p-hydroxybenzoate. It is a vigorouslipase producer, as judged by Tween 80 hydroly-sis, but gives a negative egg-yolk reaction. Thecomposite results from all of our studies indicatedthat Pseudomonas 4G-9 is a strain of P. multi-vorans.

Detection of a new intermediate formed during2-tridecanone degradation by P. multivorans. Ahexane extract, obtained by extracting 4 liters ofmedium from a culture grown for 72 hr on 0.3%2-tridecanone, was treated with NaHSO3 (seeMaterials and Methods) to remove some of theunmetabolized substrate and then was analyzedby GLC. A new component in the extract was

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TABLE 2. Reactions of Pseudomonas strain 4G-9compared with the positive characters of

greatest differential value in therecognition of P. multivorans

Reactionsof ideal Reactions of

Characters phenotype Pseudomonasof P. strain 4G-9

multivoranse

Poly-,B-hydroxybutyrate ascellular reserve material.. +b +

Utilization of D-arabinose. + +D-Fucoe.+ +Cellobiose................. + +Saccharate and mucate ... + +2, 3-Butylenegycol .......... + +Sebacate................... + +meso-Tartrate.............. + +Citraconate................ + +o-Hydroxybenzoate ........ +m-Hydroxybenzoate ..... . . + +L-Threonine ............... + +DL-Ornithine .............. + +Tryptamine................ + +

a Data from Stanier, Palleroni, and Doudoroff(15).bSymbols: +, positive; -, negative.

detected; this component was tentatively identi-fied as undecyl acetate by its retention time onseveral columns (Table 3). Rechromatography ofthe extract on each column after addition ofsynthetic undecyl acetate confirmed the presenceof the biological ester, both by its unalteredretention time and by an increase in its peakheight. Further evidence that the new inter-mediate was an ester was obtained by treatingextracts containing ester with RAPSAP (seeMaterials and Methods) and rechromatograph-ing them. In each GLC analysis, the peak cor-responding to undecyl acetate no longer appeared.

Analytical difficulties arose during the course ofthese GLC analyses because four compounds, 2-tridecanone, 2-tridecanol, undecyl acetate, and1-undecanol, which usually occurred together inculture fluid extracts could not be resolved on asingle column. For example, 1-undecanol was notresolved from 2-tridecanone either on polar Lac-2R-446 and Versamid 900 columns or on anintermediately polar Reoplex 400 column; onnonpolar SE-30 and Apiezon L columns, undecylacetate was not resolved from 2-tridecanone.We could not find a suitable nonpolar columnwith which to analyze undecyl acetate in thepresence of 2-tridecanone, 2-tridecanol, and 1-undecanol. The Reoplex 400 column had to besubstituted for a nonpolar column to obtainidentification of the ester on two columns of

TABLE 3. Gas liquid chromatographic identificationof undecyl acetate recovered from culture

fluid of P. multivorans grown on2-tridecanone'

ColumnbCompound

Versamid Reoplex FFAP900-A 400

Authentic undecyl ace-tate................ 18.1 12.0 14.1

Component present inextract.............. 18.1 12.0 14.1

a Results expressed as retention time in min-utes.bColumns and analytical conditions are de-

scribed in Materials and Methods.

TABLE 4. Gas-liquid chromatographic identificationof products recovered from culture fluid ofP. multivorans grown on 2-tridecanola

Authentic compounds Compound in extract

Columnt Column"

Compound Com-FFCAP Reoplex ponent FFAP ReoplexFFP 400 FFP 400

1-Undecanol. .. 18.0 1 18.0Undecyl ace-

tate.......... 14.1 12.0 2 14.1 12.02-Trideca-none......... 15.9 3 15.9

a Results expressed as retention time in minutes.b Columns and analytical conditions are de-

scribed in Materials and Methods.

different polarity. Ultimately, we constructed apolar FFAP column (see Materials and Methods)that could accomplish the separation required,and this column simplified subsequent analyses.

Re-examination of products formed during 2-tridecanol degradation by P. multivorans. Previ-ously, we showed that both 2-tridecanol and 1-undecanol appear as products of 2-tridecanonemetabolism and that 1-undecanol can be pro-duced from 2-tridecanol by the organism (5).These results established that 1-undecanol is acommon product of the metabolism of either2-tridecanone or 2-tridecanol, and this suggestedthat other common products should occur as well.Reassessment by GLC of the products formedduring 2-tridecanol degradation reaffirmed thepresence of 1-undecanol, and indicated thatundecyl acetate and 2-tridecanone were alsopresent in extracts of culture fluid from 2-tri-decanol-grown cells (Table 4). Analysis of the

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ester on two columns differing in polarity andco-chromatography of ester and ketone on theFFAP column with standard ester and ketoneadded individually to the extracts verified thatthese products were present. Analytical resultsfrom several experiments were the same. Thesedata underscored an apparent metabolic equiva-lence between 2-tridecanol and 2-tridecanone; thecompounds appear to be biologically intercon-verted, and both are degraded to undecyl acetateand 1-undecanol.

Direct isolation, purification, and structuralcharacterization of undecyl acetate. Our discoverythat undecyl acetate may be an additional inter-mediate in 2-tridecanone catabolism made itimperative for us to isolate the tentatively identi-fied biological ester and to determine unequivo-cally its physical structure. On both structuraland chemical grounds, as we shall discuss later,undecyl acetate would be a logical intermediatethat could explain the formation of 1-undecanolfrom 2-tridecanone in our system.

Undecyl acetate invariably occurred in very lowconcentrations in extracts of culture fluid or cells.Studies that attempted to follow production ofthe compound with time, as well as inhibitorexperiments designed to cause the compound toaccumulate, gave equivocal results. The estercould neither be detected in most, nor isolateddirectly from any, untreated extracts in which itwas present because such extracts always con-tained much higher concentrations of unusedsubstrate and other components. These com-pounds either prevented ester analysis, or causedthe failure of all attempts to separate the esterfrom the compounds by thin-layer, preparativegas-liquid, or column chromatography. Use of theFFAP column and employment of 2-tridecanol asgrowth substrate in order to yield extracts whichcontained less 2-tridecanone partially solved someof these problems. In addition, methods weredeveloped to remove the more prevalent inter-fering components from the extracts. Thesemethods were mild enough to prevent hydrolysisof the ester but permitted its nearly quantitativerecovery.-A satisfactory procedure for purification of

undecyl acetate directly from extracts of growthculture fluids was devised (see Materials andMethods) and is summarized in Table 5. Al-though the procedure resulted in a 1,000-foldpurification of the ester, only 8 mg of materialwas obtained (based on a liquid density of 1).

In Fig. 1, an infrared spectral analysis of bio-logically-derived undecyl acetate is compared withan analysis of authentic ester. By studying thetracings, the following structural features, of thecompound were recognized. (i) The carbonyl

TABLE 5. Purification of undecyl acetate fromcultures of P. multivorans grown on

2-tridecanola

Ester fraction

Ethyl ether ex-tract..........

Eluate (adsorbosil-CAB column,hexane-ethergradient) ......

NaHSO3 treatmentFirst............Second ..

Preparative thin-layer chroma-tography

First............Second .........

Eluate (Florisilcolumn, hex-ane-ether), 20:1

Volume (ml)

9.5

1.4

0.50.05-0.075

0.040.01

0.006-0.008

Concn(_fold)b

1

7

19133

238950

1,188

Esterpresent(%)b

0.084

0.57

1.612

2080

100

a Conditions for growth of the organism andprocedures for extraction and purification ofundecyl acetate are described in Materials andMethods.

I Based on complete recovery of the ester.

peak at 1,735 cm-' is lower than and has anintensity intermediate between the carbonyl peakin a ketone and the carbonyl peak in a carboxyl-containing compound. (ii) The "acetate esterband" at 1,240 cm-' represents asymmetricstretching of acetate C-CO--C. This band isusually strong and broad and is fairly constantfor this type of ester. (iii) The 1,380 cm-' peakrepresents symmetrical bending of the estermethyl group. The intensity of this band isgreatly enhanced in acetate esters so that itusually becomes stronger than the 1,460 cm-lband, the latter representing asymmetric bendingof the methyl group. (iv) The band at 1,025 cm7-represents symmetrical stretching of esterC-O--C. The acetate ester spectra representedin Fig. 1 correspond satisfactorily with each otheras well as with published Sadtler standard spectraof compounds of this class (14).The experimental ester was recovered from

KBr and was analyzed by GLC to obtain furtherinformation about its structure. Retention timeand co-chromatography with synthetic undecylacetate on the FFAP column reaffirmed theearlier identification of the compound as undecylacetate. The ester was hydrolyzed with RAPSAP(see Materials and Methods); the resulting alcoholin the organic phase and the acid in the water

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phase were identified on two columns, both byretention time and co-chromatography withstandard compounds, as 1-undecanol and aceticacid, respectively (Table 6). Standard undecylacetate, hydrolyzed and analyzed under thesame conditions, yielded the same results.

Figure 2 is included both to illustrate graphi-cally the kind of data obtained from a successfulhydrolysis experiment and to show the effective-ness of the FFAP column for analyzing undecylacetate in a mixture containing those componentswhich commonly occurred in our biological ex-

tracts. A comparison of the tracings before andafter hydrolysis clearly shows that a decrease inundecyl acetate produces a concomitant increasein 1-undecanol.

Sequential production of biological intermediatesin the P. multivorans system. Characterization ofa metabolic pathway requires that each inter-mediate, after its identification, is shown to re-sult biologically from its precursor in the path-way. Degradation of 2-tridecanone by growingcells of P. multivorans may be considered toconsist of the following four reactions:

4000 3000 2000 1500 CM-, 1000 900 800 700

0.10 C-I - - , I- ! 10.

404 i\ <

.5

o; g=---=--~~~-=~~e=--2=~~--j- ;---------0.0

3 4 5 6 7 8 9 10 11 12 13 14 15WAVELENGTH (MICRONS,

FIG. 1. Infrared absorption spectra of (A) authentic undecyl acetate, and (B) biologically derived undecylacetate.

TABLE 6. Gas-liquid chromatographic identification of cleavage products after basic hydrolysisof undecyl acetatea

Authentic compounds Cleavage products

Column Column

CompoundPp

Component inFFAP SE-30 PAR-IB Porapak FFAP SE-30 PAR-IB Porapak

1-Undecanol ....... 18.0 12.1 Organic phase.... 18.0 12.1Acetic acid 4.9 11.4 Water phase 4.9 11.4

a Results expressed as retention time in minutes. Descriptions of the hydrolysis procedure, columns,and analytical conditions appear in Materials and Methods.

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5

4

3

2

6

5

'4

qzj 2

6

5

4

3

4 8 12 16 20 2aTIME (minutes)

A13

B

WC

ltL. IA . _ ^ ^ A n O z ^ > c-.- ^.3nIn32r112

FIG. 2. Gas-liquid chromatograms on FFAP columnof: (A) standard mixture containing undecyl acetate(1), 2-tridecanone (2), 1-undecanol (3), and 2-tridecanol(4); (B) extract from cultures grown on 2-tridecanol;(C) same as (B), except after basic hydrolysis.

(1) 2-tridecanone -- 2-tridecanol + undecyl acetate

+ 1-undecanol(2) 2-tridecanol -i 2-tridecanone + undecyl acetate

+ 1-undecanol(3) undecyl acetate -- 1-undecanol + acetate(4) 1-undecanol undecanoic acid

Data already presented substantiate reactions 1and 2, whereas data that establish the biologicaloccurrence of reactions 3 and 4 are now presented.To demonstrate reaction 3, the organism was

grown for 72 hr in 100 ml of growth mediumcontaining 0.4% undecyl acetate. The culturefluid was harvested and extracted as described inMaterials and Methods, and the resulting ex-

tract was analyzed by GLC. Stability of the ester

to abiological hydrolysis was determined bytreating an uninoculated culture containing 0.4%undecyl acetate in the same manner. Analyticalresults from this experiment are presented inTable 7. 1-Undecanol was produced from un-decyl acetate by the organism. Undecyl acetatealone did not hydrolyze under the same condi-tions. Acetic acid was not detected as a product ofgrowth; however, considerable 1-undecanol, butonly a trace of undecyl acetate, remained in theculture fluid after growth of the organism on theester. Indirectly, these results show that theorganism grew mainly on the acetate moiety ofthe ester.

Reaction 4 could not be shown by growthexperiments since growth on the alcohol wouldrequire rapid utilization of the resulting acid bythe organism for carbon and energy. "Replace-ment" culturing (see Materials and Methods)was used to establish that this reaction can occurbiologically. To detect free acids, a chloroformsolution containing compounds extracted from"replacement" culture fluid was analyzed byGLC on a PAR-1 column. Methyl esters pre-pared from this solution were analyzed by GLCon both polar and nonpolar columns. Undecanoicacid was identified both as the free acid and asmethyl undecanoate (Table 7); this confirms thatundecanoic acid is produced from 1-undecanolby the organism.The foregoing data have established that reac-

tions 1 through 4, as outlined above, occur bio-logically and that the intermediate compoundsshown are produced sequentially from theirprecursors.

Identification and sequential production ofbiological intermediates in the P. aeruginosa sys-tem. In an effort to extend our study of methylketone catabolism to include other pseudo-monads, we tested P. aeruginosa strain Sol 20for its ability to utilize 2-tridecanone for carbonand energy. This pseudomonad grew more rapidlyon the substrate and produced several inter-mediates that were common to the P. multi-vorans system. Working on the assumption thatboth organisms could metabolize the substratein the same way, a comparative study of sequen-tial intermediates produced by P. aeruginosawas undertaken. Only GLC analyses were done,and no intermediates were isolated. Indeed, asthe results compiled in Table 8 attest, P. aeru-ginosa produces the same intermediate com-pounds from 2-tridecanone in a sequentialfashion as does P. multivorans. Apparently, bothorganisms degrade this methyl ketone by thesame metabolic route.

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TABLE 7. Gas-liquid chromatographic identification ofproducts recovered from culture fluidsof P. multivorans after metabolism of undecyl acetate or 1-undecanol

Retention time (min) of compoundpresent in extracts

Carbon source Compound Column"Standard Biologicalcompound compound

For growthUndecyl acetate 1 -Undecanol Versamid 900-B 19.4 19.4

Apiezon L 4.4 4.4

For replacement1 -Undecanol Undecanoic acid PAR-IA 11.9 11.9

LAC-2R-446 7.6b 7.6bSE-30 15.1 15.1

a Columns and analytical conditions are described in Materials and Methods.b Analyzed as methyl ester.

TABLE 8. Biological intermediates produced sequentially during catabolism of 2-tridecanone byP. aeruginosa

Carbon source for growtha

2-Tridecanone

CH3-(CH2) 9-CH-C-CH3110

2-Tridecanol

CH3-(CH2) 9-CH2-CH-CH3

OH

Undecyl acetateCH3-(CH2)9-CH2-O-C-CH3

11

1 -UndecanolCH3-(CH2) 9-CH2-OH

Intermediates identified"

2-Tridecanol

Undecyl acetate

1 -Undecanol

2-Tridecanone

Undecyl acetate

1 -Undecanol

1 -Undecanol

Undecanoic acid

CH3-(CH2) -CH2-CH-CH3

OHCH3-(CH2) 9-CH2-O-C-CH3

110

CH3-(CH2) 9-CH2-OH

CH3-(CH2) 9-CH2-C-CH3II0

CH3-(CH2) -CH2-O-C-CH11

CH3-(CH2) 9-CH2-OH

CH3-(CH2) 9-CH2-OH

-CH3-(CH2) ,-COOHa The organism was grown in the same manner as P. multivorans.bCompounds were recovered and analyzed under the same conditions and with the same results as

shown in Tables 3, 4, and 7.

Amended pathway for catabolism of 2-tri-decanone. We have established that undecylacetate is a metabolic product of 2-tridecanonedegradation by two different species of aerobicpseudomonads. As a result of this finding, wehave amended our earlier scheme (5) and wepropose the pathway shown in Fig. 3; this path-way is adequate, both chemically and biologically,to account for the ultimate conversion of 2-

tridecanone to provide carbon and energy. Wehave shown that biological end products of eachstep-reaction are produced during growth of bothorganisms on each precursor intermediate, eachproduct has been identified, and all products arein accord with our proposal. The substrate under-goes two distinct transformations: (i) it is reducedto form 2-tridecanol, which always appears as aproduct, and (ii) it is oxidized to form an ester,

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OXIDATIVE DEGRADATION OF METHYL KETONES

1-UndecanolCH3-tCH2t9- CH2- OH

I

Undecanoic AcidCH3-(CH219-COOH

Via /-oxidation,Tricarboxylic Acidand Glyoxalate Cyc!es

2-Tridecanone 2-TridecanolCH3-iCH2)9-CH2-C-CH3 C3-(CH2)CH3-(CH2)9 CH~~ (A)- CH2-CH-CH43

| O OH

Undecyl AcetateCH3-tCH2)- CH2-0-C-CH3

l9

Acetate+ CH3-COOH

1fVia Tricarboxylic Acidand Glyoxalate Cyctes

FIG. 3. Amended pathway for complete degradationof 2-tridecanone by two aerobic pseudomonads.

undecyl acetate, which is split into two moieties,1-undecanol and acetate, respectively. Subse-quently 1-undecanol is utilized by its oxidationto undecanoic acid which also supports growth.In crude cell-free extracts of P. aeruginosa,alcohol dehydrogenase activity that readily con-verts 1-undecanol to undecanoic acid has beenfound (F. W. Forney, unpublished data). ,8-Oxida-tion probably accounts for dissimilation ofundecanoic acid in the cell. The acetate moiety ofthe ester, on the other hand, is assumed to furnishcarbon and energy for cellular metabolism viathe tricarboxylic acid cycle and the glyoxalatebypass.Some speculations on the biochemical mecha-

nisms for 2-tridecanone degradation. Althoughthe experimental evidence is inadequate tosupport our proposed catabolic pathway for2-tridecanone at the enzyme level, the followingsuggestions may be made. When 2-tridecanolserves as a primary substrate, conversion to2-tridecanone may be catalyzed by an alcoholdehydrogenase. However, it is not clear why2-tridecanol should always be found when2-tridecanone is metabolized.The conversion of 2-tridecanone to undecyl

acetate indicates that primary attack on methylketones by bacteria indeed can occur by sub-terminal oxidation. The enzyme mediating thisreaction may be an oxygenase which belongs tothe class of mono-oxygenases (7) or to the classof mixed-function oxidases (9). The reactioncould occur by insertion of a linking or bridgingatom of oxygen into the carbon chain betweencarbon atoms 2 and 3 of the substrate, by a

mechanism analogous to that of the chemicalBaeyer-Villiger reaction of peracids with methylketones (6); this would result in the formation ofundecyl acetate. Although a Baeyer-Villiger typeof reaction has not been reported to occur

chemically with aliphatic methyl ketones longerthan eight carbon atoms (6), or biologically with

any simple, unbranched or unsubstituted ali-phatic methyl ketone, its biological counterparthas been documented. Reports of enzymaticmodification of alicyclic compounds, such assteroids (4, 8, 10, 11, 13) and camphor (1, 2), orcarbohydrates, such as inositol (3), indicate thata Baeyer-Villiger oxidation mechanism may beresponsible for either ring or side-chain cleavageduring biological degradation of these complexcompounds. Sutton (16) reported that an en-zyme derived from Mycobacterium phlei oxida-tively decarboxylated lactic acid; this appears tobe the only example of Baeyer-Villiger oxidationof a simple compound. Only two of these studiesemployed enzymes derived from bacteria; oneenzyme, camphor ketolactonase (2), was derivedfrom an aerobic pseudomonad. Our study con-stitutes the first indirect demonstration of bio-logical Baeyer-Villiger oxidation of a simple,aliphatic methyl ketone by bacteria, specifically2-tridecanone by two aerobic pseudomonads.The hydrolysis of undecyl acetate is enzymatic,

as we have demonstrated that it can occurbiologically and that the ester is very resistant toabiological splitting. We suggest that the acyl-oxygen bond is the bond that is broken and theacyl group is the group that is transferred. Thissuggestion is supported by two observations:(i) free acetic acid was never detected as a productof 2-tridecanone metabolism by whole cells, and(ii) growth on the ketone did not result in amarked drop inpH of the culture. Therefore, it isunlikely that the free form of the acid is released;it is more likely that the acetate moiety is trans-ferred, concurrent with its release and activation,to the tricarboxylic acid and glyoxylate cycleswhere it provides carbon and energy for cellgrowth. The failure of undecyl acetate to ac-cumulate in our system, which made its isolationso laborious, may be due to the presence ofhighly active esterases with lower levels of or lessactive ketone-oxygenase in cells growing on2-tridecanone. The oxygenase reaction would berate-limiting under these conditions, with theresult that ester cleavage products, rather thanthe ester intermediate, would accumulate prefer-entially.The pathway we have proposed for 2-tri-

decanone catabolism provides for early andfacile conversion of the substrate to acetate, acommon central metabolite in cellular me-tabolism. This feature of the pathway favorsmetabolic economy.

ACKNOWLEDGMENTSWe thank the Fellowship Committee of the Ameri-

can Society for Microbiology for awarding a Presi-dent's Fellowship to the senior author for study in the

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FORNEY AND MARKOVETZ

laboratory of I. C. Gunsalus during the time thiswork was in progress, and we thank Prof. Gunsalusfor his generous hospitality and stimulating discus-sions.

This investigation was supported by research grantGB-6875 from the National Science Foundation.F.W.F. is a U.S. Public Health Service predoctoralfellow awardee (2-Fl-GM 16,902-06) of the NationalInstitute of General Medical Sciences.

LITERATURE CITED1. Conrad, H. E., R. Dubus, and I. C. Gunsalus.

1961. An enzyme system for cyclic ketonelactonization. Biochem. Biophys. Res. Com-mun. 6:293-297.

2. Conrad, H. E., R. Dubus, M. J. Namtvedt, andI. C. Gunsalus. 1965. Mixed function oxida-tion. II. Separation and properties of theenzymes catalyzing camphor lactonization. J.Biol. Chem. 240:495-503.

3. Charalampous, F. C. 1960. Biochemical studieson inositol. VI. Mechanism of cleavage ofinositol to D-glucuronic acid. J. Biol. Chem.235:1286-1291.

4. Fonken, G. S., H. C. Murray, and L. M. Reinecke.1960. Pathway of progesterone oxidation byCladosporium resinae. J. Am. Chem. Soc.82:5507-5508.

5. Forney, F. W., A. J. Markovetz, and R. E. Kallio.1967. Bacterial oxidation of 2-tridecanone to1-undecanol. J. Bacteriol. 93:649-655.

6. Hassal, C. H. 1957. The Baeyer-Villiger oxida-tion of aldehydes and ketones, p. 73-106. InR. Adams (ed.), Organic reactions, vol. 9.John Wiley and Sons, Inc., New York.

7. Hayaishi, 0. 1964. Proc. Plenary Sessions, 6thIntern. Congr. Biochem., IUB vol. 33, p. 31.Federation of American Societies for Experi-mental Biology, Washington, D.C.

8 Laskin, A. 1., P. Grabowich, C. Del Meyer, andJ. Fried. 1964. Transformations of eburicoicacid. V. Cleavage of ring A by the fungusGlomerella fusarioides. J. Med. Chem. 7:406-409.

9. Mason, H. S. 1957. Mechanisms of oxygenmetabolism. Advan. Enzymol. 19:79-233.

10. Peterson, D. H. 1964. Biochemistry of industrialmicroorganisms. Academic Press, Inc., NewYork.

11. Prairie, R. L., and P. Talalay. 1963. Enzymaticformation of testololactone. Biochemistry2:203-208.

12. Radin, N. S., A. K. Hajra, and Y. Akahori. 1960.Preparation of methyl esters. J. Lipid Res.1:250-251.

13. Rahim, M. A., and C. J. Sih. 1966. Mechanismsof steroid oxidation by microorganisms. XI.Enzymatic cleavage of the pregnane side chain.J. Biol. Chem. 241:3615-3623.

14. Sadtler, S. P. 1964. The Sadtler standard spectra,vol. 2 (spectrum no. 2973) and vol. 18 (spec-trum no. 18090). Sadtler Research Laboratories,Inc., Philadelphia.

15. Stanier, R. Y., N. J. Palleroni, and M. Doudoroff.1966. The aerobic pseudomonads: a taxonomicstudy. J. Gen. Microbiol. 43:159-271.

16. Sutton, W. B. 1957. Mechanism of action andcrystallization of lactic oxidative decarboxylasefrom Mycobacterium phlei. J. Biol. Chem.226:395-405.

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