Assimilatory reduction of trimethylamine N-oxide in the yeast Sporopachydermia cereana

Appl Microbiol Biotechnol (1987) 26:277--282 Applied Microbiology

Biotechnology © Springer-Verlag 1987

Assimilatory reduction of trimethylamine N-oxide in the yeast Sporopachydermia cereana

David Whitfield* and Peter J. Large

Department of Biochemistry, University of Hull, Hull HU6 7RX, U. K.

Summary. Washed microsomal preparations (100000 xg sediment) from the yeast Sporopachy- dermia cereana that had been grown on trimethylamine N-oxide as sole nitrogen source catalysed the NAD(P)H-dependent reduction of trimethylamine N-oxide to trimethylamine. Under anaerobic conditions, this was the sole reaction product, but under aerobic conditions only small amounts of trimethylamine accumulated, most being further metabolized to methylamine and formaldehyde (no detectable dimethylamine accumulated due to its rapid turnover). In the absence of NAD(P)H, no formation of amines or formaldehyde from trimethylamine N-oxide was detected. The trimethylamine N-oxide reductase activity was inhibited by quinacrine, Cu 2+ ions, triethylamine N-oxide (apparent Ki 0.43 mM) and dimethyl sulphoxide (Ki 0.94 mM). Chlorate and nitrate failed to inhibit the enzyme. The Km for trimethylamine N-oxide was 29 IxM. Triethylamine N-oxide was also reduced by the microsomal preparation with the formation of acetaldehyde, and this reduction was sensitive to the same inhibitors as trimethylamine N-oxide, suggesting that both amine oxides are metabolized by the same enzyme(s). It is concluded that trimethylamine N- oxide is metabolized in this yeast via an NAD(P)H-dependent reductase.

* Present address: Biology Division, Chemical Defence Estab- lishment, Porton Down, Salisbury, Wilts. SP4 0JQ, U.K.

Offprint requests to: P. J. Large

Abbreviations: TMAO, trimethylamine N-oxide

Introduction

Trimethylamine N-oxide (TMAO) is an abundant constituent of the muscle and body fluids of ma- rine fish, and during bacterial decay under relatively anaerobic conditions, the odourless, non- volatile N-oxide is reduced to trimethylamine with its characteristic fishy smell (Barrett and Kwan 1985). The putrefactive bacteria are using TMAO as a terminal electron acceptor with the concomitant electron transport-dependent formation of ATP. The enzymic nature of this dissimila- tory TMAO reduction has been well characterized in a wide range of bacteria (e. g. Salmonella typhi- murium, Kwan and Barrett 1983; Escherichia coli, Shimokawa and Ishimoto 1979; Yamamoto et al. 1986; and Rhodopseudomonas eapsulata, McEwan et al. 1985). The same enzyme system is also al- most certainly responsible for the reduction of dimethyl sulphoxide (Styrvold and Strom 1984; McEwan et al. 1985). The trimethylamine formed from TMAO under these conditions is not metabolized further.

TMAO however can also function as a nitrogen or carbon source for the growth of some bacteria (Large 1981 ; Large and Green 1984), and as a nitrogen source for yeasts (Yamada et al. 1976; Green and Large 1984), and in this situation is playing an assimilatory role rather than a dis- similatory role. Utilization of TMAO by methylotrophic bacteria does not normally involve reduction, but a non-oxidative cleavage of the N-oxide to dimethylamine and formaldehyde catalysed by TMAO aldolase (E. C. 4.1.2.32) (Large 1971; Myers and Zatman 1971) (Eq. 1).

( C H 3 ) 3 N O ~ ( C H 3 ) 2 N H + H C H O (1)

As a preliminary to investigating the possible ap-

278 D. Whitfield and P. J. Large: Trimethylamine N-oxide reductase activity in a yeast

pl icat ion o f amine-uti l izing yeasts in the removal o f odours f rom fish-processing plants, it was nec- essary to determine how methyla ted amines are metabol ized by yeasts. The demethyla t ion o f di- and t r i -methylamine by mono-oxygenases in Can- dida utilis and other yeast species has previously been studied by us (Green and Large 1983 ; 1984), but the mechan i sm by which yeasts metabol ize T M A O is unknown.

Earlier work by Y a m a d a et al. (1976) and Green and Large (1984) failed to identify any enzyme, either reductase or aldolase, that could me- tabolize T M A O in various Candida species. Re- cently, however it has been found that washed microsomal prepara t ions o f the ascosporogenous yeast Sporopachydermia cereana catalyse the T M A O - d e p e n d e n t oxidat ion of N A D H or N A D P H (Whitfield and Large 1986). This reac- t ion only occur red in cells grown on T M A O as ni- t rogen source, and it had a pH op t imum of 8.0. It was extremely sensitive to inhibitors o f microsomal cy tochrome P-450 systems such as ca rbon monox ide (Ki 1 ~tM) and proadi fen (SKF 525-A). In this pape r we demonst ra te that the p roduc t o f this reaction is trimethylamine, and not dimethylamine, showing that the react ion involves a reductase, perhaps similar to E.C. 1.6.6.9 (Eq. 2).

(CH3)3NO + N A D ( P ) H + H + (CH3)3N + NAD(P) + + H20 (2)

This rules out as a possible mechan i sm the non- oxidative fo rmat ion of d imethylamine fol lowed by its NAD(P)H-dependen t conversion to methylamine and fo rmaldehyde (Green and Large 1983).

Materials and methods

Materials. Dimethyl sulphoxide was from Fisons Scientific, triethylamine N-oxide from K & K Rare and Fine Chemicals (ICN Life Sciences Group) and other materials from sources described previously (Green and Large 1984).

Growth and maintenance of cultures. Cultures of S. cereana were grown aerobically in shake flasks in medium containing 55 mM glucose, 30 mM TMAO-HCI, salts and vitamins as described previously (Whitfield and Large 1986).

Preparation of extracts and washed microsomal suspensions. These were prepared in 50 mM potassium phosphate buffer pH 7.0 containing 20 llM FAD as described by Whitfield and Large (1986).

Assay of enzyme activities. TMAO reductase activity was measured by two methods.

(a) Continuous spectrophotometric assay, following the decrease in absorbance at 340 nm in 3 ml polystyrene cuvettes (1 cm light path) at 25°C, containing 200~tmol potassium phosphate buffer pH 8.0, 0.15 ~tmol NADH (or NADPH where indicated) 0.1 mg microsomal protein and water in a fi- nal volume of 3 ml. After measurement of the significant en- dogenous NADH oxidation rate, the reaction was started by addition of 1.5 ~tmol of TMAO-HCI. One unit of enzyme is the amount required to catalyse the TMAO-dependent oxidation of 1 ~tmol of NAD(P)H/min under these conditions.

(b) Fixed time interval assay. Graduated tubes contained in a total volume of 0.75 ml, 25 Ixmol potassium phosphate buffer, pH 7.5 or 8.0, 0.05 mg microsomal protein, 1.13 Ixmol TMAO-HC1 or trimethylamine-HCl or 3.75 ~tmol dimethylamine-HCl and I 1.3 ~tmol NAD(P)H. Buffer and microsomes were preincubated at 25°C for 5 rain before addition of amine or TMAO mixed with NAD(P)H. After 5, 10, 15, 20, or 30 min, the reaction was stopped by addition of 0.375 ml of ice-cold 5% (w/v) zinc sulphate followed by storage in ice and addition of 0.375 ml of saturated Ba(OH)2. The pH of the solution was adjusted to 7.0 with 2 M NaOH or HC1 and the volume adjusted to 2.5 ml. After 5 rain centrifugation at 1000 xg, samples of the supernatant were removed for analysis in duplicate for amine or formaldehyde.

Mono-oxygenase activities were assayed similarly to spectrophotometric assay (a), but with the TMAO replaced by dimethylamine (4 ~tmol) or trimethylamine (1.5 [lmol),

Anaerobic incubations. These were performed under nitrogen which had been made oxygen-free by passing it through alkal- ine dithionite in a Nilox deoxygenator (Dixon 1971), and solu- tions were used that had been depleted in oxygen by pro- longed bubbling with the oxygen-free nitrogen.

Chem&al determinations. Methylamine, dimethylamine and trimethylamine were separated and estimated by g.l.c, at 100°C on a column (1.9 m x2 mm) of Carbopack B coated with 4% Carbowax 20 M and 0.8% KOH (Supelco Inc., Crans, Switzerland) as described by Di Corcia and Samperi (1974). Responses were linear with amine concentration, and the re- tention times for the above three amines were 1.8, 2.2 and 3.1 rain respectively. TMAO was estimated by the method of Mitchell and Ziegler (1969), correcting for any metabolically formed formaldehyde. Formaldehyde was determined by the Nash (1953) method, and other aldehydes by the method of Sawicki et al. (1961). Protein was estimated by the Bradford (1976) method.

Results

Reduction o f T M A O by washed microsomal preparations

Washed prepara t ions of microsomes from S. cereana catalysed the T M A O - d e p e n d e n t oxidat ion o f N A D H at a typical rate of approximate ly 40 - - 50 n m o l / m i n / m g protein (Table 1). When N A D P H was substituted, the rate was only 2 5 - - 37% of that with N A D H (Table 1). When the as- says were pe r fo rmed under oxygen-free ni t rogen the rates o f N A D H oxidat ion fell by 16% for TMAO, compared with 88% for d imethylamine

D. Whitfield and P. J. Large: Trimethylamine N-oxide reductase activity in a yeast 279

Table 1. Rate of oxidation of NADH or NADPH by w a s h e d microsomal preparations of S. ce reana incubated aerobically or anaerobically in the presence of amine substrates (1 raM). Rates ( n m o l / m i n / m g protein) were measured spectrophotom- etrically at 25 ° C

Substrate added Aerobic rate Anaerobic rate

Electron donor NADH NADPH NADH

Rate after reaeration of anaerobic cuvettes NADH

TMAO 53 17.4 44.5 41 Trimethylamine 46 3.8 2.6 33 Dimethylamine 150 13.5 17.7 137

2 " 0 -

E 1.5 n ~

o

0 1.0 "1" (J -r n 8 0.5

/ - I I I I 0 5 10 15 20

Time (rain)

Fig. 2. Formation of formaldehyde (xT) from TMAO (D) and NADH aerobically in the presence of washed microsomal preparations (assay method (b))

and 94% for trimethylamine (Table 1). Aeration of the cuvettes showed that only about 10--20% of this activity loss was due to inactivation of the enzyme in each case. Clearly the TMAO-dependent oxidation of NAD(P)H is not oxygen-dependent, while the oxidation of N A D H by the other two amines is.

0.7E

0.5

0.2 E

A &

E

1-C

-~ o-7~

0-5

02=" ~ /

/ I I I I I I

0 5 10 15 20 25 30

Time (rain)

Fig. 1. A. Formation of t r imethylamine (O, t ) and methylamine (/x) from TMAO and NADH in the presence of washed microsomal preparations from S. cereana . B. Formation of methylamine (/x) from trimethylamine (©, O) and NADH in the presence of washed microsomal preparations. In both graphs open symbols denote aerobic incubation, filled symbols denote anaerobic incubation (assay method (b))

Examination of the reaction products by g.l.c. showed that while both trimethylamine (Fig. 1B) and dimethylamine gave rise only to methylamine in aerobic conditions (dimethylamine does not ac- cumulate as an intermediate in trimethylamine breakdown because the rate that it is oxidized is three times as fast as the rate at which it is formed from trimethylamine, see Table 1), and no detectable volatile amine product under anaerobic conditions (Fig. 1B), TMAO gave rise under aerobic conditions to small quantities of trimethylamine and large quantities of methylamine (Fig. 1A). Under anaerobic conditions in contrast, only trimethylamine was formed (Fig. 1A). Experiments to measure TMAO disappearance under aerobic conditions showed that for each mol of TMAO consumed, one mol of N A D H was oxidized (results not shown), and about 2 mol of formaldehyde were also formed (Fig. 2). In the absence of

2OO

100

~_ 8O

~" 60 m

~ 40 _z,

"------ ii.,,,, i I

lO I I I I I • 0 10 20 30 40 50 60 70

Time (h}

Fig. 3. Loss of activity of TMAO reductase (O) on incubat ion at 4 ° C, compared with the loss of trimethylamine mono-oxygenase activity ( • ) and d imethylamine mono-oxygenase activity ( • ) (assay method (a))


NAD(P)H, no formation of formaldehyde or amines from TMAO could be detected.

Values in the region of 29 ktM for the apparent Kin and 51 n m o l / m i n / m g protein for the Vmax were determined at an NADH concentration of 0.05 mM by a direct linear plot (Eisenthal and Cornish-Bowden 1974) of rates measured in assay method (a) at various TMAO concentrations. The TMAO reductase activity was very labile. At 4°C its half-life in microsomal preparations was 35 h, similar to that for trimethylamine mono-oxygenase, and significantly less than that for dimethylamine mono-oxygenase (Fig. 3).

These experiments clearly establish that the first step in TMAO metabolism in S. cereana is its reduction to trimethylamine (Eq. 2), followed by breakdown of the latter via trimethylamine and dimethylamine mono-oxygenases (Eqs. 3 and 4) (Whitfield and Large 1986)

(CH3)3N + NAD(P)H + H + + 02 (CH3)2NH + HCHO + NAD(P) + + H20 (3)

(CH3)2NH + NAD(P)H + H ÷ + 02 CH3NH2 + HCHO + NAD(P) ÷ + H20 (4)

and rule out any mechanism involving non-oxidative cleavage of TMAO to dimethylamine and formaldehyde.

Inhibition o f TMA 0 reductase activity

I - 0 - 5

I I 0 . 5 1.0

Cohen. of t r i e t h y l o r n i n e _ N - o x i d e ( r n M )

CO,

6C

i i

Ill I I I I -1 ,0

~ 0

f m I

o12 ", o!6 o'8 ,!o COhen of d i m e t h y l su lphox ide (rnfA)

1,5

Copper ions (Kwan and Barrett 1983), nitrate (Easter et al. 1982) and quinacrine (Shimokawa and Ishimoto 1979) are known to be inhibitors of bacterial TMAO reductase. Dimethyl sulphoxide, a probable substrate of the bacterial enzyme (Styrvold and Strom 1984) and triethylamine N- oxide, a substrate analogue, were also tested as possible inhibitors. Copper ions (0.1 mM) produced 69% inhibiton of TMAO reductase (compared with 60% of trimethylamine mono-oxygenase and 36% of dimethylamine mono-oxygenase), while 1 mM quinacrine produced 50-- 70% inhibition of all three activities, presumably because it is a flavin analogue and all three activities require the presence of FAD in the enzyme buffer (Whitfield and Large 1986). Neither potassium nitrate nor potassium chlorate at 1 mM had any stimulatory or inhibitory effect on TMAO-dependent N A D H oxidation. Dimethyl sulphoxide and triethylamine N-oxide were potent competi- tive inhibitors of TMAO-dependent NADH oxidation (Fig. 4) with Ki values respectively of 0.94 and 0.43 raM.

Fig. 4. Dixon (1953) plot of inhibition of TMAO reductase activity of washed microsomal preparations by (A) triethylamine N-oxide and (B) dimethyl sulphoxide. The TMAO concentrations used were: O, 0.01 mM; 0 , 0.025 raM; /x, 0.05 mM; A, 0.1 mM; E], 0.25 mM; and II, 0.5 raM. Assay method (a) was used

Triethylamine N-oxide as a substrate

Since the Km value for TMAO was 29 ~tM, it seemed likely that in addition to being a competi- tive inhibitor at relatively high concentrations, triethylamine N-oxide might also be a substrate. This was tested for by measuring its activity as a replacement for TMAO in assay method (a) at a concentration of 1.6 mM, four times its K~ value, when it caused NADH oxidation at a rate of 12 nmol /min /mg protein, some 23% of the rate with TMAO. When tested in assay method (b), there was a linear rate of formation of aldehyde amounting to 6.4 nmo l /min /mg protein. This aldehyde reacted in the general aldehyde assay of Sawicki et al. (1961), but not in the Nash formal-

D. Whitfield and P. J. Large: Trimethylamine N-oxide reductase activity in a yeast 281

Table 2. Inhibition of the reduction of triethylamine N-oxide by washed microsomal preparations compared with that of TMAO. Assay method (a) was used and inhibitors were preincubated for 5 min with the enzyme before addition of the amine oxide. The uninhibited rates were 11 nmol /min /mg protein for triethylamine N-oxide and 40 nmol /min /mg protein for TMAO

Inhibitor Concen- Percentage inhibition tration of activity with (mM)

triethyl- trimethyl- amine amine N-oxide N-oxide

trans-2-Phenylcyclo- propylamine 0.25 89.6 95.1

Aminoacetonitrile 0.1 6.5 8.1 Benzylamine 0.05 42.9 45.1 1,10-Phenanthro- line 5.0 20.8 73.9

Disodium EDTA 2.5 33.9 17.4 KCN 0.5 33.9 37.3 n-Octylamine 1.0 63.5 76.5 Ethanol 10.0 96.8 47.5 Proadifen-HC1

(SKF 525-A) 0.5 69.1 100.0 Metyrapone 1.0 20.6 0 2-Mercaptoethanol 0.5 59.4 100.0 N-Ethylmaleimide 1.25 44.6 53.0

dehyde method, so it was concluded that it was acetaldehyde.

To determine whether the reduction of triethylamine N-oxide was due to the same enzyme as is responsible for TMAO reduction, its sensitivity to various inhibitors was tested in the continuous spectrophotometric assay (a). The results are shown in Table 2 with the data for TMAO (from Whitfield and Large 1986) for comparison. With the exception of metyrapone, the two inhibition patterns are very similar, suggesting that triethylamine N-oxide is indeed a substrate for the TMAO reductase. It is known that further metabolism of the triethylamine that is presumably formed to acetaldehyde could take place via trimethylamine and dimethylamine mono-oxygenases (Whitfield and Large 1986).

Discussion

The evidence presented here shows that S. cereana metabolizes TMAO by reduction to trimethylamine (equation 2), in confirmation'of earlier suggestions (Large and Green 1984; Green and Large 1984). Trimethylamine is then further metabolized via the two mono-oxygenases (equations 3

and 4) as already described (Whitfield and Large 1986). The methylamine and formaldehyde so formed cannot be metabolized further by microsomal preparations because formaldehyde dehy- drogenase is in the cytosol in yeasts (Sahm et al. 1975; Jenkins et al., 1985) and methylamine oxi- dase is in the peroxisomes (Green and Large 1984). The essential distinction between yeasts and methylotrophic bacteria possessing the mono-oxygenase route for trimethylamine cata- bolism is that in the former trimethylamine is an intermediate in TMAO breakdown, whereas in the latter TMAO is an intermediate in the breakdown of trimethylamine (Boulton et al. 1974).

The reductase system shows what is probably a fairly broad specificity, as it seems to catalyse the reduction of triethylamine N-oxide and possi- bly dimethyl sulphoxide also, although in Saeeha- romyces cerevisiae dimethyl sulphoxide is known to be reduced by a thioredoxin-dependent methionine sulphoxide reductase (Gibson and Large 1985). In this respect the TMAO reductase differs from TMAO aldolase in the methylotrophic bacterium Pseudomonas aminovorans, for which triethylamine N-oxide was an inhibitor but not a substrate (Large 1971).

The system shows few resemblances to the TMAO reductase of the bacterial terminal oxidation system (Barrett and Kwan 1985), thus it also uses NADPH as electron donor, which the bacterial enzyme does not (Easter et al. 1982), and nitrate is neither a substrate nor an inhibitor. It does however resemble the N-oxide reductase system found in mammalian microsomes in its sensitivity to carbon monoxide and SKF 525-A, its stimula- tion by flavins and its activity with both NADH and NADPH (Sugiura et al. 1976).

It is difficult at the present stage to determine what intermediate electron carrier(s) may lie between NAD(P)H and TMAO in S. cereana microsomes. The dependence of the system on the presence of FAD or FMN in the buffers (Whitfield and Large 1986) might imply an electron carrier role for the flavin, as was postulated for the bacterial system by Shimokawa and Ishimoto (1979) and Cox et al. (1980). The sensitivity of the system to carbon monoxide however suggests to us that the microsomal system probably consists of a flavo- protein NAD(P)H-cytochrome P-450 reductase and a cytochrome P-450, though only very low levels of carbon monoxide-reactive cytochrome have been detected in S. eereana microsomal preparations (D. Whitfield, unpublished results). A definitive answer to this will have to await purification of the TMAO reductase system, but the in-


dications are that this will be difficult because of the instability of the system.

Acknowledgements. We thank Carol Gillyon for drawing the diagrams. This work was supported by the Science and Engi- neering Research Council under grant GR/C/54777, which is gratefully acknowledged.

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Received December 30, 1986/Revised February 19, 1987

Assimilatory reduction of trimethylamine N-oxide in the yeast Sporopachydermia cereana

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