FEMS Microbiology Letters 34 (1986) 279-282 279 Published by Elsevier

FEM 02413

A complete pathway for fl-alanine and fl-amino-iso-butyrate catabolism in Pseudomonas aeruginosa

(Aminotransferase; oxidative decarboxylase; racemase; dehydrogenase; DL-alanine)

Paul Waters and W.A. Venables *

Department of Microbiology, University College, Newport Road, Cardiff, CF2 ITA, U.K.

Received 2 January 1986 Revision received 21 January 1986

Accepted 22 January 1986

1. SUMMARY

Pseudomonas aeruginosa PAO1 was found to catabolise fl-alanine and fl-amino-iso-butyrate (fl- AIB) by the following pathway: (i) transamination by fl-alanine: pyruvate aminotransferase (BAPAT) to yield L-alanine and either malonic semial- dehyde or its methyl analogue, respectively; (ii) oxidative decarboxylation of the respective semial- dehydes to acetyl CoA or propionyl CoA; (iii) regeneration of pyruvate from L-alanine by the action of oL-alanine racemase (AR) and D-alanine dehydrogenase (DAD). Mutants defective in BAPAT or DAD failed to catabolise either fl- alanine or fl-AIB, and fl-alanine was an inducer for the entire pathway.

2. INTRODUCTION

Pioli et ai. [1] showed that P. aeruginosa catabolised L-alanine by a two-step pathway in- volving racemisation to D-alanine by AR followed by oxidation to pyruvate plus ammonia by DAD. These authors suggested that AR and DAD were also involved in fl-alanine catabolism but the na-

* To whom correspondence should be addressed.

ture of their roles was unclear. Pseudomonas putida has been shown to possess a BAPAT and a malonic semialdehyde oxidative decarboxylase (MSOD) which were assumed to be involved in fl-alanine catabolism by this organism [2].

The present paper elucidates the roles of AR and DAD in fl-alanine catabolism by P. aeru- ginosa, and shows that they combine with BAPAT and MSOD to constitute a novel inducible path- way by which fl-alanine and its analogue fl-AIB can be utilised as sources of carbon, nitrogen and energy for growth.

3. MATERIALS AND METHODS

3.1. Organisms and culture methods P. aeruginosa PAO1 (ATCC15692) was used,

and mutants BAU1 and AOll were derived from it. Cultures were maintained, grown and harvested as previously described [1].

3.2. Cell breakage and subcellular fractionation Cells were broken either in a Hughes press

followed by fractionation by differential centrifu- gation into envelope and soluble fractions as de- scribed in [3], or by sonication followed by centri- fugation at 6000 × g for 10 rain which removed

0378-1097/86/$03.50 © 1986 Federation of European Microbiological Societies

280

cell debris and left a supernatant which was termed unfractionated homogenate [3].

3.3. Chemicals Ethyl-fl,fl-diethoxypropionate, for use in the

preparation of malonic semialdehyde, was a gift from Dr. A.O.T. Charles. Malonic semialdehyde, for use as a substrate in determinations of MSOD activity, was freshly prepared from ethyl-,8,fl-di- ethoxypropionate, about 1 h before use, by the method described in [4]. fl-Alanine and DL-fl- amino-iso-butyric acid (fl-AIB) were obtained from Sigma, U.K.

3.4. Enzyme assays DAD was measured as previously described [1]

and expressed as nmol 2,6-dichlorophenol-in- dophenol reduced per min. AR was measured as in [51 and expressed as nmo! D-alanine produced per min. BAPAT was measured as in [2] in which the appearance of the product, malonic semial- dehyde, is followed by coupling to a diazonium salt, and measuring absorbance at 440 nm; activ- ity was expressed as nmoi malonic semialdehyde produced per minute. MSOD was measured essen- tially as in [2] by following the formation of NADH spectrophotometrically. Activity was ex- pressed as nmol NADH produced per min. Lin- ked assays in which BAPAT was used to generate the substrate for MSOD were performed as fol- lows. Sodium pyruvate (30 lamol), pH 8.0, potas- sium phosphate buffer (100 ttmol), CoA (0.2 ~tmol), mercaptoethanol (10 #1), soluble cell frac- tion and either fl-alanine (30/ tmol) or fl-AIB (30 #mol) were added to cuvettes in a total volume of 0.9 mi. After varying times of incubation at room temperature during which malonic semialdehyde or methyl malonic semialdehyde were generated, MSOD activity was started by the addition of NAD (1.5 #moi L and appearance of NADH was followed spectrophotometrically. Protein was mea- sured as in [6].

3.5. Mutant isolation Cultures were mutagenised with nitrosoguani-

dine as described Ill. Mutants defective in fl- alanine catabolism were selected as minute col- onies on minimal plates containing fl-alanine

(0.3%) as the major carbon source and either succinate or DL-alanine (0.002%) as a supplemen- tary carbon source allowing slight growth of the desired mutants, followed by checks for lack of growth on fl-alanine. Wild-type revertants were isolated by plating 0.1 ml of overnight broth cul- tures to plates of fl-alanine medium and picking colonies which developed after 60-70 h growth. 1-5 revertants per plate were usually obtained.

4. RESULTS AND DISCUSSION

4.1. Enzymes involved in fl-alanine catabolism BAPAT and MSOD activities were both found

to be present in P. aeruginosa PAO1, and were located almost exclusively (> 95%) within the soluble cell fraction. BAPAT required both pyru- vate and fl-alanine for activity. Production of a-alanine by BAPAT was demonstrated by its isolation as in [2] and chromatographic compari- son with authentic samples, and its identity as the L-isomer was inferred from its lack of activity as a substrate for pig kidney D-amino acid oxidase. Production of malonic semialdehyde was demon- strated and measured as in MATERIALS AND METH- ODS and was the basis of routine assay. MSOD activity was dependent on NAD and CoA. Com- parison of glycerol-grown cells showed that both BAPAT and MSOD synthesis were induced 4-5- fold by growth on fl-alanine (Table 1).

Growth on fl-alanine also induced synthesis of AR and DAD, the pathway for L-alanine catabo-

Table ]

Effect of growth medium on levels of enzymes involved in fl-alanine catabolism

Cells were broken in a Hughes press and fractionated as described in MAI"ERIALS AND METHODS. o-alanine dehydro- genase was measured in the envelope and other enzymes in the .soluble fraction.

C-source in growth medium

Specific activities ( n m o l - m i n - 1 (mg protein)- i )

BAPAT MSOD AR DAD

Glycerol 230 48 2,500 6 ,8-alanine 920 200 16,200 41 DL-alanine 650 103 20,000 81 fl-AIB 1,240 210 18,400 35

[3-alanine pyruvate ~-~NH 3 TRANSAMINASE DEHYDROGENASE

malonic L-alanino semialdehyde

i I DECARBOXYLASE RACEMASE

ace ty l CoA D-alanine - -

Fig. 1. Complete pathway for the catabolism of fl-alanine by P. aeruginosa. Transaminase =fl-alanine: pyruvate aminotrans- ferase (BAPAT); decarboxylase = malonic semialdehyde oxidative decarboxylase (MSOD); dehydrogenase = o-alanine dehydrogenase (DAD); racemase = oL-alanine racemase (AR).

lism in PAO1, with activity levels of 80% and 50%, respectively of those induced by DL-alanine (Table 1). This is very significant, as catabolism of fl- alanine by the BAPAT-MSOD route (Fig. 1) would generate stoichiometric amounts of L-alanine, and the continued operation of the pathway would require a means for the regeneration of pyruvate (the amino acceptor for BAPAT) from L-alanine. The AR-DAD pathway would provide such a regeneration system, and as DAD is respiration- linked [1] would be an additional source of meta- bolic energy.

Unexpectedly, oL-alanine was found to be an inducer of BAPAT and MSOD (Table 1). This appears to be an example of gratuitous product- mediated induction as found, for example, in L- tryptophan catabolism [7].

4.2. Properties of mutants 24 mutants were isolated which failed to grow

on fl-alanine but retained the ability to grow on oL-alanine. In every case, BAPAT activity was found to be absent or reduced to less than 25% of wild-type level. MSOD, AR and DAD activities were all within wild-type ranges. R.evertants with restored ability to grow on B-alanine were iso- lated, and all had regained BAPAT activities com- parable with wild-type. Table 2 summarises the enzymology of BAU1, the mutant most studied, and its revertant BAU1-R.

Another mutant, AOl l , failed to grow on any alanine isomer. Analysis showed it to possess nor- mal levels of BAPAT, MSOD and AR, but to lack

281

Table 2

Enzyme levels in mutants

Cells were grown with 0.3~ glycerol and 0.5~ fl-alanine and broken by sonication. Assays were performed on unfractionated homogenates which accounts for the generally lower specific activities compared with Table 1. For explanation of strains see text.

Strain Specific activities (nmol • min - i (mg protein) - I )

BAPAT MSOD AR DAD

PAO1 660 126 10,100 25 BAU1 0 158 8,600 29 BAU1-R 710 134 7,800 32 A O l l 1.150 196 12,500 0

DAD activity (Table 2). As the AR-defective mutant of Pioli et al. [1] failed to grow on fl- alanine, the essential role in fl-alanine catabolism of all enzymes in the proposed pathway (Fig. 1) except MSOD has now been established by mutant isolation.

The induction of MSOD, AR and DAD by fl-alanine in BAU1 indicates that metabolism of fl-alanine is not required for their induction, and suggests that fl-alanine is itself a direct inducer of the entire pathway.

The results of enzyme induction studies and mutant studies provide very strong evidence for the catabolism of fl-alanine by the scheme shown in Fig. 1.

4. 3. Catabolism of fl-A IB fl-AIB is the a-methyl analogue of fl-alanine.

The two compounds are also metabolically related in that they are the respective products of uracil and thymine catabolism in mammalian cells [8]. They are also substrates for the same inducible transport system in P. aeruginosa [9]. Trans- aminase enzymes which utilise fl-AIB as substrate have previously been reported, e.g., [10], but we are not aware of a previously established complete pathway for fl-AIB catabolism in bacteria. The use of the fl-alanine pathway in the catabolism of fl-AIB by PAO1 is strongly supported by the following results.

PAO1 was found to utilise fl-AIB as a source of carbon and energy for growth. Lag phase after

282

o

~2oo~ A

,ooi/ •

"~ .

25 50 75 100 time of pre-incubation tmin)

Fig. 2. Effect of concentration of malonic semialdehyde and methyl malonic semialdehyde on the rate of their oxidation by malonic semialdehyde oxidative decarboxylase. Cuvenes were preincubated with fl-alanine (curve A) or fl-AIB (curve B) to permit generation of maionic semialdehyde or methyl malonic semialdehyde respectively by the action of BAPAT. Full details are given in text and in MATERIALS AND METHODS.

transfer from glycerol culture was about 2 h and mean generation time on fl-AIB minimal medium was 80 min. Oxidation of fl-AIB by resting ceils was induced by incubation with fl-alanine and vice versa (Table 3). Growth on fl-AIB induced all 4 enzymes of the fl-alanine pathway (Table 1). BAU1 showed only marginal growth on fl-AIB and negligible oxidation of this substrate. The revertant BAU1-R had restored ability to grow upon and oxidise fl-AIB. Substitution of fl-AIB for fl-alanine in the BAPAT assay still resulted in formation of t.-alanine as a reaction product. By analogy, the second product of this reaction was assumed to be methyl malonic semialdehyde, and by use of the linked assay described in MATERIALS AND METHODS it was demonstrated that it was a substrate for MSOD. Fig. 2 (curve B) shows the initial rate of CoA-dependent NAD reduction plotted against time of pre-incubation of fl-AIB and pyruvate with BAPAT. The concentration of substrate required for maximum velocity was ap- proached after a 90-min pre-incubation, and was calculated stoichiometrically from NAD reduction to be about 130 #mol / l . Reduction of NAD was negligible if fl-AIB was omitted from the pre-in- cubation or if extracts of a BAPAT-defective mutant were used. A comparable plot for the

Table 3

Oxidation of fl-alanine and fl-AIB by whole-cell suspensions of PAOI

Oxygen uptake was followed in Gilson Respirometer with 2 ml of cell suspension (E55 o = 1.0) and 0.2 mmol of substrate in each flask. Endogenous rates were 37-40 #l /h, and rates shown have not been corrected for this.

Carbon source for growth

Oxygen uptake (pl. h - i ) with substrates

fl-alanine fl-AIB

Glycerol 43 42 fl-alanine 220 165 ~-AIB 190 140

oxidation of malonic semialdehyde generated by the action of BAPAT on fl-alanine is also shown (curve A). Methyl malonic semialdehyde appears to be oxidatively decarboxylated at about one- quarter the rate of malonic semialdehyde, and presumably giving propionyl CoA instead of acetyl CoA.

As pure optical isomers of fl-AIB were not readily available, the DL-racemic mixture was used in this study. Consequently, it is not possible to say which of the two isomers is the substrate for BAPAT.

REFERENCES

[1] Pioli, D., Venables, W.A. and Franklin, F.C.H. (1976) Arch. Microbiol. 110, 287-293.

[2] Hayaishi, O., Nishizuka, Y., Tatibana, M., Takashita, M. and Kuno, S. (1961) J. Biol. Chem. 236, 781-790.

[3] Bater, A.J. and Venables, W.A. (1977) Biochim. Biophys. Acta 468, 209-226.

[4] Robinson, W.G. and Coon, M.J. (1963) in Methods in Enzymology, Vol. VI (Kolowick, S.P. and Kaplan, N.O., Eds.) pp. 549-553. Academic Press, New York and London.

[5] Julius, M., Free, C.A. and Barry, G.T. (1970) in Methods in Enzymology, Vol. XVII (Tabor, H. and Tabor, C.W., Eds.) pp. 171-176. Academic Press, New York and London.

[61 Lowry, O.H., Rosebrough, N.J., Farr, A.L. and Randall, R.J. (1951) .I. Biol. Chem. 193, 265-275.

[7] Palleroni, N.J. and Stanner, R.Y. (1964) J. Gen. Micro- biol. 35, 319-334.

[8] Yasumitsu, T., Takoa, T. and Kakimoto, Y. (1976) Bio- chem. Pharmacol. 25, 253-258.

[9] Lacoste, A.M., Cassaigne, A. and Neuzil, E. (1977) Bio- chimie 59, 789-798.

[10l Yonaha, K., Toyama, S., Yasuda, M. and Soda, K. (1976) FEBS l,ett. 71, 21-24.