The biosynthesis of ectoine

FEMS MicrobiologYLetters 71 {1990) 157-162 Published by Elsevier

157

FEMSLE 04125

The biosynthesis of ectoine Petra Peters, E.A. Galinski and H . G . Triiper Institut fiir Mikrobiologi¢ i~d BzozechnologJe, Rheimsche Friedrich-Wdhelms-Untt,ersaill Bonn, Meckenheimertlllee 168, J3 Bonn, l, F.R.G.

Received6 March 1990 Revisionreceived8 May 1990 Accepted 10 May lg90 Key words: Ectothiorhodospira halochloris; Halomonas elongata; Compatible solute; Tetrahydropyrimidine; Ectoine bios)'nthesis

1. SUMMARY The biosynthetic pathway of the novel compatible solute ectoine (1,4.5,6-tetrahydro-2-methyl-4pyrimidine carboxylic acid) was studied in the two ext~mely halophillc eubacteria Ectothiorhodo~pira halochloris and Halomonas elongata. The pathway starts with the phosphorylation of L-aspartate and shares its first two enzymatic steps with the biosynthesis of amino acids of the aspartate family: aspartokinasc and L-aspartate-fl-semiaidehyde dehydrogenasc, Evidence is presented for the presence of the enzymes L-diaminobutyric acid transaminase and L-diaminobutyric acid acetyl transferasc and for the new enzyme the ring-forming cctoine synthasc.

2, I N T R O D U C T I O N Halophilic microorganisms living in habitats of high ionic strength may pursue two different

Correspondeno, to: P. Peters. lnstitut tier Miktobiologic und

Biotcchnologi¢, Rhcinischc Friedrich-Wilhdms-Universiliit Bonn, M~kenheirner Alice 168, Bonn 1, F.R.G.

strategies to maintain osmotic balance. Halophilic archaebacteria and some anaerobic heterotrophic eubacteria accumulate various electrolytes [1,2] whereas aerobic chemoorganotrophic eubacteria and phototrophic eubacteria employ "compatible solutes" [1] such as sugars, polyols, hetaines or amino acids [3]. The compatible solute ectoine may be chemically characterized as a heterocyclic amino acid or as a partially hydrogenated pyrimidine derivative ( 1,4,5,6-tetrahydro-2-methyl-4-pyfimidinecarboxylic acid [4]). Ectoine was first discovered in the extremely halophilie phototrophic bacterium Ectothiorhodospira halochloris [4] and has been characterized by ~JC-NMR spectroscopy, mass spectrometry and infrared spectroscopy (IR). It has since been detected in a number of halophilic and halotolerant eubacteria, where it serves as an osmolyte [5]. Moreover, Inbar and Lapidot [6] discussed ectoine as a nitrogen reservoir for the production of antibiotics in Szrepzomyces parvulm'. Ectoine can be dissolved in water quite easily (up to 6 mol/kg water at 4°C), it possesses a non-ionic character at physiological pH-vahies [4] and hence meets the requirements for an organic osmolyte. First approaches towards the elucidation of the biosynthetic pathway o[ ectoine were

0378-1097/q0/$07.50 © 1990 Federation of European MicrobiologicalSocieties

158 L-aspartate

lyslne ~

~

It

asparagine

aspartylphosphate

N-acetyl-aspartate

"~-~sp~tat.e-f/-semlaldchyde

N-acetyl-aapartylphosphate

~ hom-~serin~. ~ mnthionine 4-- 4,.-

;

t h r e o n i n e .,..

3

8

L-diaminobutyric

acid

4

N - a c e t y l - a s p a r t at o- f/semialdehyde

N - a c o t y l - d l n m i n o l m t y r i c acid

;°

ecto|n~

H N.,-"O'I2.~;Hz

IIo

I

c~'L-....-C"-c00~

14 Fig. ]. Biosyntheticpathway of the compatible solute ectoine (thick arrow~). The other branch, involvingenzymes 6 8. is not established, t, aspartokinase; 2. aspanate-fl-semlaldehydedehydrogenase: 3, L-diaminobulyricacid transaminase: 4. l-diaminobulyric acid acetyltransferase; 5, eeloinesynthase; 6,1-asparlate acetyl transferase; 7. N-acetyl aspartokinase: 8, N.acetyl-aspanate-Bsemiald©hydedehydrogenase; 9, N-acetyl-aspartate-~-semialdehyde transan6nase. performed using 13C-labelled isotopes [7]. Fig. 1 displays possible pathways of ¢ctoine biosynthesis which have been proposed by Galinski I7]. The purpose of the present study was the elucidation of the enzymic reactions involved in ectoine biosynthesis,

3. MATERIALS AND METHODS 3.1. Growth media and culture conditions E. halochloris strain DSM 1059 was grown photosynthetically at 40 o C with illumination of 10 000 lux in a medium according to Imhoff and Triiper [8]. Acetate and bicarbonate were used as carbon

soula3es. Cell material of Halomonas elongata strain DSM 2581 (grown on glucose as carbon source)

was kindly supplied by A. Wohlfarth The organism was chosen because it employs e~:toine as the main compatible solute (cytoplasmic concentration between 1-2 M) [5]. This heterotrophic bacterium may therefore serve as a com'~::,ason to the phototrophic E. hah~'hloris which i :,~auces only minor amoums of ec~oin¢. Cells were harvested in the exponential ~rowth phase.

3.2. Preparation of cell-free extracts Cell-free extracts were prepared by passing the cell suspension through a French press. Ccntrifugation of the suspension (Sorval RC 5, 28000 x g, 50 rain, 4°C) and subsequent uhracentrifugation of the supernatant (Beckman L5/50, 200000 × g, 2 h, 6 ° C ) yielded an extract suitable for measure-

159 meres. Protein estimations were made according to the method of Beisenherz et at. [9].

3.3. Enzyme assays Enzyme activity is defined as U =/~mol substrate converted per rain. 3.3.1. Aspartokinase (E.C.2.L2.4.) Activity was estimated on the basis of the hydroxamate-ferriehloride-method [10].

3.3.2, L-aspartate-~-semialdehyde dehydrogenase (E.C,1.ZI.II) Activity was measured as described by Hegeman [11].

3.3.3. L-diaminobutyric acid transaminase (E.C,2.6.1.46) The assay ~ystem contained: 100 mM Tris-HCI buffer, pH 9.1; 20 mM a-oxoglutarate; 20 mM L-diaminobutyrate (L-DABA); 50 .aM pyridoxal phosphate; crude extract (1.0-3.0 mg protein) in a total volume of 1.0 ml. After 5 re.in preincubation at 37°C the reaction was initiated by the addition of substrate; 30 min later 100 #1 20% (w/v) perehloric acid were added and after centrifugation (10 min. 5000 × g) the glutamate concentration of the superuatant was measured using a test combination (Boehringer No. 139092).

3.3.4. t-diaminobutyric acid acetyl transferase The reaction mixture consisted of 20 mM LDABA; 87 #M acetyI-CoA; 160 mM Tris-HCI, pH 9.0; 10 gl crude extract (approx. 300 #g protein) and demineralized water to a total volume of 1.0 ml. After 5 min preincubation at 37°C the depletion ot the thioester bond was monitored at 250 nm in a spectrophotometer (Zeiss PM 6) until no further change in absorbance was observed. 3.3.5. Ectoine .~ynthase The assay system contained 100 mM Tris-HCl, pH 9.0, 7.2 raM a-Nacetyl-diaminobutyrie acid (n-N-Ac-DABA); crude extract (3.0 mg protein) in a total volume of 1.0 ml. After 30 rain incubation (37 ° C) the reaction was stopped by boiling in a water bath for 10 rain. Eetoine was identified in the supernatant by HPLC-analysis. The reversibility was examined by the following test: 100 mM Tris-HCl, pH 9.0; 100 td crude extract (30/:g protein), 5 mM ectoine in a volume of 1.0 ml. After 40 min (37°C) 100 /~1 20% HC104 stopped the reaction. Thin layer chromatography was used for the identification of N-Ac-DABA.

3.3.6. Aspartate acetyl transferase (E, C2.3.1.17) Enzyme activity was tested as described by Maas et al. [12] for Eschertchia coil (ATCC 9637) varying different parameters (temperature, pH, salinity).

3.4. Chemical synthesis of substrates Ozonization of oL-allylglycine as described by Black [13] yielded Dt-aspartate-fl-scmialdehyde (ASA). The reaction time was increased to 3 h b~-cause 13C-NMR spectroscopy revealed large amounts of underivatized substrate after only 100 m~n of ozonization, a- and y-N-aeetyl-diaminobutyric acids were synthesized as described by Leclerc [14]. For the purification and isoaltinn of the two derivatives a Dowex 50 W × 8 cationexehange-coinmn (2 × 15 era) in its protonated form was eluted with a linear perchloric acid gradient (0-2 M). Absorption at 200 nm was monitored by a Gilson/Abimed spectr~hrom M and conductivity by a WTW-conduetivlty meter. The fractions of interest were neutralized and the supernatant containing the a-derivative was reduced to a volume of 5 ml and applied to a Biorad AG 11 A 8 ion retardation-column (2.5 × 63 era) in order to remove inorganic ions. a- and y-NAc-DABA were identified using spectroscopic (13C-NMR spectroscopy, infrared spectroscopy (IR)) and chromatographic methods.

3..5. Identificatwn by ~~C.NMR spectroscopy and infrared spectroscopy Aqueous samples (1 ml containing at least 70 mg of substance) for natural abundance t3C-NMR spectroscopy were supplemented with 0.5 ml D2O as an internal lock signal and 20 #l acetonitrile as a n "nterna 1 I standard. 13 C-NMR spectra were recorded in the pulsed Fourier transform mode with proton noise decoupling on a Brnker (model WP80-FT) spectrometer operating at 20,115 MHz. The identification was confirmed by infrared spectroscopy (PYE Unicorn SP 1100). To distinguish between the a- and y-isomer a chromatographic method was used (see below).

3. 6. Thin layer chromatography Chromatograms were run on silica gel plates (Merck 5534) with the solvent 2-propanol : acetic

160 acid : water (70 : 30 : 20, v/v). The position of the aoctyl group of N-Ac-DABA was determined using a copper-ninhydrin reagent [15]. Moreover vanillin-potassium hydroxide was used as an unspecific reagent [16].

preparation - - starting with 0.475 g DABA - resulted in 14,6 mg pure n-N-Ac-DABA representing a yield of 3.1~. 13C-NMR spectroscopy gave the following allocation of signals for a-NAc-DABA: 177.2, 169.4, 55.1, 39.5, 32.2 and 24.4 ppm. Using the coppcr-ninhydrin-spray [15] thin layer chromatography distinguished n-N-AcDABA (Rf 0.35) and ¥-N-Ac-DABA (Rf 0.92) by their colours. The copper molecule specifically blocks the a-amino group so thal a-N-Ac-DABA can still react with ninhydrin by its free -/-amino group, whereas the y-acctylatcd derivative remains undetected. In addition both substances form yellow coloured spots after spraying with the vanillin-reagent [16].

3.7. High performance liquid chromatography (HPLC) Ectoine synthesis was confirmed on a L D C / Milton Roy HPLC instrument as described by Galinski [17]. A nucleosil 5 NH 2 (Machery and Nagel, Diiren, F.R.G.) column (200 × 8 × 4 i.d,) and 70~ acetonitrile as solvent were used,

4. RESULTS

4.2. Enzyme assays Both models for a presumative biosynthetic F:~thwa) of ectoine (Fig. 1) were examined. As L-aspartate acetyl transferase was not detected in either of the two microorganisms we concluded that the pathway shown on the right side starting with the acetylation is not established (thin arrows). The first two enzymes - - aspartokinase and aspartate-fl-semialdehyde dehydrogenase - - are common enzymes found in other biosynthetic sequences (formation of homoserine, methionine. threonine, isoleucine and diaminopimelic acid). The specific activity of the aspattokinase determined at pH 7.8 was similar for both E. halochloris and H. elongala (Table 1). The specific

4.1. Chemical synthesis of substrates The successful chemical synthesis of L-aspartate-fl-semialdehyde was proved by 13C-NMR spectroscopy. With the aid of a computer simulation (INKA, information system Karlsruhe) ASA was identified by the following signals: 192, 172.4, 53 and 43.6 ppm. L-aspartate (175.5 ppm, 173.2 ppm, 51.6 ppm, 36.1 ppm) and formaldehyde (84.2 ppm) were further products. Thin layer chromatography confirmed these results (L-aspartate Rf 0.19, ASA Rf 0.67). The purification of the a, y-N-Ac-DABA mixture separate "/-N-Ae-DABA (clution volume 36-144 ml) and a-N-Ac-DABA (elution volume 210-228 nil) from substrates and further products such as p-nitrophenol. A typical

Table l Properliesof enzymesinvolvedin ectoinebiosynthesis,measured in extractsof E. hatochlorisand H. elongata(valuesin parenthoes) n.d., not determined. +, enzymepresent but the specificactivitywas not deternuned becauseASA-concentrationwas not esfima|ed 0ee section4.1). Enzyme Aspartate~kinase(l) Asp~xtale-~-semialdehyde d©hydrogenase (2) L-Diaminobutyri¢acid transaminas¢(3) L-Diaminobutydcacid acctyl transferase(4)

Specificactivity (mU/mg protein) 31 (30) + (+ ) 54 (171)

pH-optimum n.d.

Temperature optimum ( o C) n,d.

Kin-value (raM) n,d.

8.7-8.8 (8.7-8.8) 8,2 (7,9)

40-42 (40-42) n.d. n.d.

n.d. 1,2 (I.85)

114

g,2

35-40

3.5

161 activity of the aspartate-/~-semiaidehyde dehydrogenase was not determined becauSe the unpurified ASA solution contained formaldehyde in small amounts which acts as an irreversible inhibitor of the enzyme. Moreover the concentration of ASA was not calculated because ~3C-NMR data of the unpurified solution showed that the synthesis of ASA from DL-allylglycine after Black and Wright [14] was incomplete. Therefore, specific activities were not calculated and the true activity must be higher than measured by this test (194 mU at 40°C, pH 8.8). The enzymes of both microorganisms showed a similar temperature and pH optimum (Table 1). The specific part of the ectoine synthesis, therefore, presumably employs only three characteristic enzymes: L-diaminobutyric acid transaminase, Ldiaminobutyric acid acotyl transferase and ectoine synthase (enzymes 3-5, Table 1, Fig. 1). The L-diaminobutyric acid transaminase was examined thoroughly because it represents the branching point of the ectoinc biosynthetic pathway. The enzyme can only use L-glutamate as amino group donor. The data for the transaminase of E. halochloris are shown in Table 1. In comparison with these results the enzyme of H. elongata had a higher specific activity and a lower pH-optimum (Table 1). The following enzyme catalyzes the specific formation of a-N-Ac-DABA. The final step of the ectoine synthesis was shown to be an enzyme-catalyzed reaction using a-N-Ac-DABA as a substrate. HPLC analysis and thin layer chromatography proved the formation of ectoine. The appropriate enzyme has not been described before. The name "eetoine-synthase" or "n-N-acetyl-diaminobutyric acid dehydratase" is proposed. First examinations ( E. halocMoris ) have shown that the enzyme catalyzes a reversible reaction. No spontaneous formation of eetoine was observed at physiological pH-vahies and 37 o C.

.8-semialdehyde dehydrogenase from both microorganisms under investigation are in agreement with those of other bacteria examined before [10,12,171. The subseqaent enzymes needed for eetoine synthesis seem to be similar in both microorganisms. The L-diaminobutyF.c acid transaminase activity of H. elongata is about 3.2-fold higher than in E. halocMoris. This may be explained by the different levels of ectoine in the two organisms. The main compatible solute of E. halochloris is glycine betaine [17] whereas ectoine is present at a concentration of only 200 mM [7] or 20 m g / g dry weight. H. elongata on the other uses ectoine as its major osmolyte and accumulates a 5-fold higher concentration of ectoine (about 100 m g / g dry weight [5]). Although .the L-DABA-transaminase has also been described for Xanthomonas species [19] the sole use of Lalanine as amino group donor points towards a different function of the enzyme in Xamhomonas species. L-Diaminobutyric acid is the substrate for the enzyme L-diaminobutyric acid acetyl transferase. The product, a-N-acctyl-diaminobutyric acid, is used by a new enzyme catalyzing the ring closure in a reversible and substrate-specific manner. The name ectoine-synthase or a-N-acctyl-diaminobutyric acid dehydratase is proposed. Kinetic prop¢rties of the ectoine synthase could not be exattuned because so far there are no methods to pursue the depletion of ~-N-acetyl-diaminobutydc acid or the increase of ectoine. This biosynthetic pathway represents an efficient sequence of biochemical reactions as only three additional enzymes are necessary to synthesize the compatible solute ectoine. It is therefore not surprising that ectoine, which had previously escaped detection, is widespread in nature and seems to serve as a universal osmolyte for a wide range of halophilic eubaeteria [5,19]. Farther work on ectoine biosynthesis is in progress.

5. DISCUSSION The enzymic reactions of the probable biosynthetic pathway of the compatible solute ectoine were examined in the present study. The properties of the enzymes aspartokinase and aspartat¢-

ACKNOWLEDGEMENTS We thank C. Schmidt and R. Leeves (Department of Chemistry) for performing the ~zC-NMR

162 measurements. This work was supported b y a g r a n t of the Deutsche Forschungsgemeinschaft and by the Fonds der Chemischen lndustrie.

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[15] Brov,aX D,H. and Fowden, L. (1966) Phytochemlstry 5, 881-886. [16] CMruzon,C. and Giltrow. J. (1953) Nature 172. 356. [17] Galinski, E.A. 0987) in Bioreaclors and Biotransformalions (Moody, G.W. and Baker, P.B., eds.), pp. 201-212, Elsevier, London, New York. [18] Truffa-Bachi, P., van Rapenbusch, R.. Janin, J., Gros, C. and Cohen, G.N. (1968) Eur. J. Biochem. 7. 401-407. [19[ Rajagopal Rao, R., Hariharan, K. and Vijayakshmi, K.R. (1969) Biochem. J. 114,107-113. [20] Tfdper, H.G,, Sere[in, J., Wohlfarth. A.. MUller, E. and Galinski, E.A. (1990) m General and Applied Aspects of Halophilic Microorganisms (Rodrigues-Valera. F.. ed.). Plenum Press, in press.