Báo cáo khoa học: Two beta-alanyl-CoA:ammonia lyases in Clostridium propionicum pot

Two beta-alanyl-CoA:ammonia lyases in Clostridium propionicum Gloria Herrmann 1 , Thorsten Selmer 1 , Holly J. Jessen 2 , Ravi R. Gokarn 2 , Olga Selifonova 2 , Steve J. Gort 2 and Wolfgang Buckel 1 1 Laboratorium fu ă r Mikrobiologie, Fachbereich Biologie, Philipps-Universita ă t, Marburg, Germany 2 Biotechnology Development Center, Cargill Incorporated, Minneapolis, MN, USA b-Alanine (3-aminopropionate) is formed as an end product of uracil fermentation [1] and by decarboxyla- tion of l-aspartate catalysed by the bacterial enzyme as- partate 1-decarboxlyase (a-decarboxlyase; EC 4.1.1.11) as a precursor of (R)-pantothenate and hence coen- zyme A [2,3]. Furthermore, b-alanine, which can be considered as the homologue of glycine, is a constitu- ent of homoglutathione from pea nodules and is important for nitrogen xation [4]. The hydroxyradical scavenging peptides l-carnosine and carnicine [5] as well as the antibiotics efrotomycin [6] and destruxin [7] also contain b-alanine. The only organism known to ferment this compound is Clostridium propionicum [8]. 3 b-Alanine ỵ 2H 2 O ẳ 3NH ỵ 4 ỵ Acetate ỵ CO 2 ỵ 2 Propionate 1ị where, DG ẳ )155 kJặmol acetate )1 (for DG see [9] and Results). C. propionicum has been isolated from black mud of the San Francisco Bay as an organism fermenting Keywords acryloyl-CoA; beta-alanyl-CoA; CoA- transferase; L-alanine and beta-alanine fermentation; pentamer Correspondence W. Buckel, Laboratorium fu ă r Mikrobiologie, Fachbereich Biologie, Philipps-Universita ă t, 35032 Marburg, Germany Fax: +49 64212828979 Tel: +49 6421 28 21527 E-mail: buckel@staff.uni-marburg.de (Received 15 October 2004, revised 24 November 2004, accepted 7 December 2004) doi:10.1111/j.1742-4658.2004.04518.x The fermentation of b-alanine by Clostridium propionicum proceeds via activation to the CoA-thiol ester, followed by deamination to acryloyl- CoA, which is also an intermediate in the fermentation of l-alanine. By shifting the organism from the carbon and energy source a-alanine to b-alanine, the enzyme b-alanyl-CoA:ammonia lyase is induced 300-fold (% 30% of the soluble protein). The low basal lyase activity is encoded by the acl1 gene, whereas the almost identical acl2 gene (six amino acid substi- tutions) is responsible for the high activity after growth on b-alanine. The deduced b-alanyl-CoA:ammonia lyase proteins are related to putative b-aminobutyryl-CoA ammonia lyases involved in lysine fermentation and found in the genomes of several anaerobic bacteria. b-Alanyl-CoA:ammo- nia lyase 2 was puried to homogeneity and characterized as a heteropen- tamer composed of 16 kDa subunits. The apparent K m value for acryloyl- CoA was measured as 23 4 lm, independent of the concentration of the second substrate ammonia; k cat K m was calculated as 10 7 m )1 ặs )1 . The apparent K m for ammonia was much higher, 70 5 mm at 150 lm acry- loyl-CoA with a much lower k cat K m of 4 ã 10 3 m )1 ặs )1 . In the reverse reaction, a K m of 210 30 lM was obtained for b-alanyl-CoA. The elim- ination of ammonia was inhibited by 70% at 100 mm ammonium chloride. The content of b-alanyl-CoA:ammonia lyase in b-alanine grown cells is about 100 times higher than that required to sustain the growth rate of the organism. It is therefore suggested that the enzyme is needed to bind acryloyl-CoA, in order to keep the toxic free form at a very low level. A formula was derived for the calculation of isomerization equilibra between l-alanine b-alanine or d-lactate 3-hydroxypropionate. Abbreviations acl1 and acl2, genes encoding b-alanyl-CoA:ammonia lyase 1 and 2. FEBS Journal 272 (2005) 813821 ê 2005 FEBS 813 l-alanine to ammonia, acetate, CO 2 and propionate [10,11], according to the so-called nonrandomising pathway [12,13], with acryloyl-CoA as the characteristic intermediate [14,15]. As outlined in Fig. 1, 3 mol l-alanine are oxidatively deaminated to pyruvate by the combined action of alanine transaminase (EC 2.6.1.2) and glutamate dehydrogenase (EC 1.4.1.2). Pyruvate (1 mol) is further oxidized to acetyl-CoA used for sub- strate level phosphorylation. In order to balance the redox equilibrium, 2 mol pyruvate are reduced to pro- pionyl-CoA via (R)-lactate, (R)-lactyl-CoA and acry- loyl-CoA. Propionate is liberated by CoA-transfer to (R)-lactate mediated by propionate (lactate) CoA- transferase (EC 2.8.3.1), which also uses acrylate and acetate as substrates [16]. While the NAD-dependent reduction of acryloyl-CoA to propionyl-CoA is irre- versible under physiological conditions [17], the other reactions involved in the transformation of pyruvate to acryloyl-CoA are reversible and may therefore also operate in the opposite direction. Indeed, it has been shown that washed cells of C. propionicum not only ferment pyruvate and (R)-lactate, but also acrylate to acetate, CO 2 and propionate [11,18]. Considering the ability of C. propionicum to oxidize acrylate to acetate, only two additional enzymes are required in order to allow the organism to grow on b-alanine: the substrate is activated to its CoA-thiol ester and subsequently ammonia is eliminated from b-alanyl-CoA to yield acryloyl-CoA. One of these enzymes, b-alanyl-CoA:ammonia lyase has been parti- ally purified and initially characterized with artificial substrates as an acryloyl-CoA aminase [19], while nothing is known at present about the activation of b-alanine. In this communication we report the purifi- cation of b-alanyl-CoA:ammonia lyase to homogeneity and its biochemical characterization with natural sub- strates. Two genes encoding b-alanyl-CoA:ammonia lyases and the gene of a putative b-alanine CoA-trans- ferase have been found in C. propionicum. Results b-Alanyl-CoA:ammonia lyase was purified from cells of C. propionicum grown on b-alanine as sole carbon and energy source. The decrease in the absorbance at 259 nm due to the addition of ammonia to the double bond of acryloyl-CoA (De ¼ 6.4 mm )1 Æcm )1 ) was used to monitor activity. The specific activities in cell-free extracts of b-alanine grown cells (143 UÆmg )1 ) were over 300-fold higher than in those obtained for dl-alanine grown cells (0.44 UÆmg )1 ). As shown in the SDS ⁄ PAGE of Fig. 2, a strong Coomassie-stained band below 20 kDa accompanied the high specific activity. The enzyme was purified by single anion Fig. 1. Proposed pathway of L-alanine and b-alanine fermetation. Fd and Fd – , oxidized and reduced ferredoxin, respectively; Pct, propionate (lactate) CoA-transferase; Act, b-alanine CoA-transferase; Acl, b-alanyl- CoA:ammonia lyase. Beta-alanyl-CoA:ammonia lyases G. Herrmann et al. 814 FEBS Journal 272 (2005) 813–821 ª 2005 FEBS exchange chromatography on Source 15Q in very high yields, usually more than 50 mg from 10 g of wet packed cells (Fig. 2 and Table 1). The subunit mole- cular mass of 15 999 ± 8 Da was determined by MALDI-TOF MS and the native molecular mass of 75 kDa was estimated by size exclusion chromatogra- phy, suggesting a homopentameric structure (a 5 ) of the enzyme, which has been recently confirmed by X-ray crystallography (K Reuter & T Selmer, unpublished data). The N-terminal amino acid sequence of purified b-alanyl-CoA:ammonia lyase was used to clone its gene. Surprisingly two almost identical genes were thereby detected. The gene acl1 encoded a 145 amino acid protein and acl2 encoded a 144 amino acid pro- tein. Both genes were heterologously expressed in Escherichia coli and cell-free extracts of these cells were assayed for enzymatic activity. The mass spectrometric detection of b-alanyl-CoA formed in the presence of acryloyl-CoA and ammonia strongly suggested that the proteins encoded by acl1 and acl2 are both func- tional b-alanyl-CoA:ammonia lyases (lyase 1 and 2). The observed mass of the purified enzyme (15 999 ± 8 Da) is in accord with the predicted mass of 16 004 Da for lyase 2, the protein encoded by acl2, whereas the mass of lyase 1 was calculated as 16 205 Da. Furthermore, the peptide map of the isola- ted protein also matched the sequence of lyase 2. How- ever, we cannot exclude the presence of some lyase 1 (< 0.5%) in this purified enzyme. Attempts to charac- terize the ammonia lyase from dl-alanine grown cells were only partially successful, certainly due to the low expression level of acl1. Mass spectrometry revealed the presence of a 16.2 kDa protein in the impure prep- arations, indicating that lyase 1 accounts for the basal activity in the absence of b-alanine. Both sequences of the b-alanyl-CoA:ammonia lyases from C. propionicum are very similar to those of pro- teins with unknown function found in the genomes of Fusobacterium nucleatum [20], Clostridium tetani [21], Thermoanaerobacter tengcongensis [22] and Porphyro- monas gingivalis [23] (41–46% identity). While the gene for acl1 appears to be devoid of neighbouring genes in the genomic DNA of C. propionicum, the acl2 gene is apparently expressed in context with two other genes. Downstream of the acl2 gene a second ORF (act1) was identified, encoding a 397 amino acid protein with a predicted molecular mass of 43 853 Da. The encoded protein showed significant similarities (35–51% iden- tity) to several proteins in the genomic sequences databases of unknown function and to formyl-CoA- oxalate CoA-transferase from Oxalobacter formigenes (up to 38% identity [24]). Therefore, the gene probably encodes b-alanine CoA-transferase, which is required for the initial activation of b-alanine. Downstream of the transferase gene, a third ORF was partially se- quenced, which showed some similarity to merR of Bacillus subtillis and may be the transcription initi- ator ⁄ regulator for b-alanine fermentation (bar). The coding DNA sequences for acl1, acl2 and act1 as well as the partial sequence of the regulator have been deposited in the EMBL nucleotide sequence database under the accession numbers AJ715481 (acl1) and AJ715482 (acl2, act and bar). b-Alanyl-CoA:ammonia lyase 2 from C. propionicum was used to determine the kinetic properties with the natural substrates acryloyl-CoA, ammonia and b-ala- nyl-CoA (Table 2). In order to allow a wider variation of the acryloyl-CoA concentration, the disappearance of the double bond in acryloyl-CoA was monitored at Fig. 2. SDS ⁄ PAGE of b-alanyl-CoA:ammonia lyase preparations. The lanes contained: 1, marker proteins with molecular masses (kDa); 2, cell free-extract of L-alanine grown cells (18 lg protein); 3, cell free-extract of b-alanine grown cells (18 lg protein); 4, eluate from Source 15Q (2.8 lg of pure b-alanyl-CoA ammonia lyase). Table 1. Purification of the b-alanyl-CoA:ammonia lyase from C. propionicum. Cell-free extract prepared from 2 g b-alanine grown wet cell paste; 1 U total activity ¼ amination of 1 lmol acryloyl- CoAÆmin )1 . Purification step Total Protein (mg) Total activity (U) Specific activity (UÆmg protein )1 ) Yield (%) Cell-free extract 183 22661 124 100 Source 15Q 10 10208 1033 45 G. Herrmann et al. Beta-alanyl-CoA:ammonia lyases FEBS Journal 272 (2005) 813–821 ª 2005 FEBS 815 280 nm (De ¼ 3.5 mm )1 Æcm )1 ) in potassium phosphate pH 7.5 using ammonium chloride at concentrations up to 100 mm. Irrespectively of the ammonium chloride concentration, K m ¼ 23 ± 4 lm for acryloyl-CoA was observed, while the specific activities increased with increasing ammonium chloride concentrations (up to % 1000 UÆmg )1 ); at 100 mm NH 4 Cl the k cat ⁄ K m ¼ 10 7 m )1 Æs )1 . A much higher K m ¼ 70 ± 5 mm and a 2.3 · 10 3 -times lower k cat ⁄ K m was measured for NH 4 Cl (at 150 lm acryloyl-CoA). While the addition of ammonia to the double bond of acryloyl-CoA was readily measurable, the elimination of ammonia from b-alanyl-CoA was not easily detected. Starting with b-alanyl-CoA, only trace amounts of acryloyl-CoA (< 1%) have been observed by reverse phase-HPLC. In contrast, a rapid incorporation of deuterium into b-alanyl-CoA has been found by mass spectrometry when the reaction was carried out in deuterium- enriched water (data not shown). These observations suggest that the equilibrium of the reaction strongly favours b-alanyl-CoA over acryloyl-CoA even in the initial absence of ammonia. The elimination of ammonia from b-alanyl-CoA, however, was readily measured when the acryloyl-CoA reductase complex was used to trap acryloyl-CoA [17]. In order to avoid NADH-oxidase activity of the reduc- tase complex under air, the assay was performed under strict anoxic conditions using reduced methylviologen as the electron donor. Because the reduction of acryloyl- CoA to propionyl-CoA is irreversible under physiologi- cal conditions, the acryloyl-CoA reductase allowed the ammonia lyase-dependent conversion of b-alanyl-CoA to propionyl-CoA, which was coupled to the oxidation of the dye (e 604nm ¼ 2 · 13.6 mm )1 Æcm )1 ). Using this assay, K m ¼ 210 ± 30 lm was obtained for b-alanyl- CoA. The elimination of ammonia appeared to be inhib- ited by ammonium chloride; 70% inhibiton was observed at 100 mm, whereas the K m for b-alanyl-CoA remained constant. The addition of a nucleophile to the double bond of acryloyl-CoA seems to be restricted to ammonia and methylamine. The products were exam- ined by MALDI-TOF mass spectrometry, which gave the expected masses for b-alanyl-CoA (840 Da) and b-(methylamino)propionyl-CoA (854 Da ¼ 840 + 14). Hydroxylamine, glycine, water and hydrogensulfide were not suitable substrates for lyase 2. In the incuba- tions with water, glycine and hydrogensulfide, the unchanged acryloyl-CoA could be recovered, whereas with hydroxylamine cleavage of the thiol ester was observed. As judged by enzymatic assays, the enzyme exhibited activity with both crotonyl-CoA and meth- acryloyl-CoA. The formation of the predicted products 3-aminobutyryl-CoA and 3-aminoisobutyryl-CoA was confirmed by mass spectrometry. Calculation of the Gibbs free energy of b-alanine formation (DGƒ°) The two alanine isomers differ mainly by the pK of the carboxyl group (pK A ¼ 2.35 for l-a-alanine and pK B ¼ 3.60 for b-alanine [25]) and by the stereochem- istry. Hence the equilibrium between the protonated forms dl-a-alanine and b-alanine should be close to 1.0, whereas that between chiral l-a-alanine (AH + ) and achiral b-alanine (BH + ) should be 2.0, whereby the factor 2 takes into account that the formations of l- and d-a-alanine from b-alanine are equal [26]: AH þ ! BH þ ; K ¼½BH þ =½AH þ ¼2: Equilibrium between the zwitterionic forms of l-a- alanine (A) and b-alanine (B): A ! B; K eq ¼½B=½A; with K B ¼ [B] · [H + ] ⁄ [BH + ], K A ¼ [A] · [H + ] ⁄ [AH + ] and [BH + ] ⁄ [AH + ] ¼ 2: K eq ¼ 2 K B =K A pK eq ¼Àlog2þpK B ÀpK A ¼À0:30þ3:60 À2:35 ¼þ0:95 K eq ¼ 0.11, favouring l-a-alanine. With DG° ¼ –RT ln K eq ¼ )5.7 log K eq (kJÆmol )1 )the DGƒ° of b-alanine ()366.1 kJÆmol )1 ) is more positive Table 2. Kinetic constants of b -alanyl-CoA:ammonia lyase 2 of C. propionicum. Apparent molecular mass of 16 kDa was used to calculate k cat values. Substrate Cosubstrate ⁄ Inhbitor Apparent K m (mM) V max (UÆmg )1 ) k cat (s )1 ) k cat ⁄ K m (lM )1 s )1 ) Acryloyl-CoA 50 m M NH 4 Cl 0.023 ± 0.004 730 195 8.5 Acryloyl-CoA 100 m M NH 4 Cl 0.026 ± 0.004 956 255 9.8 NH 4 Cl 150 lM Acryloyl-CoA 70 ± 5 1127 301 0.0043 b-Alanyl-CoA 0.21 ± 0.03 95 25 0.12 b-Alanyl-CoA 10 m M NH 4 Cl 0.23 ± 0.03 81 b-Alanyl-CoA 100 m M NH 4 Cl 0.16 ± 0.07 27 Beta-alanyl-CoA:ammonia lyases G. Herrmann et al. 816 FEBS Journal 272 (2005) 813–821 ª 2005 FEBS by about +5.4 kJÆmol )1 than that of l-a-alanine ()371.5 kJÆmol )1 [9]). Discussion The results presented in this work show clearly that the introduction of b-alanine into the acryloyl-CoA fermentation pathway of C. propionicum occurs at the CoA-thiol ester level. This is demonstrated by the 300-fold induction of b-alanyl-CoA:ammonia lyase 2 (Acl2) after growth of C. propionicum on b-alanine. The high lyase activity is caused by the expression of the acl2-gene, whereas the low activity found in dl-alanine grown cells stems from the acl1-gene, which is expressed only to a low level. The ammonia lyase 2 appears to be coinduced with a putative b-alanine CoA-transferase required for activation of the b-amino acid (Act). The enzyme is a class III CoA-transferase [27] different from the already known class I propion- ate CoA-transferase from C. propionicum [16], which does not accept b-alanine as substrate. Preliminary results indeed showed the expression of the act-gene in C. propionicum, but only in b-alanine grown cells (T Selmer, unpublished observations). Thus the physio- logical function of the b-alanyl-CoA ammonia lyase 1 remains to be established. Possibly the lyase 1 could provide b-alanyl-CoA from acryloyl-CoA and ammo- nia for the synthesis of unknown peptides. The synthe- sis of pantothenate requires free b-alanine [2,3], but how it is formed from the thiol ester in the absence of b-alanine CoA-transferase is not known. Among the most striking features of b-alanine fer- mentation in C. propionicum is the extraordinarily high expression level of the acl2-gene in media supplemented with b-alanine. In fact, it can be estimated that the lyase 2 accounts for 30% of the total cell protein under these conditions. It still remains an enigma why the organism produces such a high level of a functional enzyme. The cell-free extract catalyses the elimination of ammonia with the high specific activity of 144 UÆmg )1 ; therefore 100-times lower concentrations of the enzyme could eas- ily account for the growth rates observed on b-alanine. It is known that often the activity of enzymes exceeds that necessary for the observed substrate fluxes, but the occurrence of a highly active enzyme, which comprises 30% of the total soluble protein, is unusual and requires a special explanation. The product of ammonia elimin- ation, acryloyl-CoA, is a very strong electrophile, much stronger than crotonyl-CoA, and reacts readily with nucleophilic groups in essential biomolecules (thiols, enzymes and nucleic acids). The large quantities of b-alanyl CoA:ammonia lyase may be necessary to bind acryloyl-CoA, in order to keep the free form of this reactive thiol ester in the cell at a very low concentra- tion. In line with this proposal it has been shown that the K m values of acryloyl-CoA reductase (2 ± 1 lm) [17], and ammonia lyase 2 (23 ± 4 lm) (this paper), are very low, one to two orders of magnitude lower than the K m value of b-alanyl-CoA (210 ± 30 lm). On the other hand the relatively high amount of acryloyl-CoA, which may be stored by the ammonia lyase, ensures an effi- cient hydration to (R)-lactyl-CoA, necessary for the oxidative branch of the fermentation pathway (Fig. 1). The observed promiscuity of the ammonia lyase 2 with respect to the enoyl-CoA derivatives used as ammonia (and methylamine) acceptors may offer inter- esting technical applications for the enzyme. Enantio- merically pure, bifunctional b-amino C 4 -compounds like 3-aminobutyrate or 3-aminoisobutyrate are inter- esting building blocks in preparative organic chemistry. The enzyme-mediated addition of ammonia to meth- acryloyl-CoA will very likely result in only one of the two possible enantiomers of 3-aminoisobutyryl-CoA and should therefore offer a synthetic route for de novo synthesis of chiral centres starting from commercially available bulk chemicals. However, the purification and characterization of suitable CoA-transferases will be required, in order to allow the synthesis to operate on a commercially meaningful scale. The analysis of b-alanine CoA-transferase from C. propionicum may be helpful in this respect and its purification and char- acterization is currently in process in our laboratory. F. nucleatum, whose genome has been sequenced [20], is known to ferment l-lysine via 3,5-diaminohexanoate to ammonia, acetate and butyrate [28,29]. This pathway involves the reversible deamination of 3-aminobutyryl- CoA to crotonyl-CoA [30], a reaction also catalysed by b-alanine ammonia lyase. It is therefore most likely that the deduced fusobacterial protein, which is homologous to the lyases 1 and 2, functions as b-aminobutyryl-CoA ammonia lyase. Probably the deduced homologous amino lyases in C. tetani [21], T. tengcongensis [22] and P. gingivalis [23] also use b-aminobutyryl-CoA as pre- ferred substrate, as these organisms contain additional genes coding for enzymes of the lysine fermentation pathway. The Gibbs free energy DG°¢ of fermentations like that of b -alanine to ammonia, CO 2 , acetate and propionate (Eqn 1) can be calculated readily from the DGƒ° values (Gibbs free energies of formation) of the reaction partners. Unfortunately, only the DGƒ° value of l-a-alanine but not that of b-alanine is available [9]. Therefore, we assume that DGƒ° of the protonated forms of dl-a-alanine and b-alanine are equal, i.e. that the interactions of the ammonium groups with the carboxylic acid groups mainly influence the pK values G. Herrmann et al. Beta-alanyl-CoA:ammonia lyases FEBS Journal 272 (2005) 813–821 ª 2005 FEBS 817 of the carboxylates (pK a-alanine ¼ 2.35; pK b-alanine ¼ 3.60). In addition the difference in stereochemistry has to be taken into account [26]. These considerations lead towards the equilibrium between chiral l-a- and achiral b-alanine to K eq ¼ 2 · K b-alanine ⁄ K a-alanine ¼ 0.11, DGƒ° ¼ )366.1 kJÆmol )1 for b-alanine and DG°¢ ¼ )155 kJÆmol )1 acetate (Eqn 1). A similar calcu- lation can be made for the equilibrium between chiral d-lactate (pK ¼ 3.8) and achiral 3-hydroxypropionate (pK ¼ 4.5), yielding K eq ¼ 0.4. This value might be useful for the design of a 3-hydroxypropionate produc- tion pathway from carbohydrates [31]. The maximum possible amount of conserved ATP in fermentations can be calculated by dividing the DG°¢ value by )75 kJÆmol )1 [9]. Hence, in the case of b-alanine fermentation (Eqn 1) up to )155 ⁄ )75 % 2 mol ATPÆmol acetate )1 can be conserved. As shown in Fig. 1, however, only one ATP is conserved via sub- strate level phosphorylation from acetyl-CoA obtained by pyruvate oxidation. Therefore, either the other possible ATP is wasted or there is an additional elec- tron transport phosphorylation. A possible function of acryloyl-CoA as terminal acceptor in an electron trans- port phosphorylation similar to fumarate reductase [32] appears unlikely as it has been shown that acry- loyl-CoA reductase is a soluble enzyme, which uses NADH rather than menaquinol as electron donor. Growth experiments with two lactate fermenting organisms confirm the conservation of only one ATP ⁄ acetate via the acryloyl-CoA pathway [33]. Clos- tridium homopropionicum ferments lactate via acryloyl- CoA (Fig. 1) and its growth yield (2.5 g dried cellsÆmol lactate )1 ) is less than half as high that of Propionibac- terium freudenreichii (6.0 gÆmol lactate )1 ), which uses another pathway via the coenzyme B 12 -dependent methylmalonyl-CoA mutase. The latter pathway com- prises additional electron transport phosphorylation via the oxidation of reduced ferredoxin and NADH by fumarate via menaquinol [32]. Hence, the low ATP yield in the b-alanine fermentation by C. propionicum should increase the catabolic substrate flux, which in the presence of high b-alanine concentrations enhances the growth rate [33] and probably keeps the concentra- tion of the toxic acryloyl-CoA at a low level. Experimental procedures Materials Clostridium propionicum (DSM 1682) was obtained from the Deutsche Sammlung fu ¨ r Mikroorganismen und Zellkulturen (DSMZ, Braunschweig, Germany). Sequencing grade pro- teases and citrate synthase were from Roche (Mannheim, Germany). Coenzyme A (tri-lithium salt) was from ICN Biomedicals (Eschwege, Germany) and crotonyl-CoA from Sigma-Aldrich Chemie GmbH (Mu ¨ nchen, Germany). All other chemicals were of the highest available grade. Synthesis of acyl-CoA substrates Acetyl-CoA, butyryl-CoA and propionyl-CoA were syn- thesized from CoASH with a small excess of their corres- ponding anhydrides according to the method of Simon & Shemin [34]. Acryloyl-CoA and methacryloyl-CoA were prepared enzymatically from 1 mm acetyl-CoA and freshly neutralized acid (100 mm) using propionate CoA-trans- ferase [14] (10 mUÆmL )1 )in50mm potassium phosphate, pH 7.0. b-Alanyl-CoA and b-aminoisobutyryl-CoA (3-amino-2-methylpropionyl-CoA) were prepared from 1mm acetyl-CoA, 100 mm acrylate or methacrylate and ammonium chloride (100 mm) using propionate CoA- transferase (10 mUÆmL )1 ) and b-alanyl-CoA:ammonia lyase (1 UÆmL )1 )in50mm potassium phosphate, pH 7.0. All CoA derivatives were purified using a C-18 cartridge as des- cribed earlier [35], freeze-dried and stored at )80 °C. Cultivation and storage of C. propionicum The microorganism was cultivated in a complex medium containing either dl-alanine or b-alanine as major energy source as described previously [14]. Freshly prepared anoxic media were inoculated with 5–20% stationary or late exponential precultures and grown for 24–36 h at 37 °C. The cells were harvested by centrifugation and stored at )80 °C. Preparation of cell free extracts Frozen cells (2 g) were suspended in 25 mm Tris ⁄ HCl, pH 7.5, 1 mm dithiothreitol, 1 mm MgCl 2 ,1mm EDTA (buffer A). Cells were broken by sonication on ice and cell debris was removed by centrifugation for 1 h at 100 000 g at 4 °C. The clear supernatant was used immediately. Purification of b-alanyl-CoA:ammonia lyase All purification steps were carried out at 4 °C under air. Cell free extracts of b-alanine grown cells were applied on a Source 15Q (1.6 ⁄ 20) column (Amersham-Pharmacia, Freiburg, Germany) equilibrated with buffer A. The col- umn was developed using a linear gradient of 0–500 mm NaCl in buffer A. Fractions containing activity were pooled, concentrated and desalted by repetitive membrane centrifugation using Vivaspin 10 kDa cut-off concentrators (Vivascience, Hannover, Germany). The final preparation was stored at )20 °C. b-Alanyl-CoA:ammonia lyase activ- ity was measured following the decrease in absorbance of Beta-alanyl-CoA:ammonia lyases G. Herrmann et al. 818 FEBS Journal 272 (2005) 813–821 ª 2005 FEBS acryloyl-CoA at 259 nm. The assay mixture contained 100 mm triethanolamine ⁄ HCl, pH 8.0, 100 mm ammonium chloride and 20 lm acryloyl-CoA. The catalytic activity was calculated using a De 259nm ¼ 6.4 m m )1 Æcm )1 between acryloyl-CoA (22.3 mm )1 Æcm )1 ) and b-alanyl-CoA (15.9 mm )1 Æcm )1 ). N-terminal sequencing Purified protein (1 nmol) was loaded on a Supelcosil- LC3DP (4.6 · 250 mm) column (Sigma-Aldrich) equili- brated with 0.1% (v ⁄ v) trifluoroactetic acid. The column was developed using a linear gradient from 0 to 85% (v ⁄ v) acetonitrile. Elution of the protein was monitored at 280 nm. The protein was collected manually and aliqu- ots (100 pmol) were N-terminally sequenced by Edman degradation and analysed by MALDI-TOF MS. Peptide mapping HPLC-purified protein was subjected to reduction with di- thiothreitol and S-carboxymethylation with sodium iodo- acetate [36]. The proteins were desalted by size exclusion chromatography on Sephadex G25 equilibrated with 10% (v ⁄ v) acetonitrile in 50 mm ammonium acetate, pH 8.0. Aliquots (100 pmol) were digested with 1% (w ⁄ w) sequen- cing grade endoproteases (trypsin, AspN, GluC, LysC, ArgC) at 37 °C for 4 h. The samples were analysed by MALDI-TOF MS [16]. Determination of the gene sequences of b-alanyl-CoA:ammonia lyases 1 and 2 The 35 amino acid N-terminal sequence of the b-alanyl- CoA:ammonia lyase 2 was used to design degenerate prim- ers with which the corresponding DNA from genome of C. propionicium was amplified. Genomic DNA from C. pro- pionicum was isolated using the Gentra Gram-positive genomic DNA isolation procedure (Gentra Systems, Min- neapolis, MN, USA). The primers: 5¢-ATGGTWGGY AARAARGTWGT-3¢ and 5¢- TCRCCCCAYTGRTTWA CRAT-3¢ were used in a touchdown PCR program with annealing temperatures of 58 °Cto52°C. A 100 bp PCR product was purified from an agarose gel (Qiagen, Valen- cia, CA, USA), ligated into pCRII-TOPO vector, trans- formed into TOP10 E. coli cells using a TOPO cloning procedure (Invitrogen, Carlsbad, CA, USA), and sequenced using vector primers. Primers for genome walking both upstream and down- stream of the gene fragment were designed using the por- tion of the nucleic acid sequence internal to the degenerate primers. The primer sequences GSP1F: 5 ¢-GTACATCATT TAATGATGAGCGCAAAAGATG-3¢; GSP2F: 5¢-GAT GCTCACTATACTGGAAACTTAGTAAAC-3¢; GSP1R: 5¢-ATTCTAGCGCCGTTTACTAAGTTTCCAG-3¢; and GSP2R: 5¢-CCAGTATAGTGAGCATCTTTTGCGCTCA TC-3¢ were used, where GSP1F and GSP2F are primers facing downstream, GSP1R and GSP2R are primers facing upstream, and GSP2F and GSP2R are primers nested inside GSP1F and GSP1R, respectively. Genome walking libraries were constructed according to the Clontech Uni- versal GenomeWalker TM Kit User Manual (Clontech Laboratories, Palo Alto, CA, USA), with the exception that the restriction enzymes SspI and HincII were used in addi- tion to DraI, EcoRV, and PvuII. PCR was conducted in a PerkinElmer 9700 Thermocycler using the following reac- tion mix: 1· XL Buffer II, 0.2 mm each dNTP, 1.25 mm Mg(OAc) 2 , 0.2 lm each primer, 2 units of rTth DNA polymerase XL (Applied Biosystems, Foster City, CA, USA), and 1 lL of library per 50 lL reaction. Second round PCR product was separated on an agarose gel, puri- fied, and cloned as described previously: a 1.4 kb DraI band and 1.5 kb HincII band were cloned for the forward reactions, and a 0.8 kb EcoRV band and 2.0 Kb HincII band were cloned for the reverse reaction. Sequencing of the clones showed that two very homologous b-alanyl- CoA:ammonia lyase genes had been amplified. A second round of genome walking and cloning was con- ducted, as described above, to obtain the full sequence of the second b-alanyl-CoA:ammonia lyase homologue. The prim- ers GW2GSP1R, 5¢-TTATTGAGGGTGCTTTGCATCCT TGAAG-3¢, and GW2GSP2R, 5¢-AAGGCTGCCTGTTG CAGTACCACAAAG-3¢ were used. Bands (1.7 kb) were cloned from second round PCR product from the DraI and SspI libraries. The sequence of the second homolog corres- ponded to the purified b-alanyl-CoA:ammonia lyase protein. Both acl genes were amplified by PCR with the follow- ing primers: aclNdeF: 5¢-GGGAATTCCATATGGTAGG TAAAAAGGTTGTACATC-3¢, acl1BamR: 5¢-CGACG GA-TCCATTCGTCCGCTTGAATAACTAAAG-3¢, acl2- BamR: 5¢-CGACGGATCCCGAAAA-TGTCACCAAAA ATTATTGAG-3¢. The aclNdeF forward primer was the same for both genes as the genes were identical at the beginning. PCR was conducted in a PerkinElmer 2400 Thermocycler using Pfu Turbo polymerase (Stratagene, La Jolla, CA, USA) according to the manufacturer’s instructions. PCR was performed under the following conditions: initial denaturation step 94 °C for 2 min; Fol- lowed by 25 cycles of 94 °C for 30 s, 55 °C for 30 s, 72 °C for 2 min; final extension at 72 ° C for 7 min. Obtained PCR products were digested with NdeI and BamHI restriction enzymes and gel purified using a Qiagen Gel Extraction Kit (Qiagen). The genes were cloned in pET11a vector digested with NdeI and BamHI as well. Resulting plasmids pACL-1 and pACL-2 were transformed into BL21(DE3) cells to study gene expression. DNA sequence obtained downstream of the second b-alanyl-CoA:ammonia lyase homologue indicated the G. Herrmann et al. Beta-alanyl-CoA:ammonia lyases FEBS Journal 272 (2005) 813–821 ª 2005 FEBS 819 possible presence of a partial coding sequence for a CoA- transferase. A third round of genome walking was conduc- ted to obtain the full gene sequence of this downstream open reading frame. Genome walking was conducted using primers GW2GSP1F, 5¢-CTATGTAAAGCAATGGGCAG AGAGGATTTG-3¢, and GW2GSP2F, 5¢-TCCTCGTTTC AATACAAACCTGAATCGTTG-3¢. A 0.9 Kb SspI band was cloned and sequenced to obtain the full sequence of the open reading frame. Gene expression BL21(DE3) carrying pET11a (control), pACL-1 and pACL- 2 were grown in 10 mL LB medium supplemented with car- benicillin (50 lgÆmL )1 ) to an attenuance at 600 nm (D 600 )of % 0.5 and induced with 100 lm isopropyl thio-b-d-galacto- side for 4 h. The induced cells were collected by centrifuga- tion at % 2000 g in an Avanti J20 centrifuge (Beckman, Fullerton, CA, USA) and treated with Bug Buster (Nov- agen, Madison, WI, USA) according to the manufacturer’s instructions. Obtained cell extract was used for the enzyme assay that followed the conversion of acryloyl-CoA to b-alanine-CoA by mass spectrometry. The assay mixture was the same as described above for purified protein. Enzyme activities The K m values for acryloyl-CoA and ammonia were deter- mined following the decrease in absorbance of acryloyl- CoA at 280 nm (De ¼ 3.5 mm )1 Æcm )1 )in50mm potassium phosphate, pH 7.5. The concentrations of acryloyl-CoA were varied between 5 lm and 50 lm at 50 mm ammonium chloride and the ammonium chloride concentrations were varied between 10 mm and 70 mm at 150 lm acryloyl-CoA. The elimination of ammonia from b-alanyl-CoA was cou- pled to the acryloyl-CoA reductase-mediated oxidation of reduced methylviologen in 50 mm potassium phosphate at pH 7.0 under strict anoxic conditions. The assay contained 1mm methylviologen, which was titrated with 10 mm Ti(III)citrate to an absorbance at 604 nm (A 604 )of% 0.8. Considering the need of two molecules of methylviologen being oxidized per mole of acryloyl-CoA reduced, e 604 ¼ 27.2 mm )1 Æcm )1 was used to calculate activities [37]. In order to determine the specificity of the nucleophile, 0.1 mm acryloyl-CoA was incubated with ammonium chlor- ide, hydroxylammonium chloride, glycine, or methylamo- nium chloride, 100 mm each, or 5 mm sodium sulfide in 50 mm potassium phosphate pH 7.5 with b-alanyl-CoA ammonia lyase (10 UÆmL )1 ) for 30 min at ambient temperature. The resulting CoA-thiol esters were isolated as described above and subjected MALDI-TOF mass spectro- metry. In a similar way methylacryloyl-CoA or crotonyl- CoA, 0.1 mm each, were incubated with 100 mm ammonium chloride and the products were analysed by mass spectrometry. Acknowledgements This work was supported by grants from the Deutsche Forschungsgemeinschaft and the Fonds der chemis- chen Industrie. Dr Bernhard Schmidt and Klaus Neifer (Universita ¨ tGo ¨ ttingen, Germany) sequenced the N-terminus of b-alanine ammonia lyase. We thank Dr Antonio J. Pierik (Philipps-Universita ¨ t Marburg) for helpful advice. 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