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Institut für Mikrobiologie, Universität Stuttgart, Allmandring 31, 70550 Stuttgart, Germany
Correspondence
Dieter Jendrossek
dieter.jendrossek{at}imb.uni-stuttgart.de
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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A sequence alignment of AtuD, the PA1535 gene product and Megasphera elsdenii butyryl-CoA dehydrogenase is available as a supplementary figure with the online version of this paper.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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) and in the cosmetic and/or food industry. The catabolic pathway of low-molecular-mass compounds with methyl-branched carbon skeletons in micro-organisms is poorly understood. Some information exists on conversion of terpenes under anaerobic conditions (Harder & Probian, 1995; Hylemon & Harder, 1998). Acyclic terpenes such as geranylate have been described as metabolites of cyclic terpene-utilizing anaerobes (Heyen & Harder, 2000). Aerobically, citronellol can be used as a single source of carbon and energy only by Pseudomonas citronellolis (Seubert, 1960; Seubert et al., 1963; Seubert & Remberger, 1963; Seubert & Fass, 1964a, b) and a few related species such as Pseudomonas aeruginosa, Pseudomonas mendocina (Cantwell et al., 1978) and Pseudomonas delhiensis (Prakash et al., 2007). Recently, phytol-utilizing and isoprenoid wax ester-accumulating Marinobacter strains were described (Holtzapple & Schmidt-Dannert, 2007). The most information on biochemical pathways of acyclic terpenes in aerobes exists for P. aeruginosa and P. citronellolis: in our previous studies we identified the atuRABCDEFGH gene cluster of P. aeruginosa (Förster-Fromme et al., 2006; Höschle et al., 2005) and a highly similar atuRABCDEFGH gene cluster in P. citronellolis (Förster-Fromme & Jendrossek, 2006) that were essential for acyclic terpene utilization (Atu) in both species. atuC and atuF were identified to encode the two subunits of the key enzyme of the Atu pathway, geranyl-CoA carboxylase (Aguilar et al., 2006; Höschle et al., 2005). It is assumed that the atu gene cluster codes for proteins catalysing the conversion of acyclic terpenes such as citronellol into 7-methyl-3-oxo-6-octenoyl-CoA (Fig. 1). 7-Methyl-3-oxo-6-octenoyl-CoA is subsequently metabolized in P. aeruginosa and P. citronellolis by β-oxidation and the leucine/isovalerate utilization (Liu) pathway (Aguilar et al., 2006; Förster-Fromme et al., 2006). In this study we continued our investigation on catabolism of methyl-branched compounds and analysed the biochemical function of two FAD-dependent acyl-CoA dehydrogenases encoded by atuD and PA1535, respectively. Acyl-CoA dehydrogenases such as isovaleryl-CoA dehydrogenase are well-characterized enzymes from eukaryotic species (Matsubara et al., 1989) but there are few biochemical data for the bacterial counterparts. To our knowledge, acyl-CoA dehydrogenases specific for terpenoid carbon skeleton have not been described before.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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. P. aeruginosa PAO1 was routinely grown in mineral salts medium (Schlegel et al., 1961) containing different carbon sources [0.5 % (w/v) glucose, 0.6 % (w/v) succinate or 0.1 % (v/v)/(w/v) terpene (citronellol, geraniol, citronellal, geranial, sodium salts of citronellate, geranylate or isovalerate)] at 30 °C. Water-insoluble carbon sources (citronellol, geraniol, citronellal, geranial) were added in the space between the Petri dish and the agar (20 µl per plate) and the plates were sealed with Parafilm. Growth on solid media with liquid carbon sources was performed in separate incubators to avoid cross-contamination by vapours. Escherichia coli strains were grown in Luria–Bertani (LB) medium at 37 °C.
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). Correctness of the respective insertion event was verified by PCR using one gene-specific and one pKnockout-specific primer (data not shown). Polar downstream effects were reduced by selection of those mutants in which the constitutive (in P. aeruginosa) lac promoter of pKnockout was orientated collinear to the respective gene cluster.
Construction of expression plasmids.
The atuD and PA1535 genes of P. aeruginosa PAO1 were amplified using Pwo polymerase (Genaxxon) and atuDFw (5'-GGAATTCCATATGATCTTCACCCAGGAACACGAGG-3'), atuDRev (5'-CCCAAGCTTGGGGGGTTCCAGCAGCAGGGTCTCG-3'), PA1535Fw (5'-GGAATTCCATATGACTGACTTCCAGCAGTACTTCGAC-3') and PA1535Rev (5'-CCCAAGCTTGGGGGCGCATTACGACCTGGACTT-3') as primers, inserting a restriction site of NdeI and HindIII for later cloning. The PCR products were digested, cloned into pET28a and transformed into E. coli JM109. The correctness of the gene sequences was checked by DNA sequencing. The resulting constructs encoded N-terminal His-tagged AtuD and PA1535 protein and were transformed into E. coli Rosetta 2 (DE3) pLysS RARE for expression experiments.
Expression and purification of acyl-CoA dehydrogenases.
Cultures (400 ml) of E. coli Rosetta 2 (DE3) pLysS RARE/pET28a : : atuD, E. coli Rosetta 2 (DE3) pLysS RARE/pET28a : : PA1535 and E. coli Rosetta 2 (DE3) pLysS RARE/pET28a as control in LB medium were incubated at 30 °C in the presence of 1 µg riboflavin ml–1 on a rotary shaker. IPTG (1 mM) was added at an OD600 of 
0.6 for induction of gene expression and cells were collected after 3–4 h incubation by centrifugation at 4 °C and 5000 g. The cells were resuspended in 1 ml 50 mM NaH2PO4, 300 mM NaCl, 10 mM imidazole, pH 8, per g prior to disruption by 2x30 s sonification. Cell debris was removed by centrifugation at 80 000 g for 1 h at 4 °C. The supernatants were loaded on a Ni-agarose column to purify AtuD or PA1535 protein by affinity chromatography. The column (1 ml bed volume) was washed with 4 bed volumes of 50 mM NaH2PO4, 300 mM NaCl, 10 mM imidazole, pH 8, and 4 bed volumes of 50 mM NaH2PO4, 300 mM NaCl, 20 mM imidazole, pH 8. Elution was performed using the same washing buffer with increasing concentrations of imidazole (4 bed volumes each with 50, 100, 250 and 500 mM imidazole). The fractions were analysed by reducing (mercaptoethanol) SDS-PAGE and the elution fractions containing the protein of interest were desalted using PD-10 desalting columns (GE Healthcare) equilibrated with 100 mM HEPES, pH 7.5.
Assay for acyl-CoA dehydrogenases and determination of Km values.
Acyl-CoA dehydrogenase activities were measured by reduction of 2,6-dichlorophenolindophenol (DCPIP) at 600 nm and 30 °C as described by Engel (1981) with some modifications. The assay mixture contained, in 1 ml, 100 mM sodium phosphate buffer, pH 7.0, 15 µl 10 mM DCPIP, 10 µl 10 mM phenazine methosulfate and 5–10 µl enzyme solution (1.5–6.0 µg). Addition of FAD to the reaction mixture in some experiments did not change enzyme activity and was therefore omitted in routine assays. The assay was started by adding acyl-CoA at different concentrations for determination of Vmax and Km values. Calculation of the Vmax and Km values was performed using Lineweaver–Burk and Michaelis–Menten plots. The absorption coefficient of DCPIP was 22 mM–1 cm–1. One unit (U) corresponds to conversion of one µmol CoA compound per minute.
Synthesis of acyl-CoA and HPLC-(ESI)MS determination of CoA compounds.
Octanoyl-CoA and isovaleryl-CoA were from Sigma. 5-Methylhex-4-enoic acid was a gift from Chris Braddock, Imperial College, London, UK. Synthesis of citronellyl-CoA and 5-methylhex-4-enoyl-CoA was done by the mixed-anhydride method: 770 µmol acyl compound (free acid) was dissolved in 5.1 ml tetrahydrofuran and neutralized by adding an equimolar amount of triethylamine. Then 770 µmol ethylchloroformate was added and the mixture was stirred for 30 min at room temperature before the solution was filtered through glass wool. The filtrate containing the anhydride was added dropwise to 29 µmol coenzyme A (CoA, Calbiochem) that had been dissolved in 12 ml water/tetrahydrofuran (3 : 2, v/v), pH 8.0 (with solid NaHCO3). After stirring for 25 min, 4 ml water was added and the solution was adjusted to pH 3 with 2 M HCl. To remove remaining mixed anhydride the solution was extracted three times with diethyl ether. The aqueous phase was dried by lyophilization. The products were analysed by HPLC-(ESI)MS as described previously (Förster-Fromme et al., 2006).
Other methods.
2D gel electrophoresis and identification of selected protein spots were performed with citronellate-, isovalerate-, succinate- and glucose-grown cells as described previously (Förster-Fromme et al., 2006). Protein determination was done using the Bradford method (Bradford, 1976).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). The key step of the Atu pathway, the carboxylation of the branched methyl group by geranyl-CoA carboxylase to an acetate function that can be cleaved off in a lyase reaction, is catalysed by the atuC/atuF gene products (Höschle et al., 2005). The substrate for the carboxylase reaction is presumably generated by oxidation of citronellyl-CoA to geranyl-CoA via citronellyl-CoA dehydrogenase (Seubert & Fass, 1964a). The recently identified atuRABCDEFGH gene cluster harbours one gene, atuD, that had been annotated as potential acyl-CoA dehydrogenase and we speculated that atuD could encode a protein with citronellyl-CoA dehydrogenase activity. AtuD was identified in 2D gel electrophoresis gels of citronellate-grown cells but was absent or below detection limit in isovalerate- and succinate-grown cells. Interestingly, 2D gel electrophoresis revealed the presence of another spot (in addition to the Atu proteins) that was specifically expressed in citronellate-grown cells. This spot was identified as the PA1535 gene product by mass spectrometry analysis of peptide fragments generated by trypsin digestion of the isolated spot (data not shown). The PA1535 gene product is also annotated as a potential acyl-CoA dehydrogenase (http://www.pseudomonas.com) and has 51 % amino acid similarity (34 % identity) to AtuD. Apparently, two potential acyl-CoA dehydrogenases are specifically expressed in citronellate-grown P. aeruginosa cells, namely AtuD and the PA1535 gene product.
The importance of AtuD and of PA1535 for catabolism of acyclic terpenes was investigated by insertion mutagenesis. The correctness of the insertion in the mutations in atuD and PA1535 was verified by appropriate PCR analysis (not shown). Polar downstream effects of the insertion were minimized by positioning the lac promoter of pKnockout (constitutively expressed in P. aeruginosa) collinear to the genes downstream of the insertion event. Two additional independent insertion mutants in PA1535 were obtained from a P. aeruginosa transposon bank (Jacobs et al., 2003). The insertion mutant in atuD was unable to utilize any of the tested acyclic terpenes (Table 2) but grew normally on isovalerate, succinate and other unrelated substrates. Interestingly, many small single colonies appeared after 3 days incubation when the atuD insertion mutant was streaked on agar plates with citronellol or related compounds (Table 2). Apparently, spontaneous mutation events could suppress the effect of insertion in atuD. All mutants in PA1535 grew slightly more slowly on citronellol and citronellate but showed normal growth on geraniol and geranylate. It appears that only atuD is essential for acyclic terpene utilization. PA1535 somehow supports growth of P. aeruginosa on citronellol but is not essential. A double mutant in both atuD and PA1535 showed the same negative phenotype on acyclic terpenes.
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). The apparent molecular masses of the additional bands were almost the same (41±2 kDa) for both proteins investigated and corresponded to the deduced theoretical molecular masses (42.7 kDa and 42.0 kDa for AtuD and the PA1535 gene product, respectively). Both proteins were purified to homogeneity (Fig. 2B). The two purified and concentrated proteins were yellow, and spectral analysis confirmed the presence of FAD as cofactor. This result is in agreement with the finding of putative FAD-binding sites in AtuD and the PA1535 protein (see Supplementary Fig. S1, available with the online version of this paper; Dym & Eisenberg, 2001). We determined the specific acyl-CoA dehydrogenase activities and Km values of both proteins for citronellyl-CoA, 5-methylhex-4-enoyl-CoA, isovaleryl-CoA and octanoyl-CoA (Fig. 3, Table 3). 5-Methylhex-4-enoyl-CoA and isovaleryl-CoA are metabolites of pathways downstream of the Atu pathway (β-oxidation and the Liu pathway; see Fig. 1). Octanoyl-CoA was tested as a non-branched compound having the same length of carbon backbone as citronellyl-CoA. As shown in Table 3, citronellyl-CoA dehydrogenase activity of 850±37 mU mg–1 and very high affinity to citronellyl-CoA (Km 1.6±0.3 µM) were determined for purified AtuD. AtuD was inactive with all the other three CoA compounds tested, indicating high substrate specificity. We concluded that AtuD has the physiological function of a citronellyl-CoA dehydrogenase. Purified PA1535 protein showed even higher citronellyl-CoA dehydrogenase activity (2450±26 mU mg–1) than AtuD but affinity of the protein to the substrate was more than ten times lower (Km 18±1.1 µM). Purified PA1535 protein – in contrast to AtuD – exhibited significant octanoyl-CoA activity (610±17 mU mg–1; Km 130±9.8 µM) but no activity with isovaleryl-CoA or 5-methylhex-4-enoyl-CoA. This result suggested that PA1535 is a second citronellyl-CoA dehydrogenase but is less specific and has significant activity with unbranched substrates with comparable carbon skeletons.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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; Conrad et al., 1974; Massey et al., 1974; reviewed by Massey et al., 1976). In contrast, acyl-CoA dehydrogenases from eukaryotes (mammals, plants), in particular short-chain acyl-CoA dehydrogenases such as isovaleryl-CoA dehydrogenases, have been investigated in more detail (Däschner et al., 2001; Ikeda & Tanaka, 1983). It is assumed that (eukaryotic) acyl-CoA dehydrogenases with different substrate specificities have evolved from a common ancestral gene (Matsubara et al., 1989). A summary of the structure and molecular action of catalysis has been published (Djordjevic et al., 1994; Dym & Eisenberg, 2001; Thorpe & Kim, 1995). Biochemical data on acyl-CoA dehydrogenases from bacterial species based on expressed genes and biochemically characterized gene products are scant (Becker et al., 1993). However, genome sequencing in the last decade has revealed numerous potential isovaleryl-CoA and acyl-CoA dehydrogenases from many prokaryotic species. Annotation of the respective genes as acyl-CoA dehydrogenase genes was presumably based on similarity analysis of the gene products to eukaryotic isovaleryl-CoA and acyl-CoA dehydrogenases. However, to our knowledge the biochemical function and the substrate specificities have not been experimentally verified for most putative bacterial acyl-CoA dehydrogenases. Acyl-CoA dehydrogenases with specificity for substrates with terpenoid-carbon skeletons have apparently not been described at all.
Catabolism of acyclic terpenes such as citronellol requires one acyl-CoA dehydrogenase step, namely citronellyl-CoA dehydrogenase, which converts citronellyl-CoA into geranyl-CoA (Fig. 1) (Seubert & Fass, 1964a). The atuRABCDEFGH gene cluster of P. aeruginosa harbours one potential acyl-CoA dehydrogenase gene, atuD, and purified AtuD showed high specific citronellyl-CoA dehydrogenase activity (850±37 mU mg–1). Because of the low Km of AtuD for citronellyl-CoA (1.6 µM) and the inability of AtuD to utilize short- or medium-chain acyl-CoAs such as isovaleryl-CoA or 5-methylhex-4-enoyl-CoA we conclude that AtuD functions as an acyl-CoA dehydrogenase with specificity for a terpenoid carbon backbone molecule in vivo. The physiological function of the PA1535 gene product is less obvious. Although PA1535 was specifically expressed in citronellate-grown cells and purified PA1535 gene product had a specific activity with citronellyl-CoA about three times higher than that of AtuD, the Km of the protein for citronellyl-CoA is 11 times higher than that of AtuD. Moreover, the PA1535 gene product also reacted with unbranched CoA compounds such as octanoyl-CoA, and insertion mutants in PA1535 showed only slightly reduced growth on citronellol and citronellate (Table 2). This indicated that PA1535 is not essential for acyclic terpene utilization and not exclusively specific for substrates with terpenoid structure. Therefore, PA1535 may have other, as yet unidentified, physiological substrates. However, the growth reduction in PA1535 insertion mutants on citronellate and the specific expression of PA1535 gene product in citronellate-grown cells indicated at least a participation of PA1535 in catabolism of acyclic terpenes. A surprising result was the negative phenotype of an atuD insertion mutant on acyclic terpenes: if AtuD and the PA1535 gene product have an exchangeable activity (citronellyl-CoA dehydrogenase) one could expect a compensation of the mutation by expression of PA1535. However, the Km of PA1535 for citronellyl-CoA was about 11 times higher than that of AtuD and the in vivo concentration of citronellyl-CoA might be very low. Another reason for the phenotype of atuD insertion might be that the activity of the pKnockout-encoded (constitutive) lac promoter is relatively low in P. aeruginosa: Western blots of atuD insertion mutants for biotin proteins indicated constitutive but relatively weak expression of AtuF (biotin subunit of geranyl-CoA carboxylase). Geranyl-CoA carboxylase activity in terpene-grown P. aeruginosa wild-type cells is relatively low (Höschle et al., 2005). A combination of reduced citronellyl-CoA dehydrogenese activity with low geranyl-CoA carboxylase expression might result in breakdown of the complete pathway in an atuD insertion mutant. A third explanation could be that the inducer compound of PA1535 expression is unknown.
Alignment of the deduced amino acid sequences of AtuD with proteins in the database showed high similarity values only to AtuD of P. citronellolis (96 %) and to other putative AtuD proteins of Marinobacter aquaeolei (91 %) and Hahella chejuensis (90 %). The latter two strains each have a gene cluster similar to the atu gene clusters of P. aeruginosa and P. citronellolis (Förster-Fromme & Jendrossek, 2006) and phytol utilization is documented for Marinobacter strains (Holtzapple & Schmidt-Dannert, 2007). Amino acid alignment to PA1535 and to acyl-CoA dehydrogenases of other species revealed significantly lower degrees of similarity (around 50 % similarity for acyl-CoA dehydrogenases and around 35 % for isovaleryl-CoA dehydrogenases). Taken together, the biochemical data for purified AtuD and the results of the amino acid alignments show that AtuD is the first biochemically characterized member of a new subgroup of acyl-CoA dehydrogenases with high substrate specificity for terpenoid molecule structure. We predict that more terpene-specific acyl-CoA dehydrogenases will be found in the near future.
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ACKNOWLEDGEMENTS |
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Edited by: W. H. Schwarz
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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Received 31 October 2007;
revised 18 December 2007;
accepted 21 December 2007.
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