Title: Identification of the Missing Links in Prokaryotic Pentose Oxidation Pathways
Abstract: The pentose metabolism of Archaea is largely unknown. Here, we have employed an integrated genomics approach including DNA microarray and proteomics analyses to elucidate the catabolic pathway for d-arabinose in Sulfolobus solfataricus. During growth on this sugar, a small set of genes appeared to be differentially expressed compared with growth on d-glucose. These genes were heterologously overexpressed in Escherichia coli, and the recombinant proteins were purified and biochemically studied. This showed that d-arabinose is oxidized to 2-oxoglutarate by the consecutive action of a number of previously uncharacterized enzymes, including a d-arabinose dehydrogenase, a d-arabinonate dehydratase, a novel 2-keto-3-deoxy-d-arabinonate dehydratase, and a 2,5-dioxopentanoate dehydrogenase. Promoter analysis of these genes revealed a palindromic sequence upstream of the TATA box, which is likely to be involved in their concerted transcriptional control. Integration of the obtained biochemical data with genomic context analysis strongly suggests the occurrence of pentose oxidation pathways in both Archaea and Bacteria, and predicts the involvement of additional enzyme components. Moreover, it revealed striking genetic similarities between the catabolic pathways for pentoses, hexaric acids, and hydroxyproline degradation, which support the theory of metabolic pathway genesis by enzyme recruitment. The pentose metabolism of Archaea is largely unknown. Here, we have employed an integrated genomics approach including DNA microarray and proteomics analyses to elucidate the catabolic pathway for d-arabinose in Sulfolobus solfataricus. During growth on this sugar, a small set of genes appeared to be differentially expressed compared with growth on d-glucose. These genes were heterologously overexpressed in Escherichia coli, and the recombinant proteins were purified and biochemically studied. This showed that d-arabinose is oxidized to 2-oxoglutarate by the consecutive action of a number of previously uncharacterized enzymes, including a d-arabinose dehydrogenase, a d-arabinonate dehydratase, a novel 2-keto-3-deoxy-d-arabinonate dehydratase, and a 2,5-dioxopentanoate dehydrogenase. Promoter analysis of these genes revealed a palindromic sequence upstream of the TATA box, which is likely to be involved in their concerted transcriptional control. Integration of the obtained biochemical data with genomic context analysis strongly suggests the occurrence of pentose oxidation pathways in both Archaea and Bacteria, and predicts the involvement of additional enzyme components. Moreover, it revealed striking genetic similarities between the catabolic pathways for pentoses, hexaric acids, and hydroxyproline degradation, which support the theory of metabolic pathway genesis by enzyme recruitment. Pentose sugars are a ubiquitous class of carbohydrates with diverse biological functions. Ribose and deoxyribose are major constituents of nucleic acids, whereas arabinose and xylose are building blocks of several plant cell wall polysaccharides. Many prokaryotes, as well as yeasts and fungi, are able to degrade these polysaccharides, and use the released five-carbon sugars as a sole carbon and energy source. At present, three main catabolic pathways have been described for pentoses. The first is present in Bacteria and uses isomerases, kinases, and epimerases to convert d- and l-arabinose (Ara) and d-xylose (Xyl) into d-xylulose 5-phosphate (Fig. 1A), which is further metabolized by the enzymes of the phosphoketolase or pentose phosphate pathway. The genes encoding the pentose-converting enzymes are often located in gene clusters in bacterial genomes, for example, the araBAD operon for l-Ara (1Lee N. Gielow W. Martin R. Hamilton E. Fowler A. Gene (Amst.). 1986; 47: 231-244Crossref PubMed Scopus (52) Google Scholar), the xylAB operon for d-Xyl (2Rosenfeld S.A. Stevis P.E. Ho N.W. Mol. Gen. Genet. 1984; 194: 410-415Crossref PubMed Scopus (40) Google Scholar), and the darK-fucPIK gene cluster for d-Ara (3Elsinghorst E.A. Mortlock R.P. J. Bacteriol. 1994; 176: 7223-7232Crossref PubMed Google Scholar). The second catabolic pathway for pentoses converts d-Xyl into d-xylulose 5-phosphate as well, but the conversions are catalyzed by reductases and dehydrogenases instead of isomerases and epimerases (Fig. 1B). This pathway is commonly found in yeasts, fungi, mammals, and plants, but also in some bacteria (4Chiang C. Knight S.G. Nature. 1960; 188: 79-81Crossref PubMed Scopus (106) Google Scholar, 5Fossitt D. Mortlock R.P. Anderson R.L. Wood W.A. J. Biol. Chem. 1964; 239: 2110-2115Abstract Full Text PDF PubMed Google Scholar, 6Wojtkiewicz B. Szmidzinski R. Jezierska A. Cocito C. Eur. J. Biochem. 1988; 172: 197-203Crossref PubMed Scopus (8) Google Scholar). In a third pathway, pentoses such as l-Ara, d-Xyl, d-ribose, and d-Ara are metabolized non-phosphorylatively to either 2-oxoglutarate (2-OG) 4The abbreviations used are: 2-OG, 2-oxoglutarate; HSCFE, heat-stable cell-free extract; CFE, cell-free extract; Ara, arabinose; KDA, 2-keto-3-deoxy-arabinonate; DOP, 2,5-dioxopentanoate; AraDH, arabinose dehydrogenase; AraD, arabinonate dehydratase; KdaD, 2-keto-3-deoxy-d-arabinonate dehydratase; DopDH, 2,5-dioxopentanoate dehydrogenase. or to pyruvate and glycolaldehyde (Fig. 1C). The conversion to 2-OG, which is a tricarboxylic acid cycle intermediate, proceeds via the subsequent action of a pentose dehydrogenase, a pentonolactonase, a pentonic acid dehydratase, a 2-keto-3-deoxypentonic acid dehydratase, and a 2,5-dioxopentanoate dehydrogenase. This metabolic pathway has been reported in several aerobic bacteria, such as strains of Pseudomonas (7Weimberg R. Doudoroff M. J. Biol. Chem. 1955; 217: 607-624Abstract Full Text PDF PubMed Google Scholar, 8Weimberg R. J. Biol. Chem. 1961; 236: 629-635Abstract Full Text PDF PubMed Google Scholar, 9Dagley S. Trudgill P.W. Biochem. J. 1965; 95: 48-58Crossref PubMed Scopus (28) Google Scholar), Rhizobium (10Duncan M.J. J. Gen. Microbiol. 1979; 113: 177-179Crossref Scopus (23) Google Scholar, 11Duncan M.J. Fraenkel D.G. J. Bacteriol. 1979; 137: 415-419Crossref PubMed Google Scholar), Azospirillum (12Novick N.J. Tyler M.E. J. Bacteriol. 1982; 149: 364-367Crossref PubMed Google Scholar), and Herbaspirillum (13Mathias A.L. Rigo L.U. Funayama S. Pedrosa F.O. J. Bacteriol. 1989; 171: 5206-5209Crossref PubMed Google Scholar). Alternatively, some Pseudomonas and Bradyrhizobium species have been reported to cleave the 2-ke- to-3-deoxypentonic acid with an aldolase to yield pyruvate and glycolaldehyde (14Palleroni N.J. Doudoroff M. J. Bacteriol. 1957; 74: 180-185Crossref PubMed Google Scholar, 15Dahms A.S. Anderson R.L. Biochem. Biophys. Res. Commun. 1969; 36: 809-814Crossref PubMed Scopus (36) Google Scholar, 16Pedrosa F.O. Zancan G.T. J. Bacteriol. 1974; 119: 336-338Crossref PubMed Google Scholar). Despite the fact that these oxidative pathway variants have been known for more than five decades, surprisingly, the majority of the responsible enzymes and genes remain unidentified. Sulfolobus spp. are obligatory aerobic Crenarchaea that are commonly found in acidic geothermal springs. Among the Archaea, this genus is well known for its broad saccharolytic capacity, which is reflected in their ability to utilize several pentoses and hexoses, as well as oligosaccharides and polysaccharides as a sole carbon and energy source (17Grogan D.W. J. Bacteriol. 1989; 171: 6710-6719Crossref PubMed Google Scholar). Although the catabolism of hexoses is well studied (reviewed in Ref. 18Siebers B. Schonheit P. Curr. Opin. Microbiol. 2005; 8: 695-705Crossref PubMed Scopus (162) Google Scholar), the pathways for pentose degradation have neither been established in Sulfolobus solfataricus, nor in any other member of the Archaea (19Johnsen U. Schonheit P. J. Bacteriol. 2004; 186: 6198-6207Crossref PubMed Scopus (55) Google Scholar). All chemicals were of analytical grade and purchased from Sigma, unless stated otherwise. Oligonucleotide primers were obtained from MWG Biotech AG (Ebersberg, Germany). S. solfataricus P2 (DSM1617) was grown in media containing either 3 g/liter d-Ara or d-Glu as previously described (20Brouns S.J. Smits N. Wu H. Snijders A.P. Wright P.C. de Vos W.M. van der Oost J. J. Bacteriol. 2006; 188: 2392-2399Crossref PubMed Scopus (44) Google Scholar). Whole genome DNA microarrays containing gene-specific tags representing >90% of the S. solfataricus P2 genes (21Andersson A. Bernander R. Nilsson P. Bioinformatics. 2005; 21: 325-332Crossref PubMed Scopus (16) Google Scholar) were used for global transcript profiling of cultures grown on d-Ara as compared with d-Glu. Total RNA extraction, cDNA synthesis and labeling, hybridization, and scanning were performed as previously described, as were data filtration, normalization, and statistical evaluation (22Lundgren M. Andersson A. Chen L. Nilsson P. Bernander R. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 7046-7051Crossref PubMed Scopus (175) Google Scholar, 23Snijders A.P. Walther J. Peter S. Kinnman I. de Vos M.G. van de Werken H.J. Brouns S.J. van der Oost J. Wright P.C. Proteomics. 2006; 6: 1518-1529Crossref PubMed Scopus (52) Google Scholar). The proteome of S. solfataricus P2 was studied with a combination of two-dimensional gel electrophoresis, 15N metabolic labeling, and tandem mass spectrometry as previously described (24Snijders A.P. de Vos M.G. Wright P.C. J. Proteome Res. 2005; 4: 578-585Crossref PubMed Scopus (59) Google Scholar, 25Snijders A.P. de Vos M.G. de Koning B. Wright P.C. Electrophoresis. 2005; 26: 3191-3199Crossref PubMed Scopus (30) Google Scholar). Two separate growth experiments were set up: 1) S. solfataricus with d-Ara as the carbon source and (14NH4)2SO4 as the nitrogen source; and 2) S. solfataricus with d-Glu as the carbon source and (15NH4)2SO4 as the nitrogen source. Next, the 14N and 15N cultures were mixed in equal amounts on the basis of optical density (A530) measurements, proteins were extracted and separated by two-dimensional gel electrophoresis. For the localization of proteins, a previously described two-dimensional gel electrophoresis reference map was used (23Snijders A.P. Walther J. Peter S. Kinnman I. de Vos M.G. van de Werken H.J. Brouns S.J. van der Oost J. Wright P.C. Proteomics. 2006; 6: 1518-1529Crossref PubMed Scopus (52) Google Scholar). Spots were excised from the gel, and peptides were quantified on the basis of their relative intensity in the time of flight mass spectrum, according to established methods (23Snijders A.P. Walther J. Peter S. Kinnman I. de Vos M.G. van de Werken H.J. Brouns S.J. van der Oost J. Wright P.C. Proteomics. 2006; 6: 1518-1529Crossref PubMed Scopus (52) Google Scholar). d-Arabinonate was synthesized from d-Ara as previously described (26Sperber N. Zaugg H.E. Sandstrom W.M. J. Am. Chem. Soc. 1947; 69: 915-920Crossref PubMed Scopus (15) Google Scholar). The aldehyde 2,5-dioxopentanoate was synthesized from 1,4-dibromobutane according to reported procedures (27Kraus G.A. Landgrebe K. Synthesis. 1984; 1984: 885Crossref Scopus (42) Google Scholar, 28Macritchie J.A. Silcock A. Willis C.L. Tetrahedron Asymmetry. 1997; 8: 3895-3902Crossref Scopus (61) Google Scholar, 29Crestia D. Guerard C. Bolte J. Demuynck C. J. Mol. Catal. B Enzymol. 2001; 11: 207-212Crossref Scopus (15) Google Scholar). The genes araDH (Sso1300), araD (Sso3124), kdaD (Sso3118), and dopDH (Sso3117) were amplified by PCR from genomic DNA using Pfu TURBO polymerase (Stratagene) and cloned in expression vector pET24d (Novagen) (supplemental materials Table 1). The resulting plasmids were harvested from Escherichia coli HB101 transformants by Miniprep (Qiagen), sequenced by Westburg genomics (Leusden, Netherlands), and transformed to E. coli expression strain BL21(DE3) containing the tRNA accessory plasmid pRIL (Stratagene). All proteins were produced according to standard procedures in four 1-liter shaker flasks containing LB medium, but with some exceptions. When the culture A600 reached 0.5, the cultures were cold-shocked by placing them on ice for 30 min to induce host chaperones (20Brouns S.J. Smits N. Wu H. Snijders A.P. Wright P.C. de Vos W.M. van der Oost J. J. Bacteriol. 2006; 188: 2392-2399Crossref PubMed Scopus (44) Google Scholar). After that, the expression was started by adding 0.5 mm isopropyl β-d-thiogalactopyranoside, and the cultures were incubated for 12-16 h at 37 °C after which they were spun down (10 min, 5000 × g, 4 °C). At the time of induction, the arabinose dehydrogenase (AraDH) and AraD overexpression cultures were supplemented with 0.25 mm ZnSO4 (30Esposito L. Bruno I. Sica F. Raia C.A. Giordano A. Rossi M. Mazzarella L. Zagari A. Biochemistry. 2003; 42: 14397-14407Crossref PubMed Scopus (36) Google Scholar) and 20 mm MgCl2, respectively. Pelleted E. coli and S. solfataricus cells were resuspended in buffer and disrupted by sonication at 0 °C. Afterward, insoluble cell material was spun down (30 min, 26,500 × g, 4 °C) and the E. coli supernatants were subjected to heat treatment for 30 min at 75 °C. Denatured proteins were removed by centrifugation (30 min, 26,500 × g, 4 °C) yielding the heat-stable cell-free extract (HSCFE). AraDH—HSCFE in 20 mm Tris-HCl (pH 7.5) supplemented with 50 mm NaCl was applied to a 20-ml Matrex Red A affinity column (Amicon). After washing the bound protein with 2 column volumes of buffer, the recombinant protein was eluted by a linear gradient of 2 m NaCl. AraD—HSCFE in 50 mm HEPES-KOH (pH 8.0) supplemented with 50 mm NaCl was applied to a 70-ml Q-Sepharose Fast Flow (Amersham Biosciences) anion exchange column, and eluted in a 2 m NaCl gradient. Fractions containing the recombinant protein were pooled, concentrated with a 30-kDa cut-off filter (Vivaspin), and purified by size exclusion chromatography using a Superdex 200 HR 10/30 column (Amersham Biosciences) and 50 mm HEPES-KOH buffer (pH 8.0) supplemented with 100 mm NaCl as an eluent. 2-Keto-3-deoxy-d-arabinonate Dehydratase (KdaD)—HSCFE in 25 mm NaPi buffer (pH 6.8) was applied to a 70-ml Q-Sepharose Fast Flow (Amersham Biosciences) anion exchange column. Flow-through fractions containing KdaD were collected, loaded onto a 46-ml Bio-Gel hydroxyapatite column (Bio-Rad), and eluted by a linear gradient of 0.5 m NaPi buffer (pH 6.8). Fractions containing the recombinant proteins were pooled, and dialyzed overnight in 50 mm HEPES-KOH (pH 8.0) supplemented with 0.5 mm dithiothreitol (DTT). 2,5-Dioxopentanoate Dehydrogenase (DopDH)—HSCFE in 20 mm HEPES-KOH (pH 8.0) supplemented with 200 mm NaCl and 7.5 mm DTT was purified by affinity chromatography, as described for AraDH. Fractions containing the protein were pooled, concentrated using a 30-kDa cut-off membrane (Vivaspin), and purified by size exclusion chromatography as described for AraD. Unless stated otherwise, all enzymatic assays were performed in degassed 100 mm HEPES-KOH buffer (pH 7.5) at 70 °C. The optimal pH of catalysis was determined using a 30 mm citratephosphate-glycine buffer system that was adjusted in the range of pH 3-11 at 70 °C. Thermal inactivation assays were performed by incubating 50 μg/ml of enzyme at 70, 80, 85, and 90 °C and drawing aliquots at regular intervals during 2 h followed by a standard activity assay. Sugar dehydrogenase activity was determined on a Hitachi U-1500 spectrophotometer in a continuous assay using 10 mm d- and l-arabinose, d- and l-xylose, d-ribose, d-lyxose, d- and l-fucose, d- and l-galactose, d-mannose, and d-glucose as a substrate, and 0.4 mm NAD+ or NADP+ as a cofactor. Aldehyde dehydrogenase reactions were performed using 5 mm 2,5-dioxopentanoate, glycolaldehyde, dl-glyceraldehyde, acetaldehyde, and propionaldehyde in the presence of 10 mm DTT. Initial enzymatic activity rates were obtained from the increase in absorption at 340 nm (A340), and calculated using a molar extinction coefficient of 6.22 mm-1 cm-1. Standard reactions were performed using 10 mm potassium d-arabinonate in the presence of 1 mm MgCl2. The formation of 2-keto-3-deoxy-acid reaction products was determined with the thiobarbiturate assay at 549 nm using a molar extinction coefficient of 67.8 mm-1 cm-1 (31Warren L. Nature. 1960; 186: 237Crossref PubMed Scopus (134) Google Scholar, 32Kim S. Lee S.B. Biochem. J. 2005; 387: 271-280Crossref PubMed Scopus (55) Google Scholar). The effect of different divalent cations on enzymatic activity was investigated by a pre-treatment of the enzyme with 1 mm EDTA for 20 min at 70 °C, followed by a standard assay in the presence of 2 mm divalent metal ions. Enzyme reactions were performed with cell-free extract (CFE) from S. solfataricus cultures grown on either d-Ara or d-Glu, which were harvested at mid-exponential phase. The reaction was started by adding 25 μl of 3.5 mg/ml CFE to a mixture containing 10 mm potassium d-arabinonate, 1 mm MgCl2, and either 0.4 mm NAD+ or NADP+. After an incubation of 2 h at 75°C, the reactions were stopped by placing the tubes on ice. Identical reactions were set up in which the CFE was replaced by the purified enzymes AraD (4.2 μg), KdaD (13.4 μg), and DopDH (3.8 μg). The amount of 2-oxoglutarate in these mixtures was then determined by the reductive amination of 2-oxoglutarate to l-glutamate using purified recombinant Pyrococcus furiosus glutamate dehydrogenase at 60 °C (33Lebbink J.H. Eggen R.I. Geerling A.C. Consalvi V. Chiaraluce R. Scandurra R. de Vos W.M. Protein Eng. 1995; 8: 1287-1294Crossref PubMed Scopus (22) Google Scholar). The detection reaction was started by the addition of 5 units of glutamate dehydrogenase to a sample that was supplemented with 10 mm NH4Cl and 0.12 mm NADPH. The formation of pyruvate was determined at 30 °C using 4 units of chicken heart lactate dehydrogenase and 0.1 mm NADH. The conversion of 2-oxoglutarate or pyruvate was continuously monitored on a Hitachi U-1500 spectrophotometer by following the decrease in A340 until substrate depletion occurred. Changes in concentrations of NAD(P)H were calculated as described above. The oligomerization state of AraDH, AraD, KdaD, and DopDH was determined by nanoflow electrospray ionization mass spectrometry. For this, the protein was concentrated in the range of 5-15 μm and the buffer was exchanged to 50 or 200 mm ammonium acetate (pH 6.7 or 7.5) by using an Ultrafree 0.5-ml centrifugal filter device with a 5-kDa cut-off (Millipore). Protein samples were introduced into the nanoflow electrospray ionization source of a Micromass LCT mass spectrometer (Waters), which was modified for high mass operation and set in positive ion mode. Mass spectrometry experiments were performed under conditions of increased pressure in the interface region between the sample and extraction cone of 8 mbar by reducing the pumping capacity of the rotary pump (34Schmidt A. Bahr U. Karas M. Anal. Chem. 2001; 73: 6040-6046Crossref PubMed Scopus (86) Google Scholar, 35Tahallah N. Pinkse M. Maier C.S. Heck A.J. Rapid Commun. Mass Spectrom. 2001; 15: 596-601Crossref PubMed Scopus (188) Google Scholar). Capillary and sample cone voltages were optimized for the different proteins and were in the range of 1.4-1.6 kV and 75-150 V, respectively. Upstream sequences of the differentially expressed genes were extracted between -200 and +50 nucleotides relative to the open reading frame translation start site. These sequences were analyzed using the Gibbs Recursive Sampler algorithm (36Thompson W. Rouchka E.C. Lawrence C.E. Nucleic Acids Res. 2003; 31: 3580-3585Crossref PubMed Scopus (244) Google Scholar). Possible sequence motifs were checked against all upstream sequences and the complete genome of S. solfataricus. A diagram of the sequence motif was created using the WebLogo server. Protein sequences were retrieved from the National Center for Biotechnology Information (NCBI) and analyzed using PSI-BLAST on the non-redundant data base, and RPS-BLAST on the conserved domain data base. Multiple sequence alignments were built using either ClustalX or TCoffee software. Gene neighborhood analyses were performed using various webserver tools: STRING at the EMBL, Gene Ortholog Neighborhoods at the Integrated Microbial Genomes server of the Joint Genome Institute, and pinned regions at the ERGO bioinformatics suite. S. solfataricus is a model archaeon for studying metabolism and information processing systems, such as transcription, translation, and DNA replication (37She Q. Singh R.K. Confalonieri F. Zivanovic Y. Allard G. Awayez M.J. Chan-Weiher C.C. Clausen I.G. Curtis B.A. De Moors A. Erauso G. Fletcher C. Gordon P.M. Heikamp-de Jong I. Jeffries A.C. Kozera C.J. Medina N. Peng X. Thi-Ngoc H.P. Redder P. Schenk M.E. Theriault C. Tolstrup N. Charlebois R.L. Doolittle W.F. Duguet M. Gaasterland T. Garrett R.A. Ragan M.A. Sensen C.W. van der Oost J. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 7835-7840Crossref PubMed Scopus (673) Google Scholar, 38Lundgren M. Bernander R. Curr. Opin. Microbiol. 2005; 8: 662-668Crossref PubMed Scopus (31) Google Scholar). Several halophilic and thermophilic Archaea have been reported to assimilate pentose sugars, but neither the catabolic pathways for these 5-carbon sugars nor the majority of its enzymes are known (17Grogan D.W. J. Bacteriol. 1989; 171: 6710-6719Crossref PubMed Google Scholar, 19Johnsen U. Schonheit P. J. Bacteriol. 2004; 186: 6198-6207Crossref PubMed Scopus (55) Google Scholar). To close this knowledge gap, we have studied the growth of S. solfataricus on the pentose d-Ara using a multidisciplinary genomics approach, and compared the results to growth on the hexose d-Glu. Both culture media supported growth to cell densities of ∼2 × 109 cells/ml (A600 2.5) with similar doubling times of around 6 h. Several enzyme activity assays were performed with CFEs from both cultures to establish a mode of d-Ara degradation (Fig. 1). A 12.3-fold higher NADP+-dependent d-Ara dehydrogenase activity (45.5 milliunits/mg) was detected in d-Ara CFE (Table 1), which indicated the presence of an inducible d-Ara dehydrogenase. d-Ara reductase, d-arabinitol dehydrogenase, and d-Ara isomerase activity were not detected. Activity assays using d-arabinonate indicated that d-Ara CFE contained a 13.9-fold higher d-arabinonate dehydratase activity (7.4 milliunits/mg) than d-Glu CFE (Table 1). Moreover, the multistep conversion of d-arabinonate to 2-OG could readily be demonstrated with d-Ara CFE in the presence of NADP+ (Fig. 2). The formation of pyruvate as one of the products from d-arabinonate was not observed, whereas control reactions with both CFEs and d-gluconate as a substrate did yield pyruvate (data not shown), indicating that the enzymes of the Entner-Doudoroff pathway were operative. In the final step of the pathway, d-Ara CFE contained a 3.6-fold higher activity (255 milliunits/mg) toward the aldehyde 2,5-dioxopentanoate (DOP) using NADP+ as a cofactor. The data suggest that S. solfataricus employs an inducible enzyme set that converts d-Ara into the tricarboxylic acid cycle intermediate 2-OG via the pentose oxidation pathway (Fig. 1C).TABLE 1Properties of the d-Ara degrading enzymes of S. solfataricusAraDHAraDKdaDDopDHDescriptiond-Arabinose 1-dehydrogenased-Arabinonate dehydratase2-Keto-3-deoxy-d-arabinonate dehydratase2,5-Dioxopentanoate dehydrogenaseEC number1.1.1.1174.2.1.54.2.1.-1.2.1.26Locus IDSso1300Sso3124Sso3118Sso3117Uniprot IDQ97YM2Q97U96Q97UA0Q97UA1GenBank ID15898142158998301589982615899825COG1,0644,9483,9701,012PFAM00107011880155700171Specific activity in S.s extracts (milliunits/mg)45.57.4NDaND, not determined.255-Fold A/G(12.3)(13.9)(3.6)ARA-box presentYesYesYesYesSubunit size (kDa)37.342.433.152.3OligomerizationTetramerOctamerTetramerTetramerSubstrate specificity, turnover number (s−1)d-Arabinose (23.8)d-Arabinonate (1.8)ND2,5-Dioxopentanoate(8.6)l-Fucose (26.8)Glycolaldehyde(5.3)d-Ribose (17.7)dl-glyceraldehyde(4.8)l-Galactose (17.7)CofactorNADP+, Zn2+Mg2+NDNADP+Apparent pH optimum (>50% activity)8.2 (7.3-9.3)6.7 (5.2-10.2)ND7.8 (6.7-8.2)Apparent T-optimum (°C) (>50% activity)91 (74->100)85 (75-92)NDNDThermal inactivation42 min at 85 °C18 min at 85 °CNDNDHalf-life time26 min at 90 °C<10 min at 90 °Ca ND, not determined. Open table in a new tab Transcriptomics—The global transcriptional response of S. solfataricus growing exponentially on d-Ara or d-Glu was determined by DNA microarray analysis. The transcriptome comparison between both growth conditions showed that a small set of genes was differentially expressed 3-fold or more (Table 2). The highly expressed genes under d-Ara conditions included all four subunits of the Ara ABC transporter (Sso3066-3069) (39Elferink M.G. Albers S.V. Konings W.N. Driessen A.J. Mol. Microbiol. 2001; 39: 1494-1503Crossref PubMed Scopus (114) Google Scholar), a putative sugar permease for d-Ara (Sso2718), five of six subunits of the SoxM quinol oxidase complex (Sso2968-2973) (40Komorowski L. Verheyen W. Schafer G. Biol. Chem. 2002; 383: 1791-1799Crossref PubMed Scopus (52) Google Scholar), and five metabolic genes with general function predictions only (Sso1300, Sso3124, Sso3117, Sso3118, and Sso1303). The differential expression of the gene for the remaining SoxM subunit, i.e. the sulfocyanin subunit SoxE (Sso2972), was just below the threshold level (supplemental materials Table 2). Whereas the expression of the ABC-type transport system genes had been shown to be induced in Ara media previously (39Elferink M.G. Albers S.V. Konings W.N. Driessen A.J. Mol. Microbiol. 2001; 39: 1494-1503Crossref PubMed Scopus (114) Google Scholar, 41Lubelska J.M. Jonuscheit M. Schleper C. Albers S.V. Driessen A.J. Extremophiles. 2006; PubMed Google Scholar), the differential expression of the SoxM gene cluster was not anticipated.TABLE 2Differentially expressed genes (>3-fold different)Locus nameMicroarray log2 fold (q value)aq value <5 indicates statistically significant differential expression.Proteomics foldDescriptionRef.(A/G) ± S.D.High expression on d-AraSso30664.02 ± 0.58 (1.1)>20Arabinose ABC transporter, arabinose-binding protein (AraS)39Sso30683.71 ± 0.97 (1.1)NDbND, not determined.Arabinose ABC transporter, permease39Sso13003.64 ± 0.95 (1.1)>20Alcohol dehydrogenase IVcOriginal annotation. d-arabinose 1-dehydrogenase (AraDH)This studySso30673.37 ± 0.49 (1.1)NDArabinose ABC transporter, permease39Sso30692.97 ± 0.18 (2.5)NDArabinose ABC transporter, ATP binding protein39Sso29682.56 ± 1.30 (1.1)NDQuinol oxidase subunit (SoxM complex), SoxI40Sso31242.44 ± 1.13 (1.1)>20Mandelate racemace/muconate lactonizing enzymebND, not determined. d-arabinonate dehydratase (AraD)This studySso31172.39 ± 0.62 (1.1)>20Aldehyde dehydrogenasecOriginal annotation. 2,5-dioxopentanoate dehydrogenase (DopDH)This studySso29712.24 ± 1.17 (1.1)NDQuinol oxidase subunit (SoxM complex), SoxF (Rieske Fe-S protein)40Sso29732.10 ± 1.48 (2.6)NDQuinol oxidase subunit (SoxM complex), SoxM (I + III)40Sso29702.09 ± 1.53 (2.6)NDQuinol oxidase subunit (SoxM complex), SoxG (cytochrome a587)40Sso27181.99 ± 1.10 (1.1)NDPutative sugar permeaseSso30461.89 ± 0.87 (1.1)NDPutative ABC sugar transporter, ATP-binding proteinSso29691.86 ± 1.09 (1.1)NDQuinol oxidase (SoxM complex), SoxH subunit (II)40Sso31181.78 ± 0.45 (1.1)>20Conserved hypothetical proteincOriginal annotation. 2-keto-3-deoxy-d-arabinonate dehydratase (KdaD)This studySso13031.77 ± 0.57 (1.1)NDPutative pentonic acid dehydrataseSso1333NDEdNDE, not differentially expressed.3.48 ± 0.62Isocitrate lyase45Sso0527NDE3.45 ± 0.72Phosphoglycerate kinase46Sso2869NDE3.06 ± 1.06Malic enzyme47High expression on d-GluSso3073−2.59 ± 0.71 (1.1)NDPutative sugar permeaseSso3089−2.12 ± 1.12 (1.1)NDHypothetical membrane protein componentSso3104−2.04 ± 0.31 (1.1)NDHypothetical proteinSso1312−2.02 ± 0.52 (1.1)NDPutative ring oxidation complex/phenylacetic acid degradation rel. proteinSso2884−1.87 ± 0.37 (1.1)NDPutative 4-carboxymuconolactone decarboxylaseSso2657−1.77 ± 0.87 (1.1)NDQuinol oxidase (SoxABCD complex), cytochrome aa3 subunit (SoxB)42Sso3085−1.63 ± 0.86 (1.1)NDConserved hypothetical membrane proteinSso3100−1.60 ± 0.88 (1.1)NDHypothetical membrane protein componentSso2044−1.60 ± 0.48 (1.1)NDl-Glutamate dehydrogenasea q value <5 indicates statistically significant differential expression.b ND, not determined.c Original annotation.d NDE, not differentially expressed. Open table in a new tab The genes that were up-regulated under d-Glu conditions encode seven uncharacterized proteins (Sso3073, Sso3089, Sso3104, Sso1312, Sso2884, Sso3085, and Sso3100), the SoxB subunit of the SoxABCD quinol oxidase complex (Sso2657) (42Gleissner M. Kaiser U. Antonopoulos E. Schafer G. J. Biol. Chem. 1997; 272: 8417-8426Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar), and a glutamate dehydrogenase (Sso2044) (43Consalvi V. Chiaraluce R. Politi L. Gambacorta A. De Rosa M. Scandurra R. Eur. J. Biochem. 1991; 196: 459-467Crossref PubMed Scopus (70) Google Scholar) (Table 2). The Glu ABC transporter was not differentially expressed, confirming previous observations (41Lubelska J.M. Jonuscheit M. Schleper C. Albers S.V. Driessen A.J. Extremophiles. 2006; PubMed Google Scholar). The difference in gene expression of subunits SoxA (Sso2658), SoxC (Sso2656), and SoxD (Sso10828) was just below the threshold level (supplemental materials Table 2). Next to the SoxABCD genes, a small gene cluster containing the Rieske iron-sulfur cluster protein SoxL-1 (Sso2660) and Sso2661 to Sso2663 appeared to be expressed with a 2-3-fold difference (supplemental materials Table 2). It thus appears that under d-Glu conditions, the Sox-ABCD quinol oxidase complex is preferentially used, whereas under d-Ara conditions the SoxM-mediated terminal quinol oxidation is favored. Differential use of both oxidase complexes was recently a