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Novel Non-Phosphorylative Pathway of Pentose Metabolism from Bacteria

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Novel Non-Phosphorylative Pathway of Pentose Metabolism from Bacteria www.nature.com/scientificreports OPEN Novel non-phosphorylative pathway of pentose metabolism from bacteria Received: 22 March 2018 Seiya Watanabe1,2,3, Fumiyasu Fukumori4, Hisashi Nishiwaki1,2, Yasuhiro Sakurai5, Accepted: 30 September 2018 Kunihiko Tajima5 & Yasuo Watanabe1,2 Published: xx xx xxxx Pentoses, including D-xylose, L-arabinose, and D-arabinose, are generally phosphorylated to D-xylulose 5-phosphate in bacteria and fungi. However, in non-phosphorylative pathways analogous to the Entner-Dodorof pathway in bacteria and archaea, such pentoses can be converted to pyruvate and glycolaldehyde (Route I) or α-ketoglutarate (Route II) via a 2-keto-3-deoxypentonate (KDP) intermediate. Putative gene clusters related to these metabolic pathways were identifed on the genome of Herbaspirillum huttiense IAM 15032 using a bioinformatic analysis. The biochemical characterization of C785_RS13685, one of the components encoded to D-arabinonate dehydratase, difered from the known acid-sugar dehydratases. The biochemical characterization of the remaining components and a genetic expression analysis revealed that D- and L-KDP were converted not only to α-ketoglutarate, but also pyruvate and glycolate through the participation of dehydrogenase and hydrolase (Route III). Further analyses revealed that the Route II pathway of D-arabinose metabolism was not evolutionally related to the analogous pathway from archaea. Te breakdown of D-glucose is central for energy and biosynthetic metabolism throughout all domains of life. Te most common glycolytic routes in bacteria are the Embden-Meyerhof-Parnas, the Entner-Doudorof (ED), and the oxidative pentose phosphate pathways. Te distinguishing diference between the two former glyco- lytic pathways lies in the nature of the 6-carbon metabolic intermediates that serve as substrates for aldol cleav- age. For the ED pathway, this intermediate is D-2-keto-3-deoxy-6-phosphogluconate, from which pyruvate and D-glyceraldehyde 3-phosphate are formed, whereas for Embden-Meyerhof-Parnas pathway, D-glyceraldehyde 3-phosphate and dihydroxyacetone phosphate are produced from fructose-l,6-bisphosphate1. Alternatively, sev- eral hyperthermophilic and/halophilic archaea use a non-phosphorylative ED pathway that fnally yields pyruvate and D-glyceraldehyde2. Schematic sugar conversion is almost analogous to that of the ED pathway, while the equivalent metabolic enzymes possess no evolutionary relationship3–5 (see Fig. 1c). However, pentoses, includ- ing D-xylose, L-arabinose, and D-arabinose, are metabolized through three main routes, which difers from D-glucose described above. Te frst pathway is present in bacteria, and uses isomerases (EC 5.3.1.-), kinases (EC 2.7.1.-), and epimerases (EC 5.1.3.-) to yield D-xylulose 5-phosphate (Fig. 1a). In the second pathway, mainly found in yeast and fungi, pentoses are commonly converted into D-xylulose 5-phosphate by reductases, dehydro- genases instead of isomerases, and epimerases6. Te third pathway for pentoses is (partially) analogous to the non-phosphorylative ED pathway, and has been classifed into Routes I and II, in which pentoses are commonly converted into 2-keto-3-deoxypentonate (KDP) through the participation of aldose 1-dehydrogenase (EC 1.1.1.-), lactone-sugar hydrolase (lactonase) (EC 3.1.1.- ), and acid-sugar dehydratase (EC 4.2.1.-) (schematic reactions A, B, and C in Fig. 1b). Te “Route I” pathway of D-xylose and L-arabinose from bacteria and archaea7–9 is completely homologous to the non-phosphorylative ED pathway, and the KDP intermediate is cleaved through an aldolase reaction to pyruvate and glycolalde- hyde (schematic reaction H). Te analogous pathway is also found in the metabolism of deoxyhexoses, such 1Department of Bioscience, Graduate School of Agriculture, Ehime University, 3-5-7 Tarumi, Matsuyama, Ehime, 790-8566, Japan. 2Faculty of Agriculture, Ehime University, 3-5-7 Tarumi, Matsuyama, Ehime, 790-8566, Japan. 3Center for Marine Environmental Studies (CMES), Ehime University, 2-5 Bunkyo-cho, Matsuyama, Ehime, 790- 8577, Japan. 4Faculty of Food and Nutritional Sciences, Toyo University, 1-1-1 Izumino, Itakura-machi, Ora-gun, Gunma, 374-0193, Japan. 5Department of Bio-molecular Engineering, Graduate School of Science and Technology, Kyoto Institute of Technology, Matsugasaki, Sakyo-ku, Kyoto, 606-8585, Japan. Correspondence and requests for materials should be addressed to S.W. (email: [email protected]) SCIENTIFIC REPORTS | (2019) 9:155 | DOI:10.1038/s41598-018-36774-6 1 www.nature.com/scientificreports/ a b 3HC 3HC OH L-ArabinoseD-Xylose D-Arabinose L-Fucose L-Rhamnose OH OH O O OH O O O HO OH OH HO HO HO OH OH HO HO HO A 1.1.1.46(376) 1.1.1.175(179))1.1.1.1171.1.1.122 1.1.1.377(378) OH OH (Dehydrogenase)(Dehydrogenasee ) OH HO OH L-Arabinose D-Xylose D-Arabinose L-Fucose L-Rhamnose L-Arabinolactone D-Xylonolactone D-Arabinolactone L-Fuconolactone L-Rhamnonolactone Isomerase 5.3.1.4 5.3.1.5 5.3.1.25 5.3.1.14 B 3.1.1.15 3.1.1.65 (Lactonase)( L-Ribulose D-Xylulose D-Ribulose L-Fuculose L-Rhamnulose L-Arabinonate D-Xylonate D-ArabinonateL-Fuconate L-Rhamnonate KinaseKinase 2.7.1.16 2.7.1.17 2.7.1.47 2.7.1.51 2.7.1.5 C 4.2.1.25 4.2.1.82 4.2.1.68 4.2.1.68 4.2.1.90 L-Ribulose 5P D-Ribulose 5P D-Ribulose 1P L-Fuculose 1P L-Rhamnulose 1P (Dehydratase)(DD Epimerase 5.1.3.4 5.1.3.1 4.1.2.17 Aldolase 4.1.2.19 L-2-Keto-3-deoxypentonatate D-2-Keto-3-deoxypentonateeLL-2-Keto-3-deoxyfuconate-2-Keto-33-deoxyfuconatd e L-2-Keto-3-deoxL-2-Keto-3-deoxyrhamnonatxyrhamnonatee (L-KDP) (D-KDP) (L((L-KDFL-KDF)K ) (L-KD((L-KDR)DR) D-Xylulose 5P D-Xylulose 5P D-Xylulose 5P Dihydroxyacetone P Dihydroxyacetone P Dihydroxyacetone P + + + 4.2.1.141 (D) E. coli B Glycolaldehyde L-Lactaldehyde L-Lactaldehyde H 4.1.2.18 D F 4.1.2.53 1.1.1.401 (Aldolase) (Dehydratase) 4.2.1.43 (L) (Dehydrogenase) E. coli K-12 c PyruvatePyyruvata e α-Ketoglutaricc sesemialdehydmialdehydm e 5-Hy5-Hydroxy-droxy-2 2,4-dioxo-pentanonat,4-did ooxo-penentanonatet e PyruPyruvateuvata e 242,2,4-Dioxo-pentanonat 4--Dioxo--pentanontanonate + (αKG(αKGSAGSG A)A) (H(HDOP)DDOOPO ) + (D(DOPDOP) Schematic reactions COG0364 GlGlycolaldehydeycolaldehydlld e L-LactL-Lactaldehydealdehehyded Pathways Domain ABCH COG1063 E 1.2.1.26 G Glucose 6P Bacteria pfam01408 Route I (Dehydrogenase) (Hydrolase) Route I GlucoseArchaea‒ COG1028 I αKα-Keα-Ketoglutaratettoglutarata e PyruPyruvateuvate PyruPyruvatevate D-GluconateBacteria COG0667 + + L-ArabinoseArchaea‒ COG2706 GlycGlycolatcolatee L-Lactate Route Route II D-Xylose Archaea‒ COG3386 Route III Eukaryote COG3618 L-Rhamnose Route III Bacteria COG0129 d D-GalacturonateEukaryote‒ cd00308 Herbaspirillum huttiense IAM 15032 Pathways Domain ABCDE COG2721 C785_RS13670 13675 13680 13685 13690136951370013705 13710 D-GalacturonateBacteria‒ COG0800 Cluster 1 D-GlucarateBacteria‒ ‒ COG0329 GalactarateBacteria‒ ‒ COG3836 C785_RS20550 20555 Bacteria COG3970 I Cluster 2 L-Arabinose Bacteria COG1012 Archaea C785_RS21190 21195 21200 21205 21210 21215 21220 21225 21230 21235 21240 21245XylA 21250 Route I Bacteria Cluster 3 D-Xylose Archaea C785_RS00850 00855 00860 00865 00870 00875 00880 Archaea‒ D-Arabinose Cluster 4 Bacteria ‒ Bacteria ‒‒ C785_RS20900 20895 20890 20885 20880 L-Lyxonate Bacteria ‒‒ Cluster 5 Pathways Domain ABCFG Bacteria C785_RS04075 04080 04085 04090 04095 04100 04105 04110 04115 L-Rhamnose Bacteria Cluster 6 Bacteria L-Fucose Paraburkholderia mimosarum NBRC 106338 Bacteria A19U_RS0115295 01152900115285 01185000118505 Route III L-Arabinose Bacteria Cluster 1 D-Xylose Bacteria A19U_RS010065000100655 100660010066501006750100680 D-Arabinose Bacteria ?? Cluster 2 A19U_RS0104685 010469001046950104700010470501047100104715 Cluster 3 A19U_RS4322000129385 1293900129395 Cluster 4 Acidovorax avenae ATCC 19860 ACAV_RS08185 08180 08175 08170 08165 08160 08155 08150 Paraburkholderia bannensis NBRC 103871 BBA01S_RS31400 31405 31410 31415 31420 31425 31430 31435 31440 Figure 1. Pentose metabolism by microorganisms. (a) Schematic representation of phosphorylative pathways from bacteria. In the metabolism of D-arabinose, E. coli K-12 uses the same enzymes as the L-fucose pathway44, whereas E. coli strain B utilizes some of the enzymes involved in the D-ribitol pathway45. (b) Schematic representation of non-phosphorylative pathways analogous to the ED pathway. Pentose (and/or deoxyhexose) is commonly converted into 2-keto-3-deoxyacid-sugar, the subsequent metabolic fate of which is aldol-cleavage (Route I), dehydration (Route II), or dehydrogenation (Route III, in this study). (c) Comparisons of metabolic enzymes involved in non-phosphorylative pathways. Homologous genes are indicated in the same color and correspond to (d). White letters in boxes were discovered in the present study. (d) Schematic gene clusters related to non-phosphorylative sugar pathway(s). Putative genes in the box were purifed and characterized in the present study. Genes in light- and deep-gray are putative transcriptional regulators and sugar transporters, respectively. as L-rhamnose and L-fucose, by bacteria, fungi, and/or archaea10,11. In the “Route II” pathway of D-xylose12–14, L-arabinose15–18, and D-arabinose19–21 from bacteria and/or archaea, the KDP intermediate is alternatively con- verted into α-ketoglutarate via α-ketoglutaric semialdehyde (αKGSA) by KDP dehydratase (EC 4.2.1.141)22 and αKGSA dehydrogenase (EC 1.2.1.26) (schematic reactions D and E). αKGSA is also a metabolic intermediate involved in D-galacturonic acid and hexaric acids (D-glucarate and D-galactarate) pathways from bacteria23–25. On the other hand, in the modifed non-phosphorylative pathway of L-rhamnose and L-fucose from bacteria and/or archaea26–28, each L-2-keto-3-deoxyrhamnonate (L-KDR) and L-2-keto-3-deoxyfuconate intermediate (L-KDF) is converted into pyruvate and lactate via (putative) 2,4-dioxo-pentanonate by the sequential actions of dehydrogenase and hydrolase (schematic reactions F and G, the “Route III” pathway). Metabolic genes related to these non-phosphorylative pentose (and deoxyhexose) pathways ofen cluster together with the putative sugar (ABC-type) transporter genes and transcriptional regulator gene on the genomes of bacteria and archaea.
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