The nonphosphorylative Entner-Doudoroff pathway in the thermoacidophilic euryarchaeon Picrophilus torridus involves a novel 2-keto-3-deoxygluconate- specific aldolase

Abstract

The pathway of glucose degradation in the thermoacidophilic euryarchaeon Picrophilus torridus has been studied by in vivo labeling experiments and enzyme analyses. After growth of P. torridus in the presence of [1-(13)C]- and [3-(13)C]glucose, the label was found only in the C-1 and C-3 positions, respectively, of the proteinogenic amino acid alanine, indicating the exclusive operation of an Entner-Doudoroff (ED)-type pathway in vivo. Cell extracts of P. torridus contained all enzyme activities of a nonphosphorylative ED pathway, which were not induced by glucose. Two key enzymes, gluconate dehydratase (GAD) and a novel 2-keto-3-deoxygluconate (KDG)-specific aldolase (KDGA), were characterized. GAD is a homooctamer of 44-kDa subunits, encoded by Pto0485. KDG aldolase, KDGA, is a homotetramer of 32-kDa subunits. This enzyme was highly specific for KDG with up to 2,000-fold-higher catalytic efficiency compared to 2-keto-3-deoxy-6-phosphogluconate (KDPG) and thus differs from the bifunctional KDG/KDPG aldolase, KD(P)GA of crenarchaea catalyzing the conversion of both KDG and KDPG with a preference for KDPG. The KDGA-encoding gene, kdgA, was identified by matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry (MS) as Pto1279, and the correct translation start codon, an ATG 24 bp upstream of the annotated start codon of Pto1279, was determined by N-terminal amino acid analysis. The kdgA gene was functionally overexpressed in Escherichia coli. Phylogenetic analysis revealed that KDGA is only distantly related to KD(P)GA, both enzymes forming separate families within the dihydrodipicolinate synthase superfamily. From the data we conclude that P. torridus degrades glucose via a strictly nonphosphorylative ED pathway with a novel KDG-specific aldolase, thus excluding the operation of the branched ED pathway involving a bifunctional KD(P)GA as a key enzyme.

FIG. 1.

Growth of P. torridus on 25 mM glucose and 0.2% yeast extract. The cultures were incubated at pH 0.9 and 60°C in a 8,000-ml fermentor filled with 5,000 ml medium and stirred at 200 rpm. Growth on glucose and yeast extract (▪), growth on yeast extract in the absence of glucose (•), and glucose consumption (□) are shown.

FIG. 2.

SDS-PAGE of purified gluconate dehydratase (A) and KDG aldolase (B). Lane 1, molecular mass markers, lane 2, purified enzyme. The positions of molecular mass markers (in kilodaltons) are shown to the left of each gel.

FIG. 3.

Multiple amino acid sequence alignment of KDGA from P. torridus and its homologs in T. acidophilum and T. volcanii with archaeal bifunctional KD(P)GA, NAL of E. coli, and DHDPS of E. coli. The alignment was generated with ClustalX using the gonnet matrix. Consensus patterns (DHDPS_1 and DHDPS_2) are boxed. The dihydrodipicolinate synthetase family signature 1 consensus pattern is [GSA]-[LIVM]-[LIVMFY]-x(2)-G-[ST]-[TG]-G-E-[GASNF]-x(6)-[EQ] (PS00665), and the dihydrodipicolinate synthetase family signature 2 consensus pattern is Y-[DNSAH]-[LIVMFAN]-P-x(2)-[STAV]-x(2,3)-[LIVMFT]-x(13,14)-[LIVMCF]-x-[SGA]-[LIVMFNS]-K-[DEQAFYH]-[STACI] (PS00666). Signature 2 consensus pattern includes a lysine (marked by an arrow) which is involved in Schiff base formation. Catalytic residues (▾) and residues forming a putative phosphate-binding motif (#) for KDP according to references , , and are indicated. Pto-KDGA, KDGA of P. torridus; Tac-KDGA and Tvo-KDGA, homologs of Pto-KDGA in T. acidophilum and T. volcanii Ta1157 and TVN1228; Sso-KD(P)GA, S. solfataricus gi:2879782; Tte-KD(P)GA, T. tenax gi:41033593; Eco-NAL, gi:128526; Eco-DHDPS, gi:145708.

FIG. 4.

Phylogenetic relationship of KDGA with selected members of the DHDPS-like protein family from bacteria and archaea. Characterized enzymes are underlined. The numbers at the nodes are bootstrapping values according to neighbor joining (generated by using the neighbor-joining algorithm of ClustalX). Accession numbers for the proteins and enzymes from the different species are shown in parentheses as follows: for KDGA, Picrophilus torridus Pto1279, Thermoplasma volcanium GSS1 (gi:13542059) TVN1228, and Thermoplasma acidophilum DSM 1728 (gi:16082170) Ta1157; for NAL, Homo sapiens (gi:13540533), Haemophilus influenzae (Swiss Prot P44539), and Escherichia coli (Swiss Prot P06995); for KD(P)GA, Sulfolobus solfataricus (gi:2879782), Sulfolobus tokodaii strain 7 (gi:15922811), Sulfolobus acidocaldarius DSM 639 (gi:70606067), Thermoproteus tenax (gi:41033593), Pyrobaculum arsenaticum DSM 13514 (gi:145591599), and Metallosphaera sedula DSM 5348 (gi:146304062); for DHDPS, Methanocaldococcus jannaschii DSM 2661 (gi:15668419), Methanobrevibacter smithii ATCC 35061 (gi:148642891), Haloarcula marismortui ATCC 43049 (gi:55377124), Natronomonas pharaonis DSM 2160 (gi:76801395), Halorubrum lacusprofundi ATCC 49239 (gi:153895122), Haloquadratum walsbyi DSM 16790 (gi:110667468), Thermotoga maritima (gi:7531088), Escherichia coli (gi:145708), Nicotiana sylvestris (gi:14575543), and Mycobacterium tuberculosis AF2122/97 (gi:31793927); for proteins/enzymes not shown in groups, Halobacterium sp. NRC-1 (gi:15789685), Picrophilus torridus DSM 9790 (gi:48478098) Pto1026, Thermoplasma acidophilum DSM 1728 (gi:16081713) Ta0619, Thermoplasma volcanium GSS1 (gi:14324884), TVG0663048, and Ferroplasma acidarmanus Fer1 (gi:69268899). The scale bar corresponds to 0.1 substitution per site.

FIG. 5.

Proposed nonphosphorylative Entner-Doudoroff pathway in Picrophilus torridus in comparison to the proposed branched Entner-Doudoroff pathway in Sulfolobus solfataricus. Enzymes and the genes encoding these enzymes and the promiscuous activities of GDH, GAD, KDGA, KDGK, and KD(P)GA are indicated. Abbreviations: GDH, glucose dehydrogenase; GAD, gluconate dehydratase; KDG, 2-keto-3-deoxygluconate; KDGal, 2-keto-3-deoxygalactonate; KDGA, KDG-specific aldolase; GADH, glyceraldehyde dehydrogenase, GLK, glycerate kinase (2-phosphoglycerate forming); ENO, enolase; PYK, pyruvate kinase; KDGK, KDG kinase; KDPG, 2-keto-3-deoxy-6-phosphogluconate; KDPGal, 2-keto-3-deoxy-6-phosphogalactonate; KD(P)GA, bifunctional KDGKDPG aldolase; GAPN, nonphosphorylative glyceraldehyde dehydrogenase; GAOR, glyceraldehyde oxidoreductase; PGM, phosphoglycerate mutase.