A dicarboxylate/4-hydroxybutyrate autotrophic carbon assimilation cycle in the hyperthermophilic Archaeum Ignicoccus hospitalis Harald Huber*†, Martin Gallenberger*, Ulrike Jahn*, Eva Eylert‡, Ivan A. Berg§, Daniel Kockelkorn§, Wolfgang Eisenreich‡, and Georg Fuchs§ *Lehrstuhl fu¨r Mikrobiologie und Archaeenzentrum, Universita¨t Regensburg, Universitaetsstrasse 31, D-93053 Regensburg, Germany; ‡Lehrstuhl fu¨r Biochemie, Department Chemie, Technische Universita¨t Mu¨ nchen, Lichtenbergstrasse 4, D-85748 Garching, Germany; and §Lehrstuhl fu¨r Mikrobiologie, Universita¨t Freiburg, Scha¨nzlestrasse 1, D-79104 Freiburg, Germany Edited by Dieter So¨ll, Yale University, New Haven, CT, and approved April 1, 2008 (received for review February 1, 2008) Ignicoccus hospitalis is an anaerobic, autotrophic, hyperthermophilic starting from acetyl-CoA (4). On the basis of these data, pyruvate Archaeum that serves as a host for the symbiotic/parasitic Archaeum synthase and phosphoenolpyruvate (PEP) carboxylase were pos- Nanoarchaeum equitans. It uses a yet unsolved autotrophic CO2 tulated as CO2 fixing enzymes, with PEP carboxylase serving as the fixation pathway that starts from acetyl-CoA (CoA), which is reduc- only enzyme used for oxaloacetate synthesis. In addition, the tively carboxylated to pyruvate. Pyruvate is converted to phosphoe- operation of an incomplete ‘‘horseshoe-type’’ citric acid cycle, in nol-pyruvate (PEP), from which glucogenesis as well as oxaloacetate which 2-oxoglutarate oxidation does not take place, was demon- formation branch off. Here, we present the complete metabolic cycle strated. Enzyme and labeling data indicated a conventional glu- by which the primary CO2 acceptor molecule acetyl-CoA is regener- coneogenesis, but with some enzymes unrelated to those of the ated. Oxaloacetate is reduced to succinyl-CoA by an incomplete classical pathway. reductive citric acid cycle lacking 2-oxoglutarate dehydrogenase or Although the framework of central carbon metabolism was synthase. Succinyl-CoA is reduced to 4-hydroxybutyrate, which is established, the question remained how acetyl-CoA, the primary then activated to the CoA thioester. By using the radical enzyme CO2 acceptor, is regenerated. Genome analysis did not yield an 4-hydroxybutyryl-CoA dehydratase, 4-hydroxybutyryl-CoA is dehy- immediate answer to this problem. For instance, there was neither drated to crotonyl-CoA. Finally, ␤-oxidation of crotonyl-CoA leads to an indication for enzymes that form acetyl phosphate or acetyl-CoA two molecules of acetyl-CoA. Thus, the cyclic pathway forms an extra from hexose phosphates nor was an enzyme detected that could molecule of acetyl-CoA, with pyruvate synthase and PEP carboxylase regenerate acetyl-CoA from intermediates of the incomplete citric as the carboxylating enzymes. The proposal is based on in vitro acid cycle, such as malate. transformation of 4-hydroxybutyrate, detection of all enzyme activ- However, the genome of I. hospitalis contains a gene for 4-hy- 14 ities, and in vivo-labeling experiments using [1- C]4-hydroxybu- droxybutyryl-CoA dehydratase (Igni_0595), a [4Fe-4S] and FAD- 13 13 13 tyrate, [1,4- C2], [U- C4]succinate, or [1- C]pyruvate as tracers. The containing enzyme, which eliminates water from 4-hydroxybutyryl- pathway is termed the dicarboxylate/4-hydroxybutyrate cycle. It CoA by a ketyl radical mechanism yielding crotonyl-CoA (6, 7). combines anaerobic metabolic modules to a straightforward and This unique enzyme plays a role in a few 4-aminobutyrate ferment- efficient CO2 fixation mechanism. ing bacteria such as Clostridium aminobutyricum (8) and so far was considered to be restricted to the fermentative metabolism of strict ͉ ͉ 4-hydroxybutyryl-CoA dehydratase CO2 fixation pathway acetyl-CoA anaerobic bacteria. However, this enzyme recently was found to play a role in autotrophic CO2 fixation in Metallosphaera sedula gnicoccus hospitalis KIN4/IT (Desulfurococcales, Crenarchaeota) and some other Crenarchaeota (9). The encoded protein of Iis a strictly anaerobic, hyperthermophilic Archaeum with an Igni_0595 shows an amino acid sequence identity of 52% to the optimal growth temperature of 90°C (1). All Ignicoccus species 4-hydroxybutyryl-CoA dehydratase of M. sedula (Msed_1321). grow obligate chemolithoautotrophically by using the reduction of Another odd experimental finding warranted explanation: En- elemental sulfur with molecular hydrogen as the sole energy source zyme studies showed very high activities of enzymes converting and CO2 as the sole carbon source. They possess a unique ultra- oxaloacetate to succinate (4). This finding was surprising because structure of the cell envelope with an outer membrane resembling succinate or succinyl-CoA do not serve as general precursors in that of Gram-negative bacteria (2). Despite the great similarities metabolic networks except for succinyl-CoA acting as precursor of with the other Ignicoccus spp., I. hospitalis exhibits an important tetrapyrrole biosynthesis via the Shemin pathway (10). However, MICROBIOLOGY unique feature: Together with Nanoarchaeum equitans, the only the anaerobe I. hospitalis (i) requires tetrapyrroles only in very small representative of the archaeal kingdom Nanoarchaeota so far (3), amounts, and (ii) the gene for ␦-aminolevulinic acid synthase of the it forms the only known purely archaeal host-symbiont/parasite Shemin pathway appears to be absent in the genome, whereas the system. genes of the C5 pathway were present. The genome of I. hospitalis exhibits only 1,434 putative genes In this article, we show that succinate serves as a central [data available from the DOE Joint Genome Institute (http:// intermediate in a CO fixation cycle where acetyl-CoA is regener- img.jgi.doe.gov/cgi-bin/pub/main.cgi)], clearly indicating a high 2 metabolic specialization. The shortest generation time of the or- ganism grown at 90°C under autotrophic conditions (H2,CO2, and Author contributions: H.H., I.A.B., W.E., and G.F. designed research; M.G., U.J., E.E., I.A.B., elemental sulfur) is1h(1,4).Thisindicatesthat the specialized D.K., and W.E. performed research; H.H., I.A.B., W.E., and G.F. analyzed data; and H.H., U.J., metabolism also is highly streamlined. In preliminary experiments, W.E., and G.F. wrote the paper. I. pacificus and I. islandicus lacked the activities of the key enzymes The authors declare no conflict of interest. of all known autotrophic pathways (5), suggesting the operation of This article is a PNAS Direct Submission. a new autotrophic pathway. †To whom correspondence should be addressed. E-mail: [email protected]. Enzymatic analyses in vitro, combined with retrobiosynthetic This article contains supporting information online at www.pnas.org/cgi/content/full/ analyses of amino acids formed in vivo with [1-13C]acetate as a 0801043105/DCSupplemental. precursor, gave insights into the autotrophic pathway of I. hospitalis © 2008 by The National Academy of Sciences of the USA www.pnas.org͞cgi͞doi͞10.1073͞pnas.0801043105 PNAS ͉ June 3, 2008 ͉ vol. 105 ͉ no. 22 ͉ 7851–7856 Downloaded by guest on September 25, 2021 Table 1. Specific activities of the enzymes of the proposed dicarboxylate/4-hydroxybutyrate cycle in I. hospitalis Specific activity, Reaction catalyzed Assay nmol/min per (see Fig. 2) Enzyme temperature, °C mg protein* Putative gene in I. hospitalis † 1 Pyruvate synthase: methyl viologen CO2 exchange 75 115 Two possible candidates: 85 145† Igni࿝1075–1078 or Igni࿝1256–1259 2 Pyruvate:water dikinase 85 210† Igni࿝1113 3 Phosphoenolpyruvate carboxylase 85 200† Igni࿝341 4 Malate dehydrogenase (NADH/NADPH) 75 1,190/745† Igni࿝1263 5 Fumarate hydratase (class 1) 75 895† Igni࿝0678 6 Fumarate reductase (methyl viologen) 75 840† Igni࿝0276/Igni࿝0445 7 Succinate thiokinase 60 195 Igni࿝0085/Igni࿝0086 80 980 8 Succinyl-CoA reductase (methyl viologen) 60 94 Unknown 9 Succinate semialdehyde reductase 60 1,430/440 Unknown (NADH)/(NADPH) 80 3,130/2,000 10 4-Hydroxybutyryl-CoA synthetase (AMP-forming) 60 100 Igni࿝0475 11 4-Hydroxybutyryl-CoA dehydratase 40 100 Igni࿝0595 12 Crotonyl-CoA hydratase ((S)-3-hydroxybutyryl- 40 460 Igni࿝1058 CoA forming) 13 (S)-3-Hydroxybutyryl-CoA dehydrogenase 60 225/0 Igni࿝1058 (NADH/NADPH) 14 Acetoacetyl-CoA ␤-ketothiolase 60 1,100 2 possible candidates Igni࿝1401, Igni࿝0377 For technical reasons (use of mesophilic coupling enzymes and instability of some substrates), the indicated assay temperatures were used; the growth temperature was 90°C. The values are average values of several determinations; the mean deviations are 5–20%, depending mainly on the batch of cells. *Note the discrepancy in assay temperature and optimal growth temperature. Despite the lower assay temperature, the measured specific activities are already close to the requisite physiological level, although methyl viologen rather than ferredoxin is used as electron carrier of several oxidoreductase assays. †From ref. 4. ated via the dicarboxylic acids of an incomplete reductive citric acid activities of 4-hydroxybutyryl-CoA synthetase (AMP-forming), cycle and 4-hydroxybutyryl-CoA. This cycle represents the sixth 4-hydroxybutyryl-CoA dehydratase, crotonyl-CoA hydratase ((S)- autotrophic carbon fixation pathway in nature (11) and is termed 3-hydroxybutyryl-CoA forming), (S)-3-hydroxybutyryl-CoA dehy- the dicarboxylate/4-hydroxybutyrate cycle. drogenase (NADϩ), and acetoacetyl-CoA ␤-ketothiolase (Table 1). The cells that were studied here grew with a generation time of Results 2 h, which requires a specific rate of CO2 fixation of 0.4