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1 No. 8] Proc. Japan Acad., 75, Ser. B (1999) 241 Comparison of p athways for amino acid biosynthesis in archaebacteria using their genomic DNA sequences By Sadaharu HIGucHI,*) Tsuyoshi KAWASHIMA,*) and Masashi (Communicated by Setsuro EBASHI, M.J.A., Oct. 12, 1999) SuzuKI*)'**)'t) Abstract: Metabolic pathways for synthesizing amino acids in 7 archaebacteria were compared on the basis of enzymes identified using their genomic DNA sequences. No essential difference was found between the set of enzymes identified for.a heterotroph, Archeaoglobus fulgidus, and for those of two autotrophs, Methanococcus jannashii and Methanobacterium thermoautotrophicum. It is likely that these three organisms are able to synthesize all the 20 types of amino acids found in proteins in essentially the same way. From the genomes of the other four heterotrophs, Pyrococcus abyssi, Pyrococcus sp. 0T3, Thermoplasma volcanium, and Aeropyrum pernix, most of the genes coding for enzymes in the pathway for synthesizing histidine from 5-phosphoribosyl-l-pyrophosphate were missing, as were many other genes coding for enzymes in the pathways for synthesizing valine and leucine from pyruvate. The pathways for synthesizing aromatic residues from 3-phosphoglycerate seemed to be missing from P. 0T3, while in T volcanium the pathways starting from oxaloacetate were found to be significantly incomplete. Key words: Archaea, gene identification; genome analysis; homology search; metabolism. Introduction. The ultimate goal of the informatic analysis of a complete genomic DNA sequence is to predict, on the basis of the sequence, the overall organization of an organism from molecular to higher levels, and thereby to describe possible interactions of an organism with its environment. Identification of the metabolic pathways of an organism is essential for this purpose in order to clarify its dietary requirements. In our previous paper's the TCA cycles and glycolytic pathways of archaebacteria were compared on the basis of the enzymes identified using their genomic DNA sequences. In this paper, by using the genomic DNA sequences of 7 archaebacteria,2~-7) their pathways for synthesizing amino acids are compared. Materials and methods. The genomic DNA sequences were collected from databases; those of Methanococcus jannaschii2~ and Archaeoglobus fulgidus3~ from the TIGR database ( tdb/mdb/mjdb/ and respectively), that of Methanobacterium thermoau- *) AIST -NIBHT CREST Centre of Structural Biology Higashi, Tsukuba , Japan. **) Graduate School of Human and Environmental Sciences University of Tokyo, Komaba, Meguro-ku, Tokyo , Japan. t) Correspondence to: M. Suzuki at AIST-NIBHT., 1-1, totrophicum4~ from the Genome Therapeutics Corporation database ( genesequences/methanobacter/), that of Pyrococcus abyssi from the Genoscope database ( and that of Aeropyrum pernix5~ from another database ( The genomic DNA sequence of Pyrococcus sp. 0T3 was taken from our ARCHAIC database ( archaic/). This DNA sequence is essentially the same as that originally reported by another group,6~ but some minor corrections have been made. The genomic DNA sequence of Thermoplasma volcanium GSS1 has been determined by our group,7~ and this sequence is in preparation for the submission to the ARCHAIC database. The bacterial standard pathways for amino acid biosynthesis were summarized following Gottshalk8~ (Fig. 1). Enzymes that were likely to be involved in amino acid biosynthesis were identified (Fig. 2) on the basis of homology search in essentially the same was as described earlier.' Results and discussion. The standard bacterial pathways for amino acid biosynthesis8~ are essentially the same as those9} of eukaryotes. The carbon skeletons of amino acids are synthesized from 6 metabolic precursors (a-ketoglutarate, oxaloacetate, pyruvate, phospho-

242 S. HicucHI T. KAwASHIMA, and M. SUZUKI [Vol. 75(B) Fig. 1. Steps in the standard amino acid biosynthesis pathways (a), and the list of metabolic precursors in the pathways (b).

2 242 S. HicucHI T. KAwASHIMA, and M. SUZUKI [Vol. 75(B) Fig. 1. Steps in the standard amino acid biosynthesis pathways (a), and the list of metabolic precursors in the pathways (b). The same scheme as in Fig. 2 is used for the numbering of the enzymes mediating the steps. The reaction from glutamate-5-phosphate (#2) to A'pyrroline-5-carboxylate (#3) progresses spontaneously. Fourteen steps that are not mediated by enzymes identified by any of the genomic DNA sequences, and step 2 to which only one of the two subunits of the corresponding enzyme is identified, are shown by thin broken arrows. The other 9 steps that are not mediated by the enzymes identified with either of the genomic sequences of the 2 autotrophs, Methanococcus jannaschii and Met hanobacterium thermoautotrophicum, but are mediated by the enzymes identified with at least one of the genomic sequences, are shown by bold broken arrows. The 6 groups of the biosynthetic pathways starting from different precursors in the central metabolic pathways are shown by different colors.

3 No. 8] Amino acid biosynthesis in archaebacteria 243 Fig. 2. Identification of enzymes mediating the steps for the amino acid biosynthesis in Methaoococcus jannaschii (MJ), Methanobacterium thermoautotrophicum (MT), Archaeoglobus folgidus (AF), Pyrococcus abyss (PA), Pyrococcus sp. 0T3 (OT), Thermoplasma volcanium (TV), andaeropyrum peniix (AP). Circles indicate that the corresponding enzymes were identified, while crosses indicating no identification. Triangles indicate identification of only one of the two subunits of each enzyme. Double circles show that enzymes 62 and 64 are produced apparently as a single peptide ina. fulgidus.

4 244 S. Hiu CHI T. KAV ASHIAIA, and M. S~iztrxi [Vol. 75(B) enolpyruvate, 3-phosphoglycerate, and 5-phosphoribosyl-l-pyrophosphate), that are produced in the glycolytic pathway and the pentose phosphate and TCA cycles (Fig. 1). In eukaryotes phenylalanine can be converted to tyrosine, but this is not typical for bacteria, and the corresponding enzyme was not coded by the archaebacterial genomes (this step being excluded from Fig. 1). In bacteria, in general, alanine can be converted from aspartate (step 39 in Fig. 1), although this does not take place in eukaryotes. Enzymes that were expected to mediate 14 of the steps in the standard bacterial pathways were not identified with any of the genomic DNA sequences examined Fig, 3. Steps identified in the pathways of the archaebacteria. The steps that were connected by both sets of enzymes identified with the two autotrophs (MJ and MT) are grouped to those involved in the synthesis of each type of amino acid. In this grouping, steps that are involved in multiple types of amino acids are counted multiple times. The numbers of the corresponding steps connected by the enzymes of each heterotroph is listed. The numbers that are smaller than half the corresponding numbers of the two autotrophs are highlighted in bold and with circles. It is unlikely that these pathways are functional. (steps 4, 5, 7, 21, 22, 23, 34, 36, 39, 40, 53, 67, 70, and 79 in Fig. 2). Only one of the two subunits of glutamate synthase, mediating step 2, was identified. Nine other enzymes were unidentified with either of the genomic sequences of the 2 autotrophs, Methanococcus jam - naschii and Methanobacteri a m thermoautotrophicum, but were identified with at least one of the archaebacterial genomic sequences (steps 1, 6, 11, 25, 27, 35, 49, 50, and 71 in Fig. 2). The two autotrophs are expected to synthesize all the 20 types of amino acids found in proteins, and thus, new types of enzymes are expected to mediate the above 24 steps, unless biosynthesis of the corresponding amino acids are carried out in totally different ways. Each archaebacterial genome codes for several copies of amino acid aminotransferase of unknown substrate specificity. By using these enzymes, synthesis of some amino acids from the corresponding a-ketoacids might be possible-e.g. alanine from pyruvate. Alternatively, some of the 24 steps clustered in the same pathways-i.e. steps 4 and 5, steps 21-23, steps 34 and 35, steps 49 and 50, and steps 70 and 71, might be bypassed by yet unknown types of reactions. By the enzymes identified using the genomic sequences of the two autotrophs, 59 (M. jannaschii) and 57 (M. thermoautotrophicum) steps in the standard pathways were connected (Fig. 3). Of these steps 56 were connected by the enzymes of a heterotroph, Archaeoglobus fulgidus. Thus, this heterotroph might be able to synthesize all the 20 amino acids in essentially the same way as the two heterotrophs do. The three organisms showed further similarity; steps 1, 49, and 50 being unconnected. The rest of the archaebacteria, Pyrococcus abyssi, Pyrococcus sp. 0T3, Thermoplasma volcanium, and Aeropyrum pernix, are heterotrophic. The numbers of steps connected by their enzymes were smaller, 26 (P. sp. 0T3), 29 (TT volcanium), 35 (A. pernix), and 40 (P abyssi), and the pathways identified were incomplete to larger degrees (Fig. 2). In order to examine the incompleteness of these pathways further, in comparison with the pathways of the two autotrophs, the steps that were connected by both sets of enzymes identified with the two autotrophs were grouped into those involved in the synthesis of each type of amino acid. In this grouping, steps that were involved in multiple types of amino acids were counted multiple times (Fig. 3). The number of corresponding steps connected by the enzymes of each heterotroph was listed (Fig. 3). Through this process, it was found that most of the enzymes involved in the synthesis of histidine from 5-phosphoribosyl-l-pyrophosphate were missing from all of the four genomes of the heterotrophs, P abyssi, P. 0T3, TT volcanium, and A. pernix. Thus, it is unlikely that this pathway is functional in these organisms. The pathways for synthesizing valine and leucine from pyruvate, too, were identified to be largely incomplete in the four heterotrophs. In addition, in T7 volcanium the pathways for synthesizing aspartate, asparagine, methionine, threonine, isoleucine, and lysine from oxaloacetate were found to be largely incomplete. The pathway of synthesizing aromatic residues from phosphoenolpyruvate seemed to be nearly completely missing from P. 0T3, but many enzymes in

5 No. 8] Amino acid biosynthesis in archaebacteria 245 this pathway were coded by the genome of P abyssi. Thus, two species closely related in phylogeny can still have notable differences in their metabolic systems. The above description does not prove that the corresponding amino acids are not synthesized. It has been reported that branched-chain aminotransferase of another archaebacterium, Methanococcus aeolic us, has a substrate specificity broader than expected, and can synthesize aromatic residues marginally.1 This reaction might be used, for example, by P. 0T3 for synthesizing the aromatic residues. Correlation can be seen between some characteristics of the pathways identified and the dietary requirements of the archaebacteria. It has been reported that the archaebacterium, Archaeoglobus fulgidus, can produce a small amount of methane from carbon dioxide in the same way as autotrophic methanogens do." Thus, A. fulgidus might become autotrophic upon methanogenesis, although its normal mode is undoubtedly heterotrophic. The heterotrophicity of this organism is clearly reflected in its complete TCA cycle, in contrast to the TCA cycles of autotrophic methanogens, whose right half is totally missing.' The other 4 heterotrophs all require amino acids for their growth. Archaebacteria of the Pyrococcus genus were isolated from the deep sea, where the water was expected to be rich in proteins, and their absolute dependency on amino acids is reflected in their lack of some of the standard pathways for amino acid biosynthesis, as well as in some characteristics of their central metabolic pathways described in our earlier report.' Acknowledgement. This work was supported by the Core Research for Evolutional Science and Technology (CREST) program of the Japan Science and Technology Corporation (JST). References 1) Koike, H., Kawashima, T., and Suzuki, M. (1999) Proc. Japan Acad. 75B, ) Bult, C. J., White, 0., Olsen, G. J., Zhou, L., Fleischmann, R. D. et al. (1996) Science 273, ) Klenk, H.-P., Clayton, R. A., Tomb, J.-F., White, 0., Nelson, K. E, et al. (1997) Nature 390, ) Smith, D. R., Doucette-Stamm, L. A., Deloughery, C., Lee, H., Dubois, J. et al. (1997) J. Bacteriol. 179, ) Kawarabayashi, Y., Hino, Y., Horikawa, H., Yamazaki, S., Haikawa, Y. et al. (1999) DNA Res. 6, ) Kawarabayasi, Y., Sawada, M., Horikawa, H., Haikawa, Y., Hino, Y. et al. (1998) DNA Res. 5, ) Kawashima, T., Yamamoto, Y., Aramaki, H., Nunoshiba, T., Kawamoto, T. et, al. (1999) Proc. Japan Acad. 75B, ) Gottschalk, G. (1979) Bacterial Metabolism. Springer-Verlag, New York. 9) Stryer, L. (1995) Biochemistry. 4th ed., Freeman and Co. San Francisco. 10) Xing, R., and Whitman, W.B. (1992) J, Bacteriol 174, ) White, R. H. (1988) J. Bacterial. 170,

Determination of the complete genomic DNA sequence of Thermoplasma volcanium GSS 1

No. 7] Proc. Japan Acad., 75, Ser. B (1999) 213 Determination of the complete genomic DNA sequence of Thermoplasma volcanium GSS 1 By Tsuyoshi KAWASHIMA,*1l Yoshihiro YAMAMOTO,*2) Hironori ARAMAKI,*3)