Structure and function of an ancestral-type β-decarboxylating dehydrogenase from Thermococcus kodakarensis.
β-Decarboxylating dehydrogenases, which are involved in central metabolism, are considered to have diverged from a common ancestor with broad substrate specificity. In a molecular phylogenetic analysis of 183 β-decarboxylating dehydrogenase homologs from 84 species, TK0280 from Thermococcus kodakarensis was selected as a candidate for an ancestral-type β-decarboxylating dehydrogenase. The biochemical characterization of recombinant TK0280 revealed that the enzyme exhibited dehydrogenase activities toward homoisocitrate, isocitrate, and 3-isopropylmalate, which correspond to key reactions involved in the lysine biosynthetic pathway, tricarboxylic acid cycle, and leucine biosynthetic pathway, respectively. In T. kodakarensis, the growth characteristics of the KUW1 host strain and a TK0280 deletion strain suggested that TK0280 is involved in lysine biosynthesis in this archaeon. On the other hand, gene complementation analyses using Thermus thermophilus as a host revealed that TK0280 functions as both an isocitrate dehydrogenase and homoisocitrate dehydrogenase in this organism, but not as a 3-isopropylmalate dehydrogenase, most probably reflecting its low catalytic efficiency toward 3-isopropylmalate. A crystallographic study on TK0280 binding each substrate indicated that Thr71 and Ser80 played important roles in the recognition of homoisocitrate and isocitrate while the hydrophobic region consisting of Ile82 and Leu83 was responsible for the recognition of 3-isopropylmalate. These analyses also suggested the importance of a water-mediated hydrogen bond network for the stabilization of the β3-α4 loop, including the Thr71 residue, with respect to the promiscuity of the substrate specificity of TK0280.
Biotechnology Research Center, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan.
Tetsu
T
Shimizu
Biotechnology Research Center, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan.
Lulu
L
Yin
Biotechnology Research Center, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan.
Ayako
A
Yoshida
Department of Synthetic Chemistry and Biological Chemistry, Graduate School of Engineering, Kyoto University, Katsura, Nishikyo-ku, Kyoto 615-8510, Japan.
Yuusuke
Y
Yokooji
Department of Synthetic Chemistry and Biological Chemistry, Graduate School of Engineering, Kyoto University, Katsura, Nishikyo-ku, Kyoto 615-8510, Japan.
Shin-Ichi
SI
Hachisuka
Department of Synthetic Chemistry and Biological Chemistry, Graduate School of Engineering, Kyoto University, Katsura, Nishikyo-ku, Kyoto 615-8510, Japan.
Takaaki
T
Sato
Biotechnology Research Center, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan.
Takeo
T
Tomita
Biotechnology Research Center, Department of Biotechnology, Toyama Prefectural University, Imazu, Toyama 939-0398, Japan.
Hiromi
H
Nishida
Department of Synthetic Chemistry and Biological Chemistry, Graduate School of Engineering, Kyoto University, Katsura, Nishikyo-ku, Kyoto 615-8510, Japan.
Haruyuki
H
Atomi
Biotechnology Research Center, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan.
Tomohisa
T
Kuzuyama
Biotechnology Research Center, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan umanis@mail.ecc.u-tokyo.ac.jp.
Makoto
M
Nishiyama
D019843:Archaeal Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D020134:Catalytic Domain; D005816:Genetic Complementation Test; D007523:Isocitrates / Q000737:chemistry / Q000378:metabolism; D008239:Lysine / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D008293:Malates / Q000737:chemistry / Q000378:metabolism; D010088:Oxidoreductases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013329:Structure-Activity Relationship; D013379:Substrate Specificity; D019712:Thermococcus / Q000201:enzymology / Q000235:genetics; D016963:Thermus thermophilus / Q000201:enzymology / Q000235:genetics; D014233:Tricarboxylic Acids / Q000737:chemistry / Q000378:metabolism
D019843:Archaeal Proteins; D007523:Isocitrates; D008293:Malates; D011994:Recombinant Proteins; D014233:Tricarboxylic Acids; C517550:homoisocitric acid; C025313:beta-isopropylmalate; C034219:isocitric acid; D010088:Oxidoreductases; D008239:Lysine
3-isopropylmalate; Thermococcus; ancestral enzyme; homoisocitrate; isocitrate