Determinants of dual substrate specificity revealed by the crystal structure of homoisocitrate dehydrogenase from Thermus thermophilus in complex with homoisocitrate·Mg(2+)·NADH.
HICDH (Homoisocitrate dehydrogenase) is a member of the β-decarboxylating dehydrogenase family that catalyzes the conversion of homoisocitrate to α-ketoadipate using NAD(+) as a coenzyme, which is the fourth reaction involved in lysine biosynthesis through the α-aminoadipate pathway. Although typical HICDHs from fungi and yeast exhibit strict substrate specificities toward homoisocitrate (HIC), HICDH from a thermophilic bacterium Thermus thermophilus (TtHICDH) catalyzes the reactions using both HIC and isocitrate (IC) as substrates at similar efficiencies. We herein determined the crystal structure of the quaternary complex of TtHICDH with HIC, NADH, and Mg(2+) ion at a resolution of 2.5 Å. The structure revealed that the distal carboxyl group of HIC was recognized by the side chains of Ser72 and Arg85 from one subunit, and Asn173 from another subunit of a dimer unit. Model structures were constructed for TtHICDH in complex with IC and also for HICDH from Saccharomyces cerevisiae (ScHICDH) in complex with HIC. TtHICDH recognized the distal carboxyl group of IC by Arg85 in the model. In ScHICDH, the distal carboxyl group of HIC was recognized by the side chains of Ser98 and Ser108 from one subunit and Asn208 from another subunit of a dimer unit. By contrast, in ScHICDH, which lacks an Arg residue at the position corresponding to Arg85 in TtHICDH, these residues may not interact with the distal carboxyl group of shorter IC. These results provide a molecular basis for the differences in substrate specificities between TtHICDH and ScHICDH.
Biotechnology Research Center, The University of Tokyo, Japan.
Kento
K
Takahashi
Biotechnology Research Center, The University of Tokyo, Japan.
Takeo
T
Tomita
Biotechnology Research Center, The University of Tokyo, Japan.
Tomohisa
T
Kuzuyama
Biotechnology Research Center, The University of Tokyo, Japan. Electronic address: umanis@mail.ecc.u-tokyo.ac.jp.
Makoto
M
Nishiyama
D000429:Alcohol Oxidoreductases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001665:Binding Sites / Q000235:genetics; D055162:Biocatalysis; D003460:Crystallization; D018360:Crystallography, X-Ray; D006860:Hydrogen Bonding; D007700:Kinetics; D008274:Magnesium / Q000737:chemistry / Q000378:metabolism*; D008958:Models, Molecular; D009243:NAD / Q000737:chemistry / Q000378:metabolism*; D011485:Protein Binding; D000072417:Protein Domains; D055503:Protein Multimerization; D020836:Protein Structure, Quaternary; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013045:Species Specificity; D055672:Static Electricity; D013379:Substrate Specificity; D016963:Thermus thermophilus / Q000201:enzymology* / Q000235:genetics; D014233:Tricarboxylic Acids / Q000737:chemistry / Q000378:metabolism*
D001426:Bacterial Proteins; D029701:Saccharomyces cerevisiae Proteins; D014233:Tricarboxylic Acids; C517550:homoisocitric acid; D009243:NAD; D000429:Alcohol Oxidoreductases; C021368:homoisocitrate dehydrogenase; D008274:Magnesium
Crystal structure; Homoisocitrate dehydrogenase; Thermus thermophilus; β-decarboxylating dehydrogenase