Crystal structure of 3-isopropylmalate dehydrogenase in complex with NAD(+) and a designed inhibitor.
Isopropylmalate dehydrogenase (IPMDH) is the third enzyme specific to leucine biosynthesis in microorganisms and plants, and catalyzes the oxidative decarboxylation of (2R,3S)-3-isopropylmalate to alpha-ketoisocaproate using NAD(+) as an oxidizing agent. In this study, a thia-analogue of the substrate was designed and synthesized as an inhibitor for IPMDH. The analogue showed strong competitive inhibitory activity with K(i)=62nM toward IPMDH derived from Thermus thermophilus. Moreover, the crystal structure of T. thermophilus IPMDH in a ternary complex with NAD(+) and the inhibitor has been determined at 2.8A resolution. The inhibitor exists as a decarboxylated product with an enol/enolate form in the active site. The product interacts with Arg 94, Asn 102, Ser 259, Glu 270, and a water molecule hydrogen-bonding with Arg 132. All interactions between the product and the enzyme were observed in the position associated with keto-enol tautomerization. This result implies that the tautomerization step of the thia-analogue during the IPMDH reaction is involved in the inhibition.
Nango
Eriko
E
Department of Chemistry, Tokyo Institute of Technology, O-okayama, Meguro-ku, Tokyo 152-8551, Japan.
Yamamoto
Takashi
T
Kumasaka
Takashi
T
Eguchi
Tadashi
T
D050539:3-Isopropylmalate Dehydrogenase / Q000037:antagonists & inhibitors* / Q000737:chemistry* / Q000378:metabolism; D000595:Amino Acid Sequence; D001665:Binding Sites; D002262:Carboxy-Lyases / Q000378:metabolism; D018360:Crystallography, X-Ray; D004791:Enzyme Inhibitors / Q000737:chemistry* / Q000494:pharmacology*; D007930:Leucine / Q000737:chemistry / Q000378:metabolism; D008969:Molecular Sequence Data; D009243:NAD / Q000737:chemistry* / Q000378:metabolism; D011487:Protein Conformation; D016963:Thermus thermophilus / Q000201:enzymology*
D004791:Enzyme Inhibitors; D009243:NAD; D050539:3-Isopropylmalate Dehydrogenase; D002262:Carboxy-Lyases; D007930:Leucine