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 α-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 Ki = 62 nM 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 2.8 Å 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.

The crystal structure of isopropylmalate dehydrogenase derived from Thermus thermophilus in a ternary complex with NAD+ and a designed inhibitor, (2S,3S)-(−)-3-methylmercaptomalic acid, has been determined.Figure optionsDownload as PowerPoint slide