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The immunosuppressive agent mizoribine monophosphate forms a transition state analogue complex with inosine
Lu Gan1, Mohammad R Seyedsayamdost, Satoshi Shuto
1Department of Biochemistry and Chemistry and the Rosenstiel Basic Medical Sciences Research Center, Brandeis University, Waltham, Massachusetts 02454, USA.
Abstract:
Mizoribine monophosphate (MZP) is the active metabolite of the immunosuppressive agent mizoribine and a potent inhibitor of IMP dehydrogenase (IMPDH). This enzyme catalyzes the oxidation of IMP to XMP with the concomitant reduction of NAD via a covalent intermediate at Cys319 (E-XMP). Surprisingly, mutational analysis indicates that MZP is a transition state analogue although its structure does not resemble that of the expected transition state. Here we report the X-ray crystal structure of the E.MZP complex at 2.0 A resolution that reveals a transition state-like structure and solves the mechanistic puzzle of the IMPDH reaction. The protein assumes a new conformation where a flap folds into the NAD site and MZP, Cys319, and a water molecule are arranged in a geometry resembling the transition state. The water appears to be activated by interactions with a conserved Arg418-Tyr419 dyad. Mutagenesis experiments confirm that this new closed conformation is required for the hydrolysis of E-XMP, but not for the reduction of NAD. The closed conformation provides a structural explanation for the differences in drug selectivity and catalytic efficiency of IMPDH isozymes.
Insights
Mizoribine monophosphate (MZP), an immunosuppressive drug metabolite, inhibits IMP dehydrogenase (IMPDH). Its crystal structure reveals a novel conformation, explaining IMPDH
Area of Science:
- Biochemistry
- Structural Biology
- Enzymology
Background:
- Mizoribine monophosphate (MZP) is the active form of mizoribine, an immunosuppressive drug.
- MZP is a potent inhibitor of inosine monophosphate dehydrogenase (IMPDH), a key enzyme in purine biosynthesis.
- IMPDH catalyzes the oxidation of IMP to XMP, involving a covalent intermediate (E-XMP) and NAD reduction.
Purpose of the Study:
- To elucidate the mechanism of IMPDH inhibition by MZP.
- To determine the X-ray crystal structure of the IMPDH-MZP complex.
- To understand the structural basis for IMPDH enzyme selectivity and catalytic efficiency.
Main Methods:
- X-ray crystallography of the enzyme-Mizoribine monophosphate (E.MZP) complex at 2.0 Å resolution.
- Mutagenesis studies to investigate the role of specific residues and enzyme conformations.
Main Results:
- The crystal structure revealed a novel, closed conformation of IMPDH induced by MZP binding.
- In this conformation, a flap covers the NAD binding site, positioning MZP, Cys319, and a water molecule in a transition state-like geometry.
- Mutagenesis confirmed this closed conformation is essential for E-XMP hydrolysis but not NAD reduction, explaining differential drug selectivity.
Conclusions:
- The study solves the mechanistic puzzle of IMPDH inhibition by MZP, identifying it as a transition state analogue.
- The newly discovered closed conformation is critical for enzyme activity and provides structural insights into IMPDH isozyme differences.
- This structural understanding can inform the development of more selective IMPDH inhibitors for therapeutic applications.