Functional studies of rice KARI and inhibitor discovery

作者:

AV Bhumkar

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摘要:

Ketol acid reductoisomerase (KARI), a metalo-enzyme (with NADPH as cofactor), is the second common enzyme of the branched chain amino acid biosynthesis (BCAA) pathway. This enzyme catalyzes the isomerization and reduction of α-acetolactate and α-aceto-α-hydroxybutyrate and converts these to α,β-dihydroxy isovalerate and α,-β-dihydroxy-β-methylvalerate, respectively. KARI has been widely studied as a target for the development of herbicides with two potent inhibitors, N-hydroxy-N-isopropyloxamate (IpOHA) (22 pM forKARI) and 2-(dimethylphosphinoyl)-2-hydroxyacetic acid (Hoe704) (70 nM for spinach KARI) being tested as experimental herbicides. To better understand the structure andenzymatic activity of KARI a number of experiments were undertaken including, metal ion replacement and site directed mutagenesis studies. In addition to these studies, a fragment based inhibitor discovery program was also initiated using X-ray crystallography and spectrophotometric assays.Isothermal titration calorimetry was used to determine values of 0.8 μM and 4 μM for Mgand NADPH for binding to rice KARI, respectively. The Kd values for both NADPH and Mgare unaltered in the presence or absence of the other binding partner. The preliminary data from the metal ion binding study for Mnand Coshows that, Cobinds the enzyme most tightly followed by Mnand Mg. Based on metal ion replacement studies, Mnwas found to have the highest turnover for the reduction reaction followed by Coand Mgwith each having values of 290.8 min, 64.8 minand 40.6 min, respectively. Mn, Coand Mghad values of 16 μM, 43 μM and 102 μM, respectively. A pH profile study for rice KARI in the presence of Mg, Coand Mnwas undertaken. The results show that for Mg, two pKvalues (6.9 and 9.9) were observed. However, when a similar profile was determined for Co, these values shifted to 7.4 and 7.8 and for Mnwere 6.5 and 8.7. It appears that the second pKvalue is shifted the most when Mgis replaced by Coor Mn. This difference could therefore contribute to the fact that Mnand Co2+ cannot carryout the isomerization reaction. The most likely candidate to attribute this pKshift would be a water molecule coordinated to the metal ions. This is because, within the active site, there are no amino acid residues that would be likely to have an ionizable group at that pKvalue. A site directed mutagenesis study performed on Arg589 indicated that, though Arg589 is important for NADPH binding it does not affect the binding of Mgnor does it affect the turnover number. The value for NADPH decreases four- fold when Arg589 is mutated to alanine. The catalytic efficiency decreased from 1.40 minμMfor α-acetolactate to 0.63 minμMand to 1.0 minμMas observed for Arg589Ala and Arg589Glu mutants, respectively. This data supports the hypothesis that the C- terminal tail is critical for clamping shut the N- and C- domains together, resulting in a more catalytically efficient enzyme.In this study, the crystal structures of rice KARI – Mgin complex with five fragments from Zenobia library, 3-aminopyridine (ZT038), 3-hydroxypyridine (ZT042), 3-aminobenzonitrile (ZT389), 2-amino-4-methylphenol (ZT398) and 3,4-diaminotoluene (ZT393) have been solved to 1.5 - 2.5 resolution. The results show that ZT398 is bound near the active site and the other fragments bound on the surface of the protein. The binding site of ZT038 and ZT042 is near the Lys105, a part of the protein that may be important for domain motion while ZT393 binds near the entrance of the active site. In addition, through the screening of a Maybridge fragment library, two new rice KARI inhibitors, 3-[(2-thienylthio)methyl]benzoic acid (KM02425) and2-hydroxy-2-phenylacetic acid (JFD3933) have been identified with values of 212 ± 132.4 μM and 311.5 ± 122.5 μM, respectively. To better understand the mode of binding of KM02425, the crystal structure of KM02425 in complex with rice KARI – Mgenzyme was determined to 2.45 . The results show that this molecule binds within the enzyme's active site. Docking studies were also undertaken and suggest an alternative mode

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DOI:

http://espace.library.uq.edu.au/view/UQ:311382

年份:

2013

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Kari
2013

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