Kinetic analysis of site-directed mutants of methionine synthase from Candida albicans

Prasannan, Priya; Suliman, Huda S.; Robertus, Jon D.

doi:10.1016/j.bbrc.2009.03.098

Cited by 14 publications

(14 citation statements)

References 16 publications

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“…Site-directed mutagenesis was introduced by the Quikchange kit from Stratagene. The mutant GST-Met6p fusion proteins demonstrated varied enzymatic activity validating the use of this approach in the design of new antifungal drugs (Prasannan et al, 2009). …”

Section: Gene Function-essential Genesmentioning

confidence: 98%

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Site-Directed and Random Insertional Mutagenesis in Medically Important Fungi

Sturtevant¹

2013

Genetic Manipulation of DNA and Protein - Examples From Current Research

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Section: Gene Function-essential Genesmentioning

confidence: 98%

“…One such gene is MET6. In C. albicans, Prasannan et al (2009) constructed GST fusions of mutated C. albicans MET6 and expressed the fusion protein in a MET6 Saccharomyces mutant. A 3-D model that was modified from the known crystallized structure of the Arabidopsis enzyme was used to select sites for mutation in MET6.…”

Section: Gene Function-essential Genesmentioning

confidence: 99%

Site-Directed and Random Insertional Mutagenesis in Medically Important Fungi

Sturtevant¹

2013

Genetic Manipulation of DNA and Protein - Examples From Current Research

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“…Thus, the present MD calculations highlight the flexibility of the active site [62] in regulating the function of the MetH. Mutation effect on the Zn-Hcy displacement Recently, Prasannan et al [63] experimentally studied directed mutations of some selected residues in the Hcy and folate binding sites of the MetE from Candida albicans. They mutated two residues, Asp614 (which forms a salt-bridge with the ammonium group of Hcy) by Ala and Ser448 (which forms hydrogen bond with the carboxylate of Hcy) by Ala, in the binding site of Hcy.…”

mentioning

confidence: 91%

Zinc–Homocysteine binding in cobalamin‐dependent methionine synthase and its role in the substrate activation: DFT, ONIOM, and QM/MM molecular dynamics studies

Abdel‐Azeim

Chung

et al. 2011

J Comput Chem

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Cobalamin-dependent methionine synthase (MetH) is an important metalloenzyme responsible for the biosynthesis of methionine. It catalyzes methyl transfer from N(5)-methyl-tetrahydrofolate to homocysteine (Hcy) by using a zinc ion to activate the Hcy substrate. Density functional theory (B3LYP) calculations on the active-site model in gas phase and in a polarized continuum model were performed to study the Zn coordination changes from the substrate-unbound state to the substrate-bound state. The protein effect on the Zn(2+) coordination exchange was further investigated by ONIOM (B3LYP:AMBER)-ME and EE calculations. The Zn(2+)-coordination exchange is found to be highly unfavorable in the gas phase with a high barrier and endothermicity. In the water solution, the reaction becomes exothermic and the reaction barrier is drastically decreased to about 10.0 kcal/mol. A considerable protein effect on the coordination exchange was also found; the reaction is even more exothermic and occurs without barrier. The enzyme was suggested to constrain the zinc coordination sphere in the reactant state (Hcy-unbound state) more than that in the product state (Hcy-bound state), which promotes ligation of the Hcy substrate. Molecular dynamics simulations using molecular mechanics (MM) and PM3/MM potentials suggest a correlation between the flexibility of the Zn(2+)-binding site and regulation of the enzyme function. Directed in silico mutations of selected residues in the active site were also performed. Our studies support a dissociative mechanism starting with the Zn-O(Asn234) bond breaking followed by the Zn-S((Hcy)) bond formation; the proposed associative mechanism for the Zn(2+)-coordination exchange is not supported.

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“…We have previously shown that the MET6 gene is essential for fungal growth [14], and we have studied the enzyme using steady-state kinetics [1], site-directed mutagenesis [15] and X-ray crystallography [7,16]. We have been unable to crystallize wild-type Met6p from C. albicans, but we have solved the structures of several fully active mutants using surface entropy reduction [7].…”

Section: Introductionmentioning

confidence: 98%

The Cobalamin-Independent Methionine Synthase Enzyme Captured in a Substrate-Induced Closed Conformation

Ubhi

Robertus

2015

Journal of Molecular Biology

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The cobalamin-independent methionine synthase enzyme catalyzes a challenging reaction: the direct transfer of a methyl from 5-methyl-tetrahydrofolate-glutamate3 to the l-homocysteine thiol. The enzyme has a dual (βα)8 TIM barrel structure that binds, activates and brings the reactants into reaction proximity by conformational movements. In the previously observed open structures, the substrates bind too far apart to react, but we have captured a ternary complex with both substrates bound in a closed form of the enzyme. The closing is described in terms of a hinge between the N- and C-terminal TIM barrels and a rearrangement of key loops within the C domain. The substrate specificity can now be rationalized and the structure reveals His707 as the acid that protonates the THF leaving group through a water molecule trapped in the closed active site. The substrates are correctly oriented for an in-line attack by l-homocysteine on the N(5)-methyl.

show abstract

Kinetic analysis of site-directed mutants of methionine synthase from Candida albicans

Cited by 14 publications

References 16 publications

Site-Directed and Random Insertional Mutagenesis in Medically Important Fungi

Site-Directed and Random Insertional Mutagenesis in Medically Important Fungi

Zinc–Homocysteine binding in cobalamin‐dependent methionine synthase and its role in the substrate activation: DFT, ONIOM, and QM/MM molecular dynamics studies

The Cobalamin-Independent Methionine Synthase Enzyme Captured in a Substrate-Induced Closed Conformation

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