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Probing the chemical mechanism of saccharopine reductase from Saccharomyces cerevisiae using site-directed mutagenesis.

ABSTRACT: Saccharopine reductase catalyzes the reductive amination of l-?-aminoadipate-?-semialdehyde with l-glutamate to give saccharopine. Two mechanisms have been proposed for the reductase, one that makes use of enzyme side chains as acid-base catalytic groups, and a second, in which the reaction is catalyzed by enzyme-bound reactants. Site-directed mutagenesis was used to change acid-base candidates in the active site of the reductase to eliminate their ionizable side chain. Thus, the D126A, C154S and Y99F and several double mutant enzymes were prepared. Kinetic parameters in the direction of glutamate formation exhibited modest decreases, inconsistent with the loss of an acid-base catalyst. The pH-rate profiles obtained with all mutant enzymes decrease at low and high pH, suggesting acid and base catalytic groups are still present in all enzymes. Solvent kinetic deuterium isotope effects are all larger than those observed for wild type enzyme, and approximately equal to one another, suggesting the slow step is the same as that of wild type enzyme, a conformational change to open the site and release products (in the direction of saccharopine formation). Overall, the acid-base chemistry is likely catalyzed by bound reactants, with the exception of deprotonation of the ?-amine of glutamate, which likely requires an enzyme residue.

SUBMITTER: Vashishtha AK

PROVIDER: S-EPMC4596410 | biostudies-literature | 2015 Oct

REPOSITORIES: biostudies-literature

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Probing the chemical mechanism of saccharopine reductase from Saccharomyces cerevisiae using site-directed mutagenesis.

Vashishtha Ashwani K AK West Ann H AH Cook Paul F PF

Archives of biochemistry and biophysics 20150903

Saccharopine reductase catalyzes the reductive amination of l-α-aminoadipate-δ-semialdehyde with l-glutamate to give saccharopine. Two mechanisms have been proposed for the reductase, one that makes use of enzyme side chains as acid-base catalytic groups, and a second, in which the reaction is catalyzed by enzyme-bound reactants. Site-directed mutagenesis was used to change acid-base candidates in the active site of the reductase to eliminate their ionizable side chain. Thus, the D126A, C154S an ...[more]

PMID: 26342457

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Project description:BACKGROUND:Lycopene is a terpenoid pigment that has diverse applications in the food and medicine industries. A prospective approach for lycopene production is by metabolic engineering in microbial hosts, such as Escherichia coli. Isopentenyl diphosphate isomerase (IDI, E.C. 5.3.3.2) is one of the rate-limiting enzymes in the lycopene biosynthetic pathway and one major target during metabolic engineering. The properties of IDIs differ depending on the sources, but under physiological conditions, IDIs are limited by low enzyme activity, short half-life and weak substrate affinity. Therefore, it is important to prepare an excellent IDI by protein engineering. RESULTS:Directed evolution strategy (error-prone PCR) was utilized to optimize the activity of Saccharomyces cerevisiae IDI. Using three rounds of error-prone PCR; screening the development of a lycopene-dependent color reaction; and combinatorial site-specific saturation mutagenesis, three activity-enhancing mutations were identified: L141H, Y195F, and W256C. L141H, located near the active pocket inside the tertiary structure of IDI, formed a hydrogen bond with nearby ?-phosphates of isopentenylpyrophosphate (IPP). Phe-195 and Cys-256 were nonpolar amino acids and located near the hydrophobic group of IPP, enlarging the hydrophobic scope, and the active pocket indirectly. Purified IDI was characterized and the result showed that the Km of mutant IDI decreased by 10% compared with Km of the parent IDI, and Kcat was 28% fold improved compared to that of the original IDI. Results of a fermentation experiment revealed that mutant IDI had a 1.8-fold increased lycopene production and a 2.1-fold increased yield capacity compared to wild-type IDI. CONCLUSION:We prepared an engineered variant of IDI with improved catalytic activity by combining random and site directed mutagenesis. The best mutants produced by this approach enhanced catalytic activity while also displaying improved stability in pH, enhanced thermostability and longer half-life. Importantly, the mutant IDI could play an important role in fed-batch fermentation, being an effective and attractive biocatalyst for the production of biochemicals.

Dataset Information

Probing the chemical mechanism of saccharopine reductase from Saccharomyces cerevisiae using site-directed mutagenesis.

Publications

Probing the chemical mechanism of saccharopine reductase from Saccharomyces cerevisiae using site-directed mutagenesis.

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