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Characterization of biochemical penalties associated with pathogenic mutations in human methionine synthase reductase
Abstract
Human methionine synthase reductase (MSR) catalyzes an NADPH-dependent one-electron reduction of inactive cobalamin-bound to methionine synthase. This electron transfer reaction from MSR to MS is needed to maintain methionine synthase in an active form so as to generate tetrahydrofolate requested by other folate-dependent enzymes and to prevent the accumulation of the toxic metabolite, homocysteine. Derangements in methionine metabolism are primarily caused by nutritional imbalances of vitamins B12, B6 and folate and by genetic defects. Mutations in human MSR have been identified in homocystinuric patients belonging to the cblE complementation class of cobalamin/folate disorders with isolated deficiency in methionine synthase. Based on the analogy with the bacterial methionine synthase activation system, the reactivation of human methionine synthase involves the interaction of the FMN-binding domain of MSR with methionine synthase. To obtain mechanistic and structural insights into this interaction, we have investigated the biochemical penalties associated with two pathogenic mutations housed in the FMN-binding domain of MSR. For this, we have employed a combination of spectroscopic methods to assess the effects of the Val56>Met and Ala129>Thr missense mutations on the spectral, kinetic and structural properties of MSR. The existence of two common variations in MSR, Ile22Met and Ser175>Leu, prompted us to also examine the phenotype of the Val56>Met mutation in all four polymorphic backgrounds. Our results revealed that the mutation decreased the affinity of MSR for methionine synthase by a 10-fold only when expressed in the Met22 background whereas the variation at position 175 had no influence. Sequence analysis of the MSR alleles of the patients harboring the Val56>Met mutation validated our in vitro results by revealing that the patients had the Met22 polymorphism. Importantly, we have demonstrated for the first time a profound effect of an individual’s polymorphic background on the phenotypic expression of a disease-causing mutation. We have also evaluated the molecular basis for methionine synthase deficiency caused by the Ala129>Thr mutation in MSR. The mutant protein lacks a full FMN complement and the FMN-depleted Ala129>Thr mutant retains only 5% of wild-type enzyme activity. Hydrogen/deuterium exchange mass spectrometric studies indicated that the effects of Ala129>Thr are confined to the FMN-binding domain, consistent with decreased FMN binding. Partial recovery of the mutant activity was observed with exogenous FMN and suggests the potential utility of riboflavin therapy for patients harboring this mutation.
Subject Area
Biochemistry
Recommended Citation
Gherasim, Carmen, "Characterization of biochemical penalties associated with pathogenic mutations in human methionine synthase reductase" (2008). ETD collection for University of Nebraska-Lincoln. AAI3326858.
https://digitalcommons.unl.edu/dissertations/AAI3326858