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Structural and spectroscopic characterization of human cystathionine β-synthase
Abstract
Human cystathionine β-synthase (CBS), a pyridoxal 5′ -phosphate (PLP)-dependent hemeprotein, catalyzes the condensation of homocysteine and seine to give cystathionine. The CBS catalyzed reaction is the first committed step in the catabolic removal of the toxic metabolite, homocysteine, via the transsulfuration pathway. The latter also represents a pathway for the conversion of homocysteine to cysteine and ultimately to glutathione (GSH). The reaction catalyzed by CBS is of clinical importance. Mutations in CBS result in high levels of homocysteine and is the most common cause of hereditary homocystinuria. High homocysteine levels are an independent risk factor for cardiovascular diseases, neural tube defects and Alzheimer's disease. Human CBS is a homotetramer of 63 kDa subunits and contains 1 mol of heme and 1 mol of PLP per subunit. While the dependence of CBS activity on PLP can be explained by the chemical similarity of the reaction it catalyses to β-replacement reactions catalyzed by other PLP enzymes, the role of the heme and its location relative to the active site was not known when this study was initiated. In this study, we have used a combination of spectroscopic methods to provide the first structural evidence for a regulatory role for the heme. The two cofactors, heme and PLP, were estimated to be grater than 10 Å apart indicating that a catalytic role for the heme is unlikely. However, changes in the heme oxidation state were found to be transmitted to PLP in the active site consistent with a regulatory role for this cofactor. We also purified and characterized a pathogenic CBS mutant, V168M, associated with a B6-responsive phenotype in patients and showed that the cofactor content of the enzyme is regulated by interactions between the C-terminal regulatory and the N-terminal catalytic domains. The defect introduced by the V168M substitution resulted in a 7-fold lower PLP and 2-fold lower heme content with a 13-fold decrease in enzyme activity and was completely alleviated by deletion of 143 amino acids from the C-terminal. Since some CBS mutations described in patients with high levels of homocysteine due to the CBS deficiency appear to result in normal enzyme activity, we hypothesized that CBS interacts with other proteins and that these interactions are disrupted in this subclass of pathogenic mutants. In order to address this question, we have used the yeast two-hybrid system and probed a human brain library with the CBS bait. These studies identified Ubc9 and PIAS1 which represents the E2 and E3 enzymes involved in the sumoylation pathway. CBS contains three consensus ΨKXE sequences that represent targets for sumoylation. All three sequences are surface exposed in the structure of the dimeric enzyme. In fact, K102 in one of the consensus sequences, LKCE, is mutated to a glutamine in a patient and raises the question as to whether sumoylation of the protein may be impaired. Although these studies are still in progress, our efforts to observe sumoylation of CBS is discussed.
Subject Area
Biophysics
Recommended Citation
Kabil, Omer, "Structural and spectroscopic characterization of human cystathionine β-synthase" (2003). ETD collection for University of Nebraska-Lincoln. AAI3092562.
https://digitalcommons.unl.edu/dissertations/AAI3092562