title: Insights into the regulatory domain of cystathionine Beta-synthase: characterization of six variant proteins.
abstract: Cystathionine beta-synthase (CBS) catalyzes the formation of cystathionine from homocysteine and serine. CBS is allosterically activated by S-adenosylmethionine (SAM), which binds to its C-terminal regulatory domain. Mutations in this domain lead to variants with high residual activity but lacking SAM activation. We characterized six C-terminal CBS variants (p.P427L, p.D444N, p.V449G, p.S500L, p.K523Sfs*18, and p.L540Q). To understand the effect of C-terminal mutations on the functional/structural properties of CBS, we performed dynamic light scattering, differential scanning fluorimetry, limited proteolysis, enzymatic characterization, and determination of SAM-binding affinity. Kinetic data confirm that the enzymatic function of these variants is not impaired. Although lacking SAM activation, the p.P427L and p.S500L were able to bind SAM at a lower extent than the wild type (WT), confirming that SAM binding and activation can be two independent events. At the structural level, the C-terminal variants presented various effects, either showing catalytic core instability and increased susceptibility toward aggregation or presenting with similar or higher stability than the WT. Our study highlights as the common feature to the C-terminal variants an impaired binding of SAM and no increase in enzymatic activity with physiological concentrations of the activator, suggesting the loss of regulation by SAM as a potential pathogenic mechanism.
authors.affiliation: Metabolism and Genetics Group, Research Institute for Medicines, Faculty of Pharmacy, University of Lisbon, Lisbon, Portugal; Metabolic Unit, Department of Clinical Chemistry, VU University Medical Centre, Amsterdam, The Netherlands.
authors.forename: Marisa I S
authors.initials: MI
authors.lastname: Mendes
authors.forename: Ana Sofia
authors.initials: AS
authors.lastname: Santos
authors.forename: Desirée E C
authors.initials: DE
authors.lastname: Smith
authors.forename: Paulo Roque
authors.initials: PR
authors.lastname: Lino
authors.forename: Henrique G
authors.initials: HG
authors.lastname: Colaço
authors.forename: Isabel Tavares
authors.initials: IT
authors.lastname: de Almeida
authors.forename: João B
authors.initials: JB
authors.lastname: Vicente
authors.forename: Gajja S
authors.initials: GS
authors.lastname: Salomons
authors.forename: Isabel
authors.initials: I
authors.lastname: Rivera
authors.forename: Henk J
authors.initials: HJ
authors.lastname: Blom
authors.forename: Paula
authors.initials: P
authors.lastname: Leandro
mesh_terms: D000495:Allosteric Site; D000595:Amino Acid Sequence; D020134:Catalytic Domain; D003541:Cystathionine beta-Synthase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D006712:Homocystinuria / Q000201:enzymology* / Q000235:genetics; D006801:Humans; D007700:Kinetics; D009154:Mutation; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012436:S-Adenosylmethionine / Q000378:metabolism
chemical_list: D011994:Recombinant Proteins; D012436:S-Adenosylmethionine; D003541:Cystathionine beta-Synthase
keywords: CBS; S-adenosylmethionine; cystathionine beta-synthase; differential scanning fluorimetry; dynamic light scattering; homocystinuria