Cystathionine b-synthase (CBS) is a unique hemecontaining enzyme that catalyzes a pyridoxal 5¢-phosphate (PLP)-dependent condensation of serine and homocysteine to give cystathionine. De®ciency of CBS leads to homocystinuria, an inherited disease of sulfur metabolism characterized by increased levels of the toxic metabolite homocysteine. Here we present the X-ray crystal structure of a truncated form of the enzyme. CBS shares the same fold with O-acetylserine sulfhydrylase but it contains an additional N-terminal heme binding site. This heme binding motif together with a spatially adjacent oxidoreductase active site motif could explain the regulation of its enzyme activity by redox changes. Keywords: cystathionine b-synthase/cysteine biosynthesis/heme protein/pyridoxal 5¢-phosphate/X-ray crystal structure
Cystathionine beta-synthase (CBS), condensing homocysteine and serine, represents a key regulatory point in the biosynthesis of cysteine via the transsulfuration pathway. Inherited deficiency of CBS causes homocystinuria. CBS is activated by S-adenosyl-L-methionine (AdoMet) by inducing a conformational change involving a noncatalytic C-terminal region spanning residues 414-551. We report the purification of two patient-derived C-terminal mutant forms of CBS, S466L and I435T, that provide new insight into the mechanism of CBS regulation and indicate a regulatory function for the "CBS domain". Both of these point mutations confer catalytically active proteins. The I435T protein is AdoMet inducible but is 10-fold less responsive than wild-type (WT) CBS to physiologically relevant concentrations of this compound. The S466L form does not respond to AdoMet but is constitutively activated to a level intermediate between those of WT CBS in the presence and absence of AdoMet. Both mutant proteins are able to bind AdoMet, indicating that their impairment is related to their ability to assume the fully activated conformation that AdoMet induces in WT CBS. We found that I435T and WT CBS can be activated by partial thermal denaturation but that the AdoMet-stimulated WT, S466L, and a truncated form of CBS lacking the C-terminal region cannot be further activated by this treatment. Tryptophan and PLP fluorescence data for these different forms of CBS indicate that activation by AdoMet, limited proteolysis, and thermal denaturation share a common mechanism involving the displacement of an autoinhibitory domain located in the C-terminal region of the protein.
Antibody drug conjugates (ADCs) are monoclonal antibodies designed to deliver a cytotoxic drug selectively to antigen expressing cells. Several components of an ADC including the selection of the antibody, the linker, the cytotoxic drug payload and the site of attachment used to attach the drug to the antibody are critical to the activity and development of the ADC.The cytotoxic drugs or payloads used to make ADCs are typically conjugated to the antibody through cysteine or lysine residues. This results in ADCs that have a heterogeneous number of drugs per antibody. The number of drugs per antibody commonly referred to as the drug to antibody ratio (DAR), can vary between 0 and 8 drugs for a IgG1 antibody. Antibodies with 0 drugs are ineffective and compete with the ADC for binding to the antigen expressing cells. Antibodies with 8 drugs per antibody have reduced in vivo stability, which may contribute to non target related toxicities.In these studies we incorporated a non-natural amino acid, para acetyl phenylalanine, at two unique sites within an antibody against Her2/neu. We covalently attached a cytotoxic drug to these sites to form an ADC which contains two drugs per antibody.We report the results from the first direct preclinical comparison of a site specific non-natural amino acid anti-Her2 ADC and a cysteine conjugated anti-Her2 ADC. We report that the site specific non-natural amino acid anti-Her2 ADCs have superior in vitro serum stability and preclinical toxicology profile in rats as compared to the cysteine conjugated anti-Her2 ADCs. We also demonstrate that the site specific non-natural amino acid anti-Her2 ADCs maintain their in vitro potency and in vivo efficacy against Her2 expressing human tumor cell lines. Our data suggests that site specific non-natural amino acid ADCs may have a superior therapeutic window than cysteine conjugated ADCs.
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