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Heme interaction of the intrinsically disordered N-terminal peptide segment of human cystathionine-?-synthase.

ABSTRACT: Cystathionine-?-synthase (CBS) belongs to a large family of pyridoxal 5'-phosphate (PLP)-dependent enzymes, responsible for the sulfur metabolism. The heme-dependent protein CBS is part of regulatory pathways also involving the gasotransmitter hydrogen sulfide. Malfunction of CBS can lead to pathologic conditions like cancer, cardiovascular and neurodegenerative disorders. Truncation of residues 1-40, absent in X-ray structures of CBS, reduces but does not abolish the activity of the enzyme. Here we report the NMR resonance assignment and heme interaction studies for the N-terminal peptide stretch of CBS. We present NMR-spectral evidence that residues 1-40 constitute an intrinsically disordered region in CBS and interact with heme via a cysteine-proline based motif.

SUBMITTER: Kumar A

PROVIDER: S-EPMC5802807 | biostudies-literature | 2018 Feb

REPOSITORIES: biostudies-literature

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Heme interaction of the intrinsically disordered N-terminal peptide segment of human cystathionine-β-synthase.

Kumar Amit A Wißbrock Amelie A Goradia Nishit N Bellstedt Peter P Ramachandran Ramadurai R Imhof Diana D Ohlenschläger Oliver O

Scientific reports 20180206 1

Cystathionine-β-synthase (CBS) belongs to a large family of pyridoxal 5'-phosphate (PLP)-dependent enzymes, responsible for the sulfur metabolism. The heme-dependent protein CBS is part of regulatory pathways also involving the gasotransmitter hydrogen sulfide. Malfunction of CBS can lead to pathologic conditions like cancer, cardiovascular and neurodegenerative disorders. Truncation of residues 1-40, absent in X-ray structures of CBS, reduces but does not abolish the activity of the enzyme. Her ...[more]

PMID: 29410458

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Project description:Cystathionine beta-synthase (CBS) plays a central role in homocysteine metabolism, and malfunction of the enzyme leads to homocystinuria, a devastating metabolic disease. CBS contains a pyridoxal 5'-phosphate (PLP) cofactor which catalyzes the synthesis of cystathionine from homocysteine and serine. Mammalian forms of the enzyme also contain a heme group, which is not involved in catalysis. It may, however, play a regulatory role, since the enzyme is inhibited when CO or NO are bound to the heme. We have investigated the mechanism of this inhibition using fluorescence and resonance Raman spectroscopies. CO binding is found to induce a tautomeric shift of the PLP from the ketoenamine to the enolimine form. The ketoenamine is key to PLP reactivity because its imine C horizontal lineN bond is protonated, facilitating attack by the nucleophilic substrate, serine. The same tautomer shift is also induced by heat inactivation of Fe(II)CBS, or by an Arg266Met replacement in Fe(II)CBS, which likewise inactivates the enzyme; in both cases the endogenous Cys52 ligand to the heme is replaced by another, unidentified ligand. CO binding also displaces Cys52 from the heme. We propose that the tautomer shift results from loss of a stabilizing H-bond from Asn149 to the PLP ring O3' atom, which is negatively charged in the ketoenamine tautomer. This loss would be induced by displacement of the PLP as a result of breaking the salt bridge between Cys52 and Arg266, which resides on a short helix that is also anchored to the PLP via H-bonds to its phosphate group. The salt bridge would be broken when Cys52 is displaced from the heme. Cys52 protonation is inferred to be the rate-limiting step in breaking the salt bridge, since the rate of the tautomer shift, following CO binding, increases with decreasing pH. In addition, elevation of the concentration of phosphate buffer was found to diminish the rate and extent of the tautomer shift, suggesting a ketoenamine-stabilizing phosphate binding site, possibly at the protonated imine bond of the PLP. Implications of these findings for CBS regulation are discussed.

Dataset Information

Heme interaction of the intrinsically disordered N-terminal peptide segment of human cystathionine-?-synthase.

Publications

Heme interaction of the intrinsically disordered N-terminal peptide segment of human cystathionine-β-synthase.

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