Human cystathionine ␤-synthase is a pyridoxal 5-phosphate enzyme containing a heme binding domain and an S-adenosyl-L-methionine regulatory site. We have investigated by single crystal microspectrophotometry the functional properties of a mutant lacking the S-adenosylmethionine binding domain. Polarized absorption spectra indicate that oxidized and reduced hemes are reversibly formed. Exposure of the reduced form of enzyme crystals to carbon monoxide led to the complete release of the heme moiety. This process, which takes place reversibly and without apparent crystal damage, facilitates the preparation of a heme-free human enzyme. The heme-free enzyme crystals exhibited polarized absorption spectra typical of a pyridoxal 5-phosphate-dependent protein. The exposure of these crystals to increasing concentrations of the natural substrate L-serine readily led to the formation of the key catalytic intermediate ␣-aminoacrylate. The dissociation constant of L-serine was found to be 6 mM, close to that determined in solution. The amount of the ␣-aminoacrylate Schiff base formed in the presence of L-serine was pH independent between 6 and 9. However, the rate of the disappearance of the ␣-aminoacrylate, likely forming pyruvate and ammonia, was found to increase at pH values higher than 8. Finally, in the presence of homocysteine the ␣-aminoacrylate-enzyme absorption band readily disappears with the concomitant formation of the absorption band of the internal aldimine, indicating that cystathionine ␤-synthase crystals catalyze both ␤-elimination and ␤-replacement reactions. Taken together, these findings demonstrate that the heme moiety is not directly involved in the condensation reaction catalyzed by cystathionine ␤-synthase.High plasmatic levels of homocysteine have recently been associated with an increased risk of cardiovascular disease (1). Homocysteine is formed from S-adenosylhomocysteine and is either removed by cystathionine ␤-synthase (EC 4.2.1.22, CBS) 1 in the trans-sulfuration pathway or remethylated to methionine in the methionine cycle. Deficiency of CBS is the major cause of inherited homocystinuria. CBS is a pyridoxal 5Ј-phosphate (PLP)-dependent enzyme catalyzing the synthesis of cystathionine from homocysteine and L-serine. The reaction proceeds via a ␤-replacement mechanism, similar to that of tryptophan synthase and O-acetylserine sulfhydrylase (2). These enzymes belong to the ␤-family and fold II type within the PLP-dependent enzymes classification (3, 4). Other members of the ␤-family are serine and threonine dehydratases. The human CBS is a 63-kDa homotetramer containing one PLP and one heme per subunit (5). Whereas the functional role of PLP is known, the role of the heme is less clear. It was demonstrated that the heme redox state affects the affinity of the enzyme for the substrates (6). Moreover, the catalytic activity of CBS is controlled by S-adenosylmethionine, which specifically binds to a C-terminal site. The trypsinolysis of a 18 kDa C-terminal fragment leads to a dimeric form, 2-fold m...

The cystine lyase (C-DES) of Synechocystis is a pyridoxal-5-phosphate-dependent enzyme distantly related to the family of NifS-like proteins. The crystal structure of an N-terminal modified variant has recently been determined. Herein, the reactivity of this enzyme variant was investigated spectroscopically in solution and in the crystalline state to follow the course of the reaction and to determine the catalytic mechanism on a molecular level. Using the stopped-flow technique, the reaction with the preferred substrate cystine was found to follow biphasic kinetics leading to the formation of absorbing species at 338 and 470 nm, attributed to the external aldimine and the ␣-aminoacrylate; the reaction with cysteine also exhibited biphasic behavior but only the external aldimine accumulated. The same reaction intermediates were formed in crystals as seen by polarized absorption microspectrophotometry, thus indicating that C-DES is catalytically competent in the crystalline state. The three-dimensional structure of the catalytically inactive mutant C-DES K223A in the presence of cystine showed the formation of an external aldimine species, in which two alternate conformations of the substrate were observed. The combined results allow a catalytic mechanism to be proposed involving interactions between cystine and the active site residues Arg-360, Arg-369, and Trp-251*; these residues reorient during the ␤-elimination reaction, leading to the formation of a hydrophobic pocket that stabilizes the enolimine tautomer of the aminoacrylate and the cysteine persulfide product.

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Sulfur mobilization represents one of the key steps in ubiquitous Fe-S clusters assembly and is performed by a recently characterized set of proteins encompassing cysteine desulfurases, assembly factors, and shuttle proteins. Despite the evolutionary conservation of these proteins, some degree of variability among organisms was observed, which might reflect functional specialization. L-Cyst(e)ine lyase (C-DES), a pyridoxal 5-phosphatedependent enzyme identified in the cyanobacterium Synechocystis, was reported to use preferentially cystine over cysteine with production of cysteine persulfide, pyruvate, and ammonia. In this study, we demonstrate that C-DES sequences are present in all cyanobacterial genomes and constitute a new family of sulfur-mobilizing enzymes, distinct from cysteine desulfurases. The functional properties of C-DES from Synechocystis sp. PCC 6714 were investigated under pre-steady-state and steady-state conditions. Single wavelength and rapid scanning stopped-flow kinetic data indicate that the internal aldimine reacts with cystine forming an external aldimine that rapidly decays to a transient quinonoid species and stable tautomers of the ␣-aminoacrylate Schiff base. In the presence of cysteine, the transient formation of a dipolar species precedes the selective and stable accumulation of the enolimine tautomer of the external aldimine, with no formation of the ␣-aminoacrylate Schiff base under reducing conditions. Effective sulfur mobilization from cystine might represent a mechanism that allows adaptation of cyanobacteria to different environmental conditions and to light-dark cycles.

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