The PatB protein of Bacillus subtilis is a C-S-lyase S. Auger 1 , M.P. Gomez, A. Danchin, I. Martin-Verstraete * Unité de Génétique des Génomes Bactériens, Institut Pasteur, URA CNRS 2171, 28, rue du Docteur Roux, 75724 Paris cedex 15, France Received 17 June 2004; accepted 13 September 2004 Available online 01 October 2004 Abstract The PatB protein of Bacillus subtilis had both cystathionine b-lyase and cysteine desulfhydrase activities in vitro. The apparent K m value of the PatB protein for cystathionine was threefold higher than that of the MetC protein, the previously characterized cystathionine b-lyase of B. subtilis. In the presence of cystathionine as sole sulfur source, the patB gene present on a multicopy plasmid restored the growth of a metC mutant. In addition, the patB metC double mutant was unable to grow in the presence of sulfate or cystine while the patB or metC single mutants grew similarly to the wild-type strains in the presence of the same sulfur sources. In a metC mutant, the PatB protein can replace the MetC enzyme in the methionine biosynthetic pathway. © 2004 Elsevier SAS. All rights reserved. Keywords: Sulfur metabolism; Cystathionine b-lyase; Cysteine desulfhydrase; Methionine biosynthesis 1. Introduction A number of enzymes involved in the metabolism of cys- teine, homocysteine and methionine are paralogous [1]. Cys- tathionine c-synthase, cystathionine b-lyase, cystathionine c-lyase, O-acetylhomoserine sulfhydrylase and methionine c-lyase constitute a protein family [2,3]. These enzymes of about 400 amino acids use the same cofactor, pyridoxal-5′- phosphate (PLP), which is attached to a lysine residue [4]. In Bacillus subtilis, the transsulfuration pathway allows the con- version of homocysteine into cysteine via the intermediary formation of cystathionine (Fig. 1). This requires the sequen- tial action of cystathionine c-synthase (EC 4.2.99.9), the metI gene product, and cystathionine b-lyase (EC 4.4.1.8), the metC gene product [5]. Cystathionine b-lyases catalyse mainly the cleavage of L-cystathionine to homocysteine, pyruvate and ammonia via an a/b-elimination reaction. The enzyme splits the aC-N (deaminating) and bC-S bonds [6]. Homocysteine is subsequently methylated to methionine by a unique methionine synthase, MetE [7]. Mutants disrupted in the metI or the metE genes are methionine auxotrophs. A mutant inac- tivated in the metC gene grows poorly on cystathionine as sole sulfur source but is still able to grow in the presence of sulfate or cysteine [5]. These results suggest that a second homocysteine biosynthetic pathway exists in B. subtilis. As the MetI enzyme has both cystathionine c-synthase and O-acetylhomoserine sulfhydrylase activities in vitro, it was proposed that MetI could participate to the sulfhydrylation pathway, which bypasses the formation of cystathionine to form homocysteine [5]. A second class of enzymes involved in the degradation of cystathionine, cysteine and cystine has also been described [8–10]. These proteins have C-S-lyase activity. In this fam- ily, the PatC polypeptide from Lactobacillus delbrueckii is similar to the potential aminotransferase PatB protein from B. subtilis [8,11]. Moreover, a thermostable S-alkylcysteine a,b-lyase from a thermophile Bacillus species has been puri- fied [12]. Its N-terminal amino acids sequence shares simi- larities to the N-terminal part of PatB. The biological func- tion of the PatB polypeptide is still uncharacterized. In this work, we tested the involvement of PatB in the degradation of cystathionine and cysteine and its possible role in the B. subtilis methionine biosynthetic pathway. * Corresponding author. Tel.: +33-1-40-61-35-61; fax: +33-1-45-68-89-48. E-mail address: iverstra@pasteur.fr (I. Martin-Verstraete). 1 Present address. Laboratoire de Microbiologie et Génétique Molécu- laire, INRA-CNRS URA1925, 78850 Thiverval-Grignon, France. Biochimie 87 (2005) 231–238 www.elsevier.com/locate/biochi 0300-9084/$ - see front matter © 2004 Elsevier SAS. All rights reserved. doi:10.1016/j.biochi.2004.09.007