Mutagenic analysis of Thr-232 in rhodanese from Azotobacter vinelandii highlighted the di¡erences of this prokaryotic enzyme from the known sulfurtransferases Silvia Pagani a ; *, Fabio Forlani a , Aristodemo Carpen a , Domenico Bordo b , Rita Colnaghi a a Dipartimento di Scienze Molecolari Agroalimentari, Universita © di Milano, Via Celoria n. 2, 20133 Milan, Italy b Advanced Biotechnology Center, IST, University of Genova, Genoa, Italy Received 26 January 2000; received in revised form 4 April 2000 Edited by Takashi Gojobori Abstract Azotobacter vinelandii RhdA uses thiosulfate as the only sulfur donor in vitro, and this apparent selectivity seems to be a unique property among the characterized sulfurtransferases. To investigate the basis of substrate recognition in RhdA, we replaced Thr-232 with either Ala or Lys. Thr-232 was the target of this study since the corresponding Lys-249 in bovine rhodanese has been identified as necessary for catalytic sulfur transfer, and replacement of Lys-249 with Ala fully inactivates bovine rhodanese. Both T232K and T232A mutants of RhdA showed significant increase in thiosulfate-cyanide sulfurtransferase activity, and no detectable activity in the presence of 3- mercaptopyruvate as the sulfur donor substrate. Fluorescence measurements showed that wild-type and mutant RhdAs were overexpressed in the persulfurated form, thus conferring to this enzyme the potential of a persulfide sulfur donor compound. RhdA contains a unique sequence stretch around the catalytic cysteine, and the data here presented suggest a possible divergent physiological function of A. vinelandii sulfurtransferase. z 2000 Federation of European Biochemical Societies. Key words: Sulfurtransferase ; Site-directed mutagenesis ; Sulfur donor substrate; Azotobacter vinelandii rhodanese 1. Introduction Azotobacter vinelandii rhodanese (RhdA) is the only pro- karyotic sulfurtransferase structurally and functionally char- acterized [1^3]. RhdA, the product of A. vinelandii rhdA gene, which was cloned and overexpressed in Escherichia coli [3], catalyzes in vitro the sulfur transfer either to cyanide or to the dithiol dihydrolipoate in the presence of thiosulfate as donor substrate. To date, the best studied rhodanese is that from bovine liver which represents the reference enzyme among sulfurtransferases [4^7]. The active site of bovine rhodanese is characterized by the presence of a cysteine resi- due (Cys-247), which promotes formation of a persul¢de in- termediate during the catalytic cycle [5,6,8,9]. The catalytic cysteine is considered a structural feature common to all sul- furtransferases, including 3-mercaptopyruvate sulfurtransfer- ase (3-MST), claimed to be evolutionarily related to mito- chondrial rhodanese [10,11]. Crystallographic investigations have shown that in bovine rhodanese the catalytic cysteine is surrounded by polar and apolar residues which are deemed important for substrate speci¢city [5,6]. The residues in the active site pocket of the bovine enzyme [6] are fully conserved in all vertebrate rhodaneses [12^15], all showing a high degree of similarity to the bovine enzyme. In RhdA, on the other hand, the only conserved residue is the catalytic cysteine (the only cysteine in this protein), which is surrounded by residues that are entirely di¡erent from those found in the vertebrate enzymes [3,16]. In bovine rhodanese, the cationic residues Arg-186 and Lys-249 have been identi¢ed as catalytic requirements for the sulfur transfer function [17]. The critical role of Lys-249 in determining sulfur donor selectivity (thio- sulfate, for the rhodanese reaction) has been assessed by site- directed mutagenesis experiments on bovine rhodanese, rat liver rhodanese and 3-MST [17,10,11]. In A. vinelandii RhdA, the corresponding residues are glutamic acid (Glu- 173) and threonine (Thr-232), and thiosulfate is the only sul- fane sulfur donor used for catalysis in vitro [3]. This apparent selectivity seems to be a unique property among characterized sulfurtransferases, since rat 3-MST and rhodanese show both sulfurtransferase activities [10,11]. To investigate the basis of substrate recognition in A. vine- landii RhdA, and to determine the role of Thr-232 in catalysis and substrate(s) binding, a study was carried out on selec- tively engineered RhdAs. The amino acid substitutions were designed taking into account that: (i) cationic side chains are crucial for thiosulfate binding and not essential for 3-mercap- topyruvate binding [10,11,17] ; (ii) the replacement of Lys-249 with a hydrophobic residue (Ala) knocks out bovine rhoda- nese ability to transfer sulfane sulfur from thiosulfate to cya- nide [17]; (iii) the replacement of Ser-249 with Lys in rat liver 3-MST does not alter the binding of 3-mercaptopyruvate [11]. Thr-232 was replaced with Lys and Ala. The biochemical characterization of the mutant RhdAs highlighted di¡erences between A. vinelandii sulfurtransferase and vertebrate rhodan- eses, thus suggesting possible divergent functions. 2. Materials and methods 2.1. DNA manipulation and sequencing E. coli 71-18 [18] and M15 (Qiagen) strains and their trans- formed derivatives were grown at 37³C in Luria^Bertani medium [19]. Antibiotics for the selection of E. coli transformants were used at the following concentration: 100 Wg/ml (ampicillin); 30 Wg/ml (ka- namycin). All enzymes used for DNA manipulation were from Boehr- inger Mannheim, New England Biolabs and Pharmacia. Oligonucleo- tide primers were synthesized by Boehringer Mannheim. The `Silver 0014-5793 / 00 / $20.00 ß 2000 Federation of European Biochemical Societies. All rights reserved. PII:S0014-5793(00)01477-0 *Corresponding author. Fax: (39)-2-70633062. E-mail: silvia.pagani@unimi.it FEBS 23611 FEBS Letters 472 (2000) 307^311