FEBS 23611 FEBS Letters 472 (2000) 307^311 View metadata, citation and similar papers at core.ac.uk brought to you by CORE Mutagenic analysis of Thr-232 in rhodanese from Azotobacterprovided vinelandii by Elsevier - Publisher Connector highlighted the di¡erences of this prokaryotic enzyme from the known sulfurtransferases Silvia Pagania;*, Fabio Forlania, Aristodemo Carpena, Domenico Bordob, Rita Colnaghia aDipartimento di Scienze Molecolari Agroalimentari, Universita© di Milano, Via Celoria n. 2, 20133 Milan, Italy bAdvanced Biotechnology Center, IST, University of Genova, Genoa, Italy Received 26 January 2000; received in revised form 4 April 2000 Edited by Takashi Gojobori have shown that in bovine rhodanese the catalytic cysteine is Abstract Azotobacter vinelandii RhdA uses thiosulfate as the only sulfur donor in vitro, and this apparent selectivity seems to surrounded by polar and apolar residues which are deemed be a unique property among the characterized sulfurtransferases. important for substrate speci¢city [5,6]. The residues in the To investigate the basis of substrate recognition in RhdA, we active site pocket of the bovine enzyme [6] are fully conserved replaced Thr-232 with either Ala or Lys. Thr-232 was the target in all vertebrate rhodaneses [12^15], all showing a high degree of this study since the corresponding Lys-249 in bovine rhodanese of similarity to the bovine enzyme. In RhdA, on the other has been identified as necessary for catalytic sulfur transfer, and hand, the only conserved residue is the catalytic cysteine replacement of Lys-249 with Ala fully inactivates bovine (the only cysteine in this protein), which is surrounded by rhodanese. Both T232K and T232A mutants of RhdA showed residues that are entirely di¡erent from those found in the significant increase in thiosulfate-cyanide sulfurtransferase vertebrate enzymes [3,16]. In bovine rhodanese, the cationic activity, and no detectable activity in the presence of 3- residues Arg-186 and Lys-249 have been identi¢ed as catalytic mercaptopyruvate as the sulfur donor substrate. Fluorescence measurements showed that wild-type and mutant RhdAs were requirements for the sulfur transfer function [17]. The critical overexpressed in the persulfurated form, thus conferring to this role of Lys-249 in determining sulfur donor selectivity (thio- enzyme the potential of a persulfide sulfur donor compound. sulfate, for the rhodanese reaction) has been assessed by site- RhdA contains a unique sequence stretch around the catalytic directed mutagenesis experiments on bovine rhodanese, rat cysteine, and the data here presented suggest a possible divergent liver rhodanese and 3-MST [17,10,11]. In A. vinelandii physiological function of A. vinelandii sulfurtransferase. RhdA, the corresponding residues are glutamic acid (Glu- z 2000 Federation of European Biochemical Societies. 173) and threonine (Thr-232), and thiosulfate is the only sul- fane sulfur donor used for catalysis in vitro [3]. This apparent Key words: Sulfurtransferase; Site-directed mutagenesis; selectivity seems to be a unique property among characterized Sulfur donor substrate; Azotobacter vinelandii rhodanese sulfurtransferases, since rat 3-MST and rhodanese show both sulfurtransferase activities [10,11]. To investigate the basis of substrate recognition in A. vine- 1. Introduction landii RhdA, and to determine the role of Thr-232 in catalysis and substrate(s) binding, a study was carried out on selec- Azotobacter vinelandii rhodanese (RhdA) is the only pro- tively engineered RhdAs. The amino acid substitutions were karyotic sulfurtransferase structurally and functionally char- designed taking into account that: (i) cationic side chains are acterized [1^3]. RhdA, the product of A. vinelandii rhdA gene, crucial for thiosulfate binding and not essential for 3-mercap- which was cloned and overexpressed in Escherichia coli [3], topyruvate binding [10,11,17]; (ii) the replacement of Lys-249 catalyzes in vitro the sulfur transfer either to cyanide or to with a hydrophobic residue (Ala) knocks out bovine rhoda- the dithiol dihydrolipoate in the presence of thiosulfate as nese ability to transfer sulfane sulfur from thiosulfate to cya- donor substrate. To date, the best studied rhodanese is that nide [17]; (iii) the replacement of Ser-249 with Lys in rat liver from bovine liver which represents the reference enzyme 3-MST does not alter the binding of 3-mercaptopyruvate [11]. among sulfurtransferases [4^7]. The active site of bovine Thr-232 was replaced with Lys and Ala. The biochemical rhodanese is characterized by the presence of a cysteine resi- characterization of the mutant RhdAs highlighted di¡erences due (Cys-247), which promotes formation of a persul¢de in- between A. vinelandii sulfurtransferase and vertebrate rhodan- termediate during the catalytic cycle [5,6,8,9]. The catalytic eses, thus suggesting possible divergent functions. cysteine is considered a structural feature common to all sul- furtransferases, including 3-mercaptopyruvate sulfurtransfer- 2. Materials and methods ase (3-MST), claimed to be evolutionarily related to mito- chondrial rhodanese [10,11]. Crystallographic investigations 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- *Corresponding author. Fax: (39)-2-70633062. inger Mannheim, New England Biolabs and Pharmacia. Oligonucleo- E-mail: [email protected] 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 FEBS 23611 20-4-00 308 S. Pagani et al./FEBS Letters 472 (2000) 307^311 Sequence DNA Sequencing System' from Promega was used for non- 5 and 3 nm, respectively. Emission spectra were recorded from 300 to radioactive DNA sequence analysis, which was performed according 400 nm 1 min after reagent addition, and the samples were continu- to the supplier's instructions. ously stirred. In the titration experiments changes in £uorescence in- tensity at 336 nm (Fobs) are given as vF (%): 2.2. Site-directed mutagenesis and overexpression of His-tagged F 3F proteins vF % obs oU100 The P£MI-KpnI fragment containing rhdA from plasmid pRC9189 F o [3], was cloned into overexpression vector pQE32 (Qiagen) after trim- ming the P£MI site with T4 DNA polymerase and ¢lling in the Bam- where Fo is the original £uorescence intensity of the studied RhdAs. HI site on the vector. In the resulting plasmid (named pQER1), there are eight additional codons upstream of the ATG starting codon of 3. Results and discussion rhdA: the ATC codon for isoleucine, the GGG codon for glycine and six histidine codons. Site-directed mutagenesis of rhdA was performed Overexpression of A. vinelandii rhdA gene led to a signi¢- by subcloning the 1 kb EcoRI/HindIII fragment from pQER1 into pTZ18 (resulting in the plasmid pMC1). Single stranded pMC1 DNA cant increase in thiosulfate-cyanide sulfurtransferase activity was prepared by using the bacteriophage M13K07 (Pharmacia) as in cell-free extracts, and a further increase in rhodanese activ- superinfecting helper phage and used as template for elongation of ity was found when the gene was mutated (Table 1). No sig- mutagenic primers. Replacement of Thr-232 with Lys (T232K) and ni¢cant changes in 3-MST activity were observed following Ala (T232A) was achieved by using two mutagenic primers: CG- TCACCCACTGCCAGaaACATCACCGCTCCGG and CGTCACC- overexpression of either wild-type or mutant RhdAs (Table CACTGCCAGgcACATCACCGCTCCGG, respectively, where lower 1). The residual sulfurtransferase activities in cells not over- case letters indicate the mutated bases. expressing RhdA can be ascribed to the presence of other The `Gene Editor in vitro Site-Directed Mutagenesis System' sulfurtransferases in the E. coli host strain [23,24]. The over- (Promega) was used for rapid screening of the mutated recombinant expressed RhdA proteins were puri¢ed in only one fast chro- plasmids. The accuracy of mutagenesis and cloning were checked by sequencing the mutated recombinant plasmids (pMC1A and pMC1B). matography step, taking advantage of the inserted histidine For overexpression, the 1 kb EcoRI/HindIII fragments from pMC1A tag. None of the puri¢ed RhdAs showed detectable activity in and pMC1B were subcloned into pQE32, giving rise to plasmids the presence of 3-mercaptopyruvate as the sulfur donor (Table pQER3 and pQER4, respectively. The recombinant plasmids contain- 2). The thiosulfate-cyanide sulfurtransferase activity of His- ing the wild-type or mutant RhdAs were transformed into E. coli tagged wild-type RhdA was indistinguishable from that of M15, and protein overexpression was rapidly induced by addition of 1 mM isopropyl-thio-L-D-galactoside to a mid-log culture the wild-type enzyme not carrying the histidine tag, and pu- (OD600 = 0.600). ri¢ed by conventional gel-exclusion chromatography. The ability to transfer sulfane sulfur from thiosulfate to cyanide 2.3. Puri¢cation of overexpressed His-tagged proteins increased about three-fold in both mutant RhdAs, compared Cell-free extracts were prepared from 500 ml of culture. After 4 h of induction cells were harvested by centrifugation, and resuspended in to that of the wild-type enzyme.