Short communications. Nucleotide sequence of katg of Salmonella typhimurium LT2 and characterization of its product, hydroperoxidase I

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1 Mol Gen Genet (1990) 224: Springer-Verlag 1990 Short communications Nucleotide sequence of katg of Salmonella typhimurium LT2 and characterization of its product, hydroperoxidase I Peter C. Loewen t and George V. Stauffer 2 Department of Microbiology, University of Manitoba, Winnipeg, MB R3T 2N2, Canada 2 Department of Microbiology, University of Iowa, Iowa City, IA 52242, USA Received May 2, 1990 Summary. The nucleotide sequence of katg from Salmonella typhimurium was determined revealing an open reading frame of 2181 bp that could encode a 727 amino acid protein. The predicted sequence of the encoded hydroperoxidase I (HPI) was found to be 90% similar to HPI from Escherichia coli and was one amino acid longer. The physical and enzymatic properties of HPI from both Salmonella typhimurium and Escherichia eoli were found to be virtually identical despite the 10% divergence in sequence. Key words: katg sequence - catalase - peroxidase - HPI Escheriehia coli K12 produces two catalases or hydroperoxidases: the bifunctional catalase-peroxidase HPI and the monofunctional catalase HPII. Both enzymes are unusual when compared with the 'normal' catalase from eucaryotic and some bacterial sources, which is a tetramer of Da subunits and four protoheme IX groups (Deisseroth and Dounce 1970). HPI has been characterized as a tetramer of Da subunits and two protoheme IX groups (Claiborne and Fridovich 1979) while HPII has been characterized as a hexamer of Da subunits and six heme d-like groups (Loewen and Switala 1986; Chiu et al. 1989). The genes encoding the two enzymes, katg (HPI) and kate (HPII), have been cloned (Triggs-Raine and Loewen 1987; Mulvey et al. 1988) and sequenced, revealing a significant divergence from the eucaryotic sequence in the case of katg (Triggs-Raine et al. 1988). The E. coli metfgene is located immediately upstream from katg and cloning and sequencing of metf from Salmonella typhimurium (Stauffer and Stauffer 1988) has revealed an open reading frame (ORF) beginning 168 bp downstream from the end of the metf gene, which is approximately 80% similar to the E. coli katg sequence. Offprint requests to: P. Loewen Subclones of pgs139 (pstg1 and pstg2; Stauffer and Stauffer 1988) containing the S. typhimurium katg gene were generated from the 7.3 kb EcoRI fragment and the 3.1 kb DraI-EeoRI fragment respectively (Fig. 1). Using these three plasmids, a 2340 bp sequence including katg from S. typhimurium was determined, which is shown in Fig. 2. a The 2181 bp ORF beginning at the ATG codon at base + 1 and ending with a UAA codon at position is 3 bp longer than the ORF of E. coli katg and is the result of an extra CTC at position The nucleotides that differ in the E. coli sequence are indicated below the S. typhimurium sequence. When the coding region is compared with the corresponding region from E. coli, the degree of similari- 1 EMBL Accession number: X53001 E ED D D S E J II I. I [. [ pgs ED D D S E II I.I I. I pstg1 o i S 6 metf D S E [... I J pstg2 o -i, 3 katg Fig. 1. DNA fragments containing katg from Salmonella typhimurium in pgs139 (Stauffer and Stauffer 1988), pstg1 and pstg2. the 7.3 kb EcoRI segment and the 3.0 kb EcoRI-DraI segment from pgs139 were ligated into the M13 Bluescript vector (Stratagene Cloning Systems) to generate pstgi and pstg2. The locations of the sites recognized by restriction enzymes are indicated by E (EcoRI), D (DraI) and S (SalI). The numbers represent distances in kilobase pairs. The locations and directions of transcription of metf and katg are indicated by arrows D

2 148 o 8 - N u ',.. ''-'-...., N N N : N o : e e. _ ". - : e..- N o g _.... _ o :. o- "g N u g 2 - U'o o ; 1

3 149 ty is 81.9% while the similarity in the non-coding regions is only 68.3%. A potential -10 sequence indicated in Fig. 2 is identical to the equivalent sequence from E. coli and a potential -35 sequence differs in two of the six bases. In neither case is the -35 sequence similar to the consensus -35 sequence TTGACA (Harley and Reynolds 1987) providing a possible explanation of why OxyR protein is required for optimal expression. A potential Shine-Dalgarno sequence (GGAG) is indicated 8 bp upstream from the ATG start codon in a slightly different position from the corresponding E. coli sequence. The S. typhimurium katg sequence predicted a 727 amino acid protein with a molecular weight of 80042, very similar to the molecular weight of for the 726 amino acid E. coli protein. The predicted amino acid sequence is also shown in Fig. 2 along with the amino acids that differ between the E. coli and S. typhimurium proteins. The similarity between the E. coli and S. typhimurium HPI amino acid sequences was 90.1% including conservative replacements. The additional amino acid in the S. typhimurium enzyme was the result of the three additional bases (CTC) at position +216 which were not inserted in frame and caused the incorporation of a serine residue at position 73 and a glycine to alanine change in the following position. A comparison of codon usage in the two genes and of the predicted amino acid compositions of the two HPI proteins revealed few differences, the most noteworthy being the presence of a second cysteine replacing a serine at residue 700. Comparison of hydrophilicity plots of the two HPI proteins (Hopp and Woods 1981) also revealed only minor differences, which is consistent with the extensive homology. Determination of catalase levels encoded by pgs139 and pstg1 in E. coli strain UM262 (lacking HPI and HPII; Loewen etal. 1990) revealed levels (375 and 338 units/mg dry cell weight respectively; Richter and Loewen 1981; Rorth and Jensen 1967) very similar to catalase levels (331 units/rag dry cell weight) encoded <. Fig. 2. Nucleotide sequence of katg from Salmonella typhimurium and deduced amino acid sequence of the encoded HPI subunit. The pstg1 and pstg2 plasmids were transformed into strains NM522 (Mead et al. 1985) or JM101 (Yanisch-Perron et al. 1985) and grown in LB medium (Miller 1974). Single-strand DNA was prepared, and sequenced by a combination of the methods of Maxam and Gilbert (1980) and Sanger et al. (1977). The S. typhimurium katg sequence is given and the nucleotides in the Escheriehia eoli katg sequence that differ are indicated immediately below. The putative -35, -10 and Shine-Dalgarno (S.D.) sequences are underlined. The predicted amino acid sequence is indicated in italics immediately above the nucleotide sequence and the amino acids in the E. coli HPI sequence that differ are indicated below, also in italics. The differing amino acids that represent conserved changes are contained in parentheses. The nucleotide sequence is numbered on the right and the amino acid sequence is numbered in italies on the left. The dashes under nucleotides +216 to +218 indicate that those nucleotides are not present in the E. coli sequence. Similarly, the dash in the amino acid sequence at residue 73 indicates that the single amino acid is not present in the E. coli sequence A HPI B-- a E S b c d E S E S E S, < iiiiiiiil!!ii17! Fig. 3 a-d. Polyacrylamide gel electrophoresis of purified HPI from Eseherichia coli (E) and Salmonella typhimurium (S). Gels a, b and e were run under non-denaturing conditions (Davis 1964) and stained for a catalase (Clare et al. 1984; Loewen and Switala 1986), b peroxidase (Gregory and Fridovich 1974), and e protein (with Coomassie brilliant blue dye). The bands of HPI isoenzymes A and B are indicated by arrows. Gel d was run under denaturing conditions (Laemmli 1970; Weber et al. 1972) and stained for protein. The locations of size markers run alongside d, fl-galactosidase (116000), phosphorylase b (97400) and bovine albumin (66000), are indicated on the right by pbt22, the E. coli katg clone (Triggs-Raine and Loewen 1987) indicating that the S. typhimurium katg gene was expressed as well in E. coli as the E. coil gene. Even pstg2, in which the putative -10 and -35 regions had been deleted during construction, directed the synthesis of catalase (227 units/rag dry cell weight) at levels nearly equivalent to those expressed from pgs139 and pstg1. The KatG protein (HPI) encoded by plasmid pstg2 and expressed in UM262 was purified, resulting in an enzyme with catalase and peroxidase specific activities of 1740 and 1.68 units/rag protein respectively, only slightly lower than the 2480 and 1.97 units/rag protein observed for the catalase and peroxidase activities of its E. coli counterpart. The proteins had identical subunit sizes as determined on SDS-polyacrylamide gels (Fig. 3d). Electrophoresis on non-denaturing gels also resulted in similar electrophoretic patterns for the two isoenzyme forms, HPIA and HPIB, stained for catalase, peroxidase and protein (Fig. 3a-c). The only evident physical difference between the S. typhimurium and E. coli HPIs was in the relative proportions of HPIA and B, there being more A-form in the E. coli preparation and more B-form in that from S. typhimurium (Fig. 3 a- c). Kinetic analysis of the effect of increasing H202 on catalase activity revealed that 50% of apparent maximal activity was attained at 3.8 and 3.5 mm for the E. coli and S. typhimurium enzymes, respectively. The narrow

4 150 ph optimum between 6 and 7, atypical for catalase but previously reported for the E. coli (Claiborne and Fridovich 1979) and Rhodopseudomonas capsulata (Hochman and Shemesh 1987) HPIs, was also observed for the S. typhimuriurn enzyme (data not shown). Incubation at 50 C for 30 rain resulted in 32% and 34% inactivation respectively for the E. coli and S. typhimurium enzymes. TRIS and imidazole have been shown to activate the catalase but not the peroxidase activity of HPI from E. coli B (Claiborne and Fridovich 1979). We have confirmed that these reagents enchance the catalase activities of both the E. coli K12 and the S. typhimurium LT2 HPIs by 2.5- to 3.0-fold and the peroxidase activities by 1.2- to 1.6-fold. Azide (1 mm), cyanide (0.5 mm) and hydroxylamine (5 gm) are known inhibitors of normal catalases and their effects on S. typhimurium HPI (58%, 57% and 67% inhibition respectively for azide, cyanide and hydroxylamine) revealed few differences from the E. coli enzyme with the catalase activity being more sensitive than the peroxidase activity. During this work it was observed that low cyanide concentrations (< 50 I-tM) activated the catalase but not the peroxidase activity of HPI by as much as 60% for the S. typhimurium enzyme and 80% for the E. coli enzyme. Despite the additional cysteine residue in the S. typhimurium enzyme, it did not exhibit any additional sensitivity to/mercaptoethanol as compared with the E. coli enzyme. The katg gene does not exhibit any homology with known catalase sequences at the gene or protein levels but the recently reported sequence of the Bacillus stearothermophilus peroxidase gene (pera) revealing 48% similarity between the deduced sequences of PerA and KatG proteins (Loprasert et al. 1989) suggests that HPI is more closely related to the family of peroxidases than to the family of catalases. This suggestion was further supported by the spectral properties of the two proteins, which were found to be very similar to those reported earlier for the E. coli B HPI (Claiborne and Fridovich 1979) and for the R. capsulata bifunctional catalase-peroxidase (Hochman and Shemesh 1987). Specifically, the major peak at 540 nm in the cyanide spectrum was in the 538 to 540 nm region typical of most peroxidases (Keilin and Hartree 1951; Blumberg et al. 1968; Yonetani et al. 1966). Despite the apparent activation of the enzyme by low concentrations of cyanide, the only observable changes in the spectrum were a 14% decrease in intensity of the Soret band and a slight shift from 407 to 410 nm. As the concentration of cyanide was increased, the shift in the Soret continued to its final position at 421 nm but there was no further decrease in the intensity of the band. Acknowledgements. This work was supported by a grant (A9600) from the Natural Sciences and Engineering Research Council (to PCL) and a Public Health Service grant (GM26878) from the National Institute of General Medical Sciences (to GVS). References Blumberg WE, Peisach J, Wittenberg BA, Wittenberg JB (1968) The electronic structure of protoheme proteins. I. An electron paramagnetic resonance and optical study of horseradish peroxidase and its derivatives. 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