Escherichia coli TehB requires S-adenosylmethionine as a cofactor to mediate tellurite resistance

Abstract

The Escherichia coli chromosomal determinant for tellurite resistance consists of two genes (tehA and tehB) which, when expressed on a multicopy plasmid, confer resistance to K(2)TeO(3) at 128 microg/ml, compared to the MIC of 2 microg/ml for the wild type. TehB is a cytoplasmic protein which possesses three conserved motifs (I, II, and III) found in S-adenosyl-L-methionine (SAM)-dependent non-nucleic acid methyltransferases. Replacement of the conserved aspartate residue in motif I by asparagine or alanine, or of the conserved phenylalanine in motif II by tyrosine or alanine, decreased resistance to background levels. Our results are consistent with motifs I and II in TehB being involved in SAM binding. Additionally, conformational changes in TehB are observed upon binding of both tellurite and SAM. The hydrodynamic radius of TehB measured by dynamic light scattering showed a approximately 20% decrease upon binding of both tellurite and SAM. These data suggest that TehB utilizes a methyltransferase activity in the detoxification of tellurite.

FIG. 1

Alignment of sequences of E. coli TehB, H. influenzae TehB, and some non-nucleic acid methyltransferases to demonstrate the three motifs. The three homologous regions (motifs) of the methyltransferases are shown in the one-letter amino acid code. The numbers represent the residue number spacings between the motifs. The sequences are from the National Center for Biotechnology Information database. The names are abbreviated, and their accession numbers are in parentheses. EctehB, protein encoded by E. coli tellurite resistance gene tehB (M38696); HitehB, protein encoded by an H. influenzae tellurite resistance gene tehB homologue (U32807); RPIMT, rat protein l-isoaspartyl carboxyl methyltransferase (D11475); HPIMT, human protein l-isoaspartyl carboxyl methyltransferase (P22061); EcPIMT, E. coli protein l-isoaspartyl carboxyl methyltransferase (P24206); RDHPBMT, rat dihydroxypolyprenylbenzoate methyltransferase (L20427); EcUbiG, E. coli ubiquinone biosynthesis-related protein (M87509); YCoq3, Saccharomyces cerevisiae 3,4-dihydroxy-5-hexaprenylbenzoate methyltransferase (M73270); EcCFA, E. coli cyclopropane fatty acid synthetase (M98330); SaNMT, Streptomyces anulatus N-methyltransferase (X92429); EcBioC, E. coli biotin synthesis protein (P12999); EhBioC, Erwinia herbicola biotin synthesis protein; RbGNMT, rabbit glycine N-methyltransferase (D13307); RtGNMT, rat glycine N-methyltransferase (X06150); SgStsG, Streptomyces griseus methyltransferase involved in the N-methyl-l-glucosamine pathway (Y08763); RGAMT, rat guanidinoacetate methyltransferase (J03588).

FIG. 2

SDS-polyacrylamide gel electrophoresis analysis of the purification of E. coli TehB. Lane 1, protein molecular size standards with molecular masses of (from top to bottom) 97.4, 66.2, 45.0, 31.0, 21.5, and 14.4 kDa; lane 2, cell extract of induced E. coli C41(DE3)/pTWT124; lane 3, cell extract of uninduced E. coli C41(DE3)/pTWT124; lane 4, freshly purified TehB from the nickel-nitrilotriacetic acid affinity resin; lane 5, TehB after storage at 4°C for 2 weeks, showing the presence of a high-molecular-mass aggregate protein (∗).