Substitution of the conserved tryptophan 31 in Escherichia coli thioredoxin by site-directed mutagenesis and structure-function analysis

G Krause,A Holmgren

doi:10.1016/s0021-9258(20)64285-5

Abstract

All prokaryotic and eukaryotic thioredoxins contain a conserved tryptophan residue, exposed at the active site disulfide/dithiol. The role of this W31 in Escherichia coli thioredoxin (Trx) was studied by site-directed mutagenesis. Four mutant Trx with W31Y, W31F, W31H, and W31A replacements were characterized. Very low tryptophan fluorescence emission from the remaining W28 was observed in all mutant Trx; reduction resulted in large, but variable increases (up to 11-fold) of fluorescence, to levels higher than in native or denatured wild-type Trx, demonstrating a previously postulated change involving W28. All W31 mutant Trx were good substrates for E. coli thioredoxin reductase. Compared with wild type, the apparent Km values were increased less than 2-fold for the W31A, W31H, and W31F Trx and the W31Y Trx showed even slightly higher catalytic efficiency (kcat/Km value). Functions of reduced Trx with ribonucleotide reductase and in reduction of insulin disulfides were more strongly influenced by the W31 replacements, in particular at low pH for A and H residues. T7 DNA polymerase activity generated by T7 gene 5 protein and reduced Trx was lowered by large factors for W31Y, W31A, or W31H compared with W31F or the wild-type protein. The in vivo function of Trx was studied by using pUC118-trxA expression in an E. coli trxA- background. The trxA genes with W31Y and W31F substitutions restored, fully and partly, the methionine sulfoxide utilization of a trxA- metE- test strain; W31A and W31H mutations resulted in no growth. Propagation of M13 was moderately impeded by W31Y and W31F or severely by W31A and W31H replacements. Growth of a phage T3/7 hybrid was possible only with the W31Y and W31F substitutions reflecting the in vitro results for T7 DNA polymerase.

Highlights

T7 DNA polymerase activity generated by T7 gene 5 protein andreduced Trx was lowered by large factors for W31Y, W31A, or W31H compared with W31F or the wild-type protein
Propagationof M13 was moderately impeded et al (1986), utilizing a thioredoxins containdoxin gene (trxA),metE- mutant grown on miniby W31Y and W31F or severely by W31A and W31H mal medium with methionine sulfoxide
Protein concentrations were determined from the absorbance at 280 nm using molar extinction coefficients of 13,700 M” cm” for wild-type thioredoxin, andvalues of 8,200 for the W31A and W31H, 8,300 for the W31F, and 10,400 for the W31Y mutants of thioredoxin

Summary

To whom correspondence should be addressed

Dept. of Physio- 5,5’-dithio-bis(2-nitrobenzoiaccid); TR,thioredoxin reductase; logical Chemistry, Karolinska Institutet, Box 60 400, S-104 01 Stock- HEPES, 4-(2-hydroxyethyl)-l-piperazineethanesulfoniaccid; g5p, holm, Sweden. Tryptophan[31] Mutants of E. coli Thioredoxin terized and can be studied in vitro. Chemical modification studies (Holmgren, 1981) and comparison to yeast and calf thymus thioredoxins (M6rola et Expression of Mutant Thioredoxins al., 1989)attributed this rise in fluorescenceemission to W28, Phage DNA of the subclone in M13mp[19] (Fig. l ) , containing the i.e. the other of the 2 tryptophans present in E. coli thioredoxin. Particles obtained during single-stranded DNA preparatiboyn helper phage infection of E. coli CU9276 containing the mutant pUC118-. KabiGen, Stockholm, kindly supplied the mutagenesis host strain E. coli CU9276 (hsdRl7, mcr AB, recAl, A(1ac-proAB), (F’traD36, proAB+,lacIqZAM15)) and the Purification of Mutant Thioredoxins Frozen cells were taken up in 10 times their wet weight of 50 mM test template and primer for the mutagenesis system according to Vandeyar et al (1988).

Analytical Methods

RESULTS

W31Y v c Y ij eo

Findings

DISCUSSION