Abstract
The periplasmic DMSO reductase from Rhodobacter sphaeroides f. sp. denitrificans has been expressed in Escherichia coli BL21(DE3) cells in its mature form and with the R. sphaeroides or E. coli N-terminal signal sequence. Whereas the R. sphaeroides signal sequence prevents formation of active enzyme, addition of a 6x His-tag at the N terminus of the mature peptide maximizes production of active enzyme and allows for affinity purification. The recombinant protein contains 1.7-1.9 guanines and greater than 0.7 molybdenum atoms per molecule and has a DMSO reductase activity of 3.4-3.7 units/nmol molybdenum, compared with 3.7 units/nmol molybdenum for enzyme purified from R. sphaeroides. The recombinant enzyme differs from the native enzyme in its color and spectrum but is indistinguishable from the native protein after redox cycling with reduced methyl viologen and Me2SO. Substitution of Cys for the molybdenum-ligating Ser-147 produced a protein with DMSO reductase activity of 1.4-1.5 units/nmol molybdenum. The mutant protein differs from wild type in its color and absorption spectrum in both the oxidized and reduced states. This substitution leads to losses of 61-99% of activity toward five substrates, but the adenosine N1-oxide reductase activity increases by over 400%.
Highlights
The periplasmic DMSO reductase from Rhodobacter sphaeroides f. sp. denitrificans has been expressed in Escherichia coli BL21(DE3) cells in its mature form and with the R. sphaeroides or E. coli N-terminal signal sequence
The majority of molybdoenzymes contain one or more strongly chromophoric prosthetic groups in addition to molybdenum [1, 2]. The properties of their molybdenum centers have been studied by EPR and EXAFS,1 other techniques such as MCD, resonance Raman spectroscopy (RR), and UV-visible absorption spectroscopy have not been applicable to these enzymes because the weakly absorbing molybdenum centers are masked by the other stronger chromophores
Since differences in the signal sequence requirements of the two organisms could result in improper processing, we have examined the effects of deletion of the N-terminal signal sequence as well as the effects of substitution of the R. sphaeroides DMSO reductase signal sequence with that of E. coli TMAO reductase on the expression of active DMSO reductase
Summary
Materials—T4 polynucleotide kinase, T4 DNA ligase, calf intestinal alkaline phosphatase, 1-kilobase pair DNA ladder standards, Miller’s LB broth, and competent DH5␣ and DM1 E. coli cells were from Life Technologies, Inc. Glycerol-tolerant DNA sequencing gel mix was from Amersham Pharmacia Biotech. The Transformer Site-directed Mutagenesis Kit and BMH 71–18 mutS cells were from CLONTECH. The pET-28 and pET-29 expression systems, BL21(DE3) E. coli cells, induction control plasmid F, pLysE, and Perfect Protein Standards were from Novagen. Electrophoresis reagents, protein minigels, prestained low range SDS-polyacryamide gel electrophoresis standards, and gelatin were from Bio-Rad. Goat anti-rabbit horseradish peroxidase-conjugated IgG was from Boehringer Mannheim. Site-directed mutagenesis of R. sphaeroides DMSO reductase with the CLONTECH Transformer Site-directed Mutagenesis Kit was carried out on doublestranded DNA according to the manufacturer’s protocol. The pJH115 construct (Table I), containing the DMSO reductase coding sequence, free of NcoI and NdeI restriction sites, in pBluescript II KS(ϩ), was subjected to mutagenesis with selection primer BSXbaD677 (Table II) and mutagenic primers NdeA122 or NdeA-2 to create pJH118 and pJH119, respectively.
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