Cloning, Expression and Nucleotide Sequence of a Gene Encoding a Second Thioredoxin from Corynebacterium Nephridii

Lim, Chang‐Jin; Fuchs, James A.; McFarlan, Sara C.; Hogenkamp, Harry P. C.

doi:10.1007/978-1-4613-0749-5_4

Cited by 8 publications

(6 citation statements)

References 26 publications

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“…The consensus sequence WCGPC found so far in nearly all thioredoxins is substituted by "GCVPC" in E. acidaminophilum. An exchange of the conserved Gly33 to Ala has so far only been observed for the second thioredoxin present in Corynebacterium nephridii [40] and in all thioredoxins from anaerobic bacteria for which the N-terminal amino acids have been sequenced (C. sporogenes, C. sticklandii, C. litorale [7] (and Meyer et al, unpublished results). In most cases, Val was found to replace Gly.…”

Section: Discussionmentioning

confidence: 98%

Components of glycine reductase from Eubacterium acidaminophilum

Lübbers

Andreesen

1993

European Journal of Biochemistry

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The genes encoding thioredoxin reductase (trxB), thioredoxin (trxA), protein PA of glycine reductase (&A) and the first 23 amino acids of the large subunit of protein P, of glycine reductase ( g r d o belonging to the reductive deamination systems present in Eubacterium acidaminophilum were cloned and sequenced. The proteins were products of closely linked genes with 314 codons (thioredoxin reductase), 110 codons (thioredoxin), and 158 codons (protein PA). The protein previously called 'atypically small lipoamide dehydrogenase' or 'electron transferring flavoprotein' could now conclusively be identified as a thioredoxin reductase (subunit mass of 34781 Da) by the alignment with the enzyme of Escherichia coli showing the same typical order of the corresponding domains. The thioredoxin (molecular mass of 11 742 Da) deviated considerably from the known consensus sequence, even in the most strongly conserved redox-active segment WCGPC that was now GCVPC. The selenocysteine of protein PA (molecular mass of 16609 Da) was encoded by TGA. The protein was highly similar to those of Clostridium purinolyticum and Clostridium sticklundii involved in glycine reductase. Thioredoxin reductase and thioredoxin of E. acidaminophilum could be successfully expressed in E. coli.Eubacterium acidaminophilum utilizes glycine as both electron donor and electron acceptor, whereas sarcosine and betaine are exclusively used as electron acceptor [l]. Due to this fact, this organism is well suited to compare the biochemistry of the unusual reductive deaminations and the energy conservation associated with it. Glycine reductase, sarcosine reductase, and betaine reductase seem to be generally composed of three proteins: selenoprotein PA, protein Ps, and protein Pc, of which only P, is substrate-specific [2-101 (Fig. 1). In the first step the substrate is bound to protein P, that is specific for glycine, sarcosine, or betaine (M. Meyer and J. R. Andreesen, unpublished results). In all three reductases, the P,-activated substrate is proposed to be nucleophilically attacked by the selenol anion of protein PA to transform it to a carboxymethylated selenoether [3] and the respective amine [4]. By action of protein Pc, acetyl phosphate is formed in all cases leaving the selenoprotein PA in its oxidized state [3, 9, lo]. Finally the selenoprotein PA becomes reduced by the thioredoxin system to start a new catalytic cycle of reductive deamination [7, 81 (Fig. 1). In E. aciduminophilum the NADPH-derived electrons are transferred to protein PA by two proteins, a thioredoxin-reductase-like flavoprotein and an atypical thioredoxin [7,

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Section: Discussionmentioning

confidence: 98%

Components of glycine reductase from Eubacterium acidaminophilum

Lübbers

Andreesen

1993

European Journal of Biochemistry

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“…Anabaena sp. PCC7119 and Corynehacterium nephridii C2 thioredoxin (Lim et al, 1986(Lim et al, , 1987 do not support T7 replication; a comparison of their amino acid sequence to those of E. coli and R. sphaeroides (Table 1), shows that residue Gly74, as already proposed, probably has a structural role in the interaction between thioredoxin and the viral DNA polymerase. Structural genes encoding thioredoxi n have been cloned from many sources including the trxA gene of R. sphaernides (Pille et a]., 1990).…”

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confidence: 78%

The Gly74→Ser and Ser3→Ala Mutations in Rhodobacter sphaeroides Y Thioredoxin

Pille

Assemat

Breton

et al. 1996

European Journal of Biochemistry

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In this study, we report the effects of two different substitutions in Rhodohacter ,vphmroidf,.s thioredoxin on two regions of the protcin : the N-terminus end and thc hydrophobic area implicated in protein/ protein interactions. We have produced by site-dirccterl mutagcnesis R. ,sphnevoides thiorcdoxin single and double mutants in which the glycine residue at position 74 is changed to a scrine and the serinc :it position 3 is changed to an alanine; the three mutant proteins have been purified. Thc two substitutions are not equivalent. Substitution of serine by aliinine increascd the pl from 5.2 to 6.1 ; this pl value was the same in the double-mutated protein, which dcmonstrates the presence of a local cotiformational change. 111 vivo studies showed that the Gly74-.Ser substitution completely prevented phage T3/7 growth whereas the Ser34Ala substitution had no effect. This finding was corroborated by the large decrease ( IOO-fold) of polymerase activity for the double niutant in the in vilm measurement of phage T7 DNA polymcrase activity with the corresponding pure proteins. Although trxirginal (within a factor of two), the effects of the two substitutions on the calalytic activities of the thioredoxin reductase rciiction con finned their difference. Substitution of serine by alanine had no effect on the K,,, and resulted in iin improvement in the catalytic efficiency. In contrast, the second substitution increased the K,,, value, without improving the catalytic efficiency. The following can be concludcd (a) glyciiie74 of R. sp/iwroidt<.s thiaredoxin has a direct role in the binding of T7 gene 5 protein iind thc hydrophobic area of thioredoxin: (b) the Nterminus plays a role in tnaintaining the conforniiitional integrity of the active site; (c) the flcxibility of Gly74 in the hydrophobic region involved in protcin/protein interaction is the operative factor in the case of the activity of thioredoxin in the T7 DNA polymcrase.Keywords: thioredoxin; T7 DNA polymerase ; site-directed niutagenesis; kinetic constants; stt-iictui-c/ fuiiction relationships.Thioredoxin i s a small, heat-stable rcdox protein with the active-center amino acid sequence -Trp-Cys-C;ly-Pro-Cyshighly conserved in all but 1 of the 34 sequences currently rcported (Swiss-Prot data bank). This protein seetiis to be present in all living organisms from archaebactcria to humans and hiis now heen characterized from a wide variety of procaryotic iind eucaryotic cells (Holmgren, 1985 ;Gleason and Holmgren, 1988;Eklund et al., 1991;Pille, 1992). Thioredoxin has diverse functions in the cell, several of which depcnd on redox activity via thiol-disulfidc interchange reactions. In addition to thcir redox functions, some members of this class have structural roles in coliphages such as f l and M i 3 (Russel and Model, 198s; Litn et al., 1986;Russel, 1991) and as a subunit of T7 DNA polymerasc (Modrich and Richardson, 1975). The structurc of the reduced form is required for the function of thioredoxin in coliphages although the redox property is not otherw...

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“…The location of these mutations at or adjacent to the residues identified by Eklund et al (10) suggests that this surface area is critical for thioredoxin interaction with the gene 5 protein (14). Comparison of the amino acid sequence of R. sphaeroides thioredoxin with other known thioredoxin sequences (19)(20)(21)34) shows that, at the position corresponding to residue 74 of E. coli thioredoxin, Anabaena PCC7119 and C. nephridii C-2 have Ser and Asn, respectively, whereas both E. coli and R. sphaeroides have a Gly residue. Moreover, residues 73 to 76 are identical in the R. sphaeroides and E. coli thioredoxins.…”

Section: Discussionmentioning

confidence: 99%