| 1. |
- Hederstedt, Lars, et al.
(författare)
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New properties of Bacillus subtilis succinate dehydrogenase altered at the active site
- 1989
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Ingår i: Biochemical Journal. - : Portland Press Ltd.. - 0264-6021 .- 1470-8728. ; 260:2, s. 491-497
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Tidskriftsartikel (refereegranskat)abstract
- Mammalian and Escherichia coli succinate dehydrogenase (SDH) and E. coli fumarate reductase apparentlycontain an essential cysteine residue at the active site, as shown by substrate-protectable inactivation withthiol-specific reagents. Bacillus subtilis SDH was found to be resistant to this type of reagent and containsan alanine residue at the amino acid position equivalent to the only invariant cysteine in the flavoproteinsubunit of E. coli succinate oxidoreductases. Substitution of this alanine, at position 252 in the flavoprotein subunit of B. subtilis SDH, by cysteine resulted in an enzyme sensitive to thiol-specific reagents and protectable by substrate. Other biochemical properties of the redesigned SDH were similar to those of the wild-type enzyme. It is concluded that the invariant cysteine in the flavoprotein of E. coli succinate oxidoreductases corresponds to the active site thiol. However, this cysteine is most likely not essential for succinate oxidation and seemingly lacks an assignable specific function. An invariant arginine in juxtaposition to Ala-252 in the flavoprotein of B. subtilis SDH, and to the invariant cysteine in the E. coli homologous enzymes, is probably essential for substrate binding.
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| 2. |
- Lewin, Allison, et al.
(författare)
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Effects of substitutions in the CXXC active-site motif of the extracytoplasmic thioredoxin ResA
- 2008
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Ingår i: Biochemical Journal. - 0264-6021. ; 414, s. 81-91
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Tidskriftsartikel (refereegranskat)abstract
- The thiol-disulfide oxidoreductase ResA from Bacillus subtilis fulfils a reductive role in cytochrome c maturation. The pK(a) values for the CEPC (one-letter code) active-site cysteine residues of Ill are unusual for thioredoxin-like proteins ill that they are both high (> 8) and within 0.5 unit of each other. To determine the contribution of the inter-cysteine dipeptide of ResA to its redox and acid-base properties, three variants (CPPC, CEHC and CPHC) were generated representing a stepwise conversion into the active-site sequence of the high-potential DsbA protein from Escherichia coli. The substitutions resulted in large decreases in the pK(a) values of both the active-site cysteine residues: in CPHC (DsbA-type) ResA, Delta pK(a) values of -2.5 were measured for both cysteine residues. Increases in midpoint reduction potentials were also observed, although these were comparatively small: CPHC (DsbA-type) ResA exhibited all increase of +40mV compared with the wild-type protein. Unfolding studies revealed that, despite the observed differences in the properties of the reduced proteins, changes in stability were largely confined to file oxidized state. High-resolution structures of two of the variants (CEHC and CPHC ResA) in their reduced states were determined and are discussed in terms of the observed changes ill properties. Finally, the in vivo functional properties of CEHC RcsA are shown to be significantly affected compared with those of the wild-type protein.
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| 3. |
- Schröder, Ingrid, et al.
(författare)
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Glutamyl-tRNA reductase activity in Bacillus subtilis is dependent on the hemA gene product
- 1992
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Ingår i: Biochemical Journal. - : Portland Press Ltd.. - 0264-6021 .- 1470-8728. ; , s. 843-850
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Tidskriftsartikel (refereegranskat)abstract
- The Bacillus subtilis hemAXCDBL operon encodes enzymes for the synthesis of 5-aminolaevulinic acid via the C5 pathway (hemA and hemL) and uroporphyrinogen III (hemB, hemC and hemD). B. subtilis HemA protein (molecular mass 50 kDa) was overexpressed in hemA mutants of both Escherichia coli and B. subtilis. A mutant B. subtilis HemA protein with a Cys to Tyr change at position 105 was also overexpressed. Both wild-type and mutant HemA proteins migrated as oligomers (molecular mass greater-than-or-equal-to 230 kDa) on gel-filtration columns. All column fractions containing wild-type HemA protein had glutamyl-tRNA reductase activity. No glutamyl-tRNA reductase activity was found with the mutant HemA protein. It is concluded that the B. subtilis hemA gene product is identical to, or part of, the glutamyl-tRNA reductase of the C5 pathway.
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