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A residue outside the active site CXXC motif regulates the catalytic efficiency of Glutaredoxin 3.

ABSTRACT: The glutaredoxin (Grx) family of oxidoreductases has a conserved residue at position 8 that varies between Arginine in Grx1 and Lysine in Grx3. It has been proposed that this Arg/Lys change is the main cause for the 35 mV difference in redox potential between the two enzymes. To gain insights into the catalytic machinery of Grx3 and directly evaluate the role of residue 8 in the catalysis of thiol-disulfide exchange by this enzyme, we synthesized the "wild type" enzyme (sGrx3), and four analogues substituting the lysine at position 8 with arginine, ornithine (Orn), citrulline (Cit) and norvaline (Nva). The redox potential and equilibration kinetics with thioredoxin (Trx1) were determined for each enzyme by fluorescence intensity. While minor effects on redox potential were observed, we found that residue 8 had a more marked effect on the catalytic efficiency of this enzyme. Surprisingly, truncation of the functional group resulted in a more efficient enzyme, Lys8Nva, exhibiting rate constants that are an order of magnitude higher than sGrx3 for both forward and reverse reactions. These observations pose the question why would a residue that reduces the rate of enzyme turnover be evolutionarily conserved? The significant changes in the kinetic parameters suggest that this position plays an important role in the thiol-disulfide exchange reaction by affecting the nucleophilic thiolate through electrostatic or hydrogen bonding interactions. Since the reduced Grx has an exposed thiol that could easily be alkylated, either Arg or Lys could act as a gatekeeper that deters unwanted electrophiles from attacking the active site thiolate.

SUBMITTER: Shekhter T

PROVIDER: S-EPMC3820274 | biostudies-literature | 2010 Jan

REPOSITORIES: biostudies-literature

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A residue outside the active site CXXC motif regulates the catalytic efficiency of Glutaredoxin 3.

Shekhter Talia T Metanis Norman N Dawson Philip E PE Keinan Ehud E

Molecular bioSystems 20090922 1

The glutaredoxin (Grx) family of oxidoreductases has a conserved residue at position 8 that varies between Arginine in Grx1 and Lysine in Grx3. It has been proposed that this Arg/Lys change is the main cause for the 35 mV difference in redox potential between the two enzymes. To gain insights into the catalytic machinery of Grx3 and directly evaluate the role of residue 8 in the catalysis of thiol-disulfide exchange by this enzyme, we synthesized the "wild type" enzyme (sGrx3), and four analogue ...[more]

PMID: 20024086

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Project description:Endoxylanase with high specific activity, thermostability, and broad pH adaptability is in huge demand. The mutant library of GH11 endoxylanase was constructed via DNA shuffling by using the catalytic domain of Bacillus amyloliquefaciens xylanase A (BaxA) and Thermomonospora fusca TF xylanase A (TfxA) as parents. A total of 2,250 colonies were collected and 756 of them were sequenced. Three novel mutants (DS153: N29S, DS241: S31R and DS428: I51V) were identified and characterized in detail. For these mutants, three residues of BaxA were substituted by the corresponding one of TfxA_CD. The specific activity of DS153, DS241, and DS428 in the optimal condition was 4.54, 4.35, and 3.9 times compared with the recombinant BaxA (reBaxA), respectively. The optimum temperature of the three mutants was 50°C. The optimum pH for DS153, DS241, and DS428 was 6.0, 7.0, and 6.0, respectively. The catalytic efficiency of DS153, DS241, and DS428 enhanced as well, while their sensitivity to recombinant rice xylanase inhibitor (RIXI) was lower than that of reBaxA. Three mutants have identical hydrolytic function as reBaxA, which released xylobiose-xylopentaose from oat spelt, birchwood, and beechwood xylan. Furthermore, molecular dynamics simulations were performed on BaxA and three mutants to explore the precise impact of gain-of-function on xylanase activity. The tertiary structure of BaxA was not altered under the substitution of distal residues (N29S, S31R, and I51V); it induced slightly changes in active site architecture. The distal impact rescued the BaxA from native conformation ("closed state") through weakening interactions between "gate" residues (R112, N35 in DS241 and DS428; W9, P116 in DS153) and active site residues (E78, E172, Y69, and Y80), favoring conformations with an "open state" and providing improved activity. The current findings would provide a better and more in-depth understanding of how distal single residue substitution improved the catalytic activity of xylanase at the atomic level.

Dataset Information

A residue outside the active site CXXC motif regulates the catalytic efficiency of Glutaredoxin 3.

Publications

A residue outside the active site CXXC motif regulates the catalytic efficiency of Glutaredoxin 3.

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