Consensus mutagenesis reveals that non-helical regions influence thermal stability of horseradish peroxidase

Ryan, Barry J.; O’Connell, Mary J.; Ó’Fágáin, Ciarán

doi:10.1016/j.biochi.2008.04.009

Cited by 13 publications

(15 citation statements)

References 51 publications

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“…1). There are few reports with improved kinetic properties of HRP by means of single mutations, for example, an increased k cat of the enzyme ratio for 2,2′-Azino-bis (3-Ethylbenzthiazoline-6-Sulfonic Acid) (ABTS) resulted from a single S35K substitution [34], a decreased K m value for ABTS was obtained from a combination of five mutations [35] and an increased reactivity toward ABTS in E238Q and E239Q substitution was achieved [14]. An N70D substitution also decreased guaiacol oxidation rates [36].…”

Section: Resultsmentioning

confidence: 98%

Investigating the Structural and Functional Effects of Mutating Asn Glycosylation Sites of Horseradish Peroxidase to Asp

Asad

Khajeh

Ghaemi

2010

Appl Biochem Biotechnol

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Horseradish peroxidase (HRP) has long attracted intense research interest and is used in many biotechnological fields, including diagnostics, biosensors, and biocatalysis. Enhancement of HRP catalytic activity and/or stability would further increase its applications. One of the problems with heterologus expression of HRP especially in prokaryotic host is lack of glycosylation that affects it's stability toward H(2)O(2) and thermal inactivation. In this study, two asparagine residues which constitute two of the eight glycosylation sites in native HRP (Asn 13 and 268) with respectively 83% and 65% surface accessibility were substituted with aspartic acid in recombinant HRP. Both mutant proteins expressed in Escherichia coli showed increased stabilities against heat (increase in t (1/2) from 20 min in native rHRP to 32 and 67 min in N13D and N268D) and H(2)O(2) (up to threefold). Unexpectedly, despite the distance of the mutated positions from the active site, notable alterations in steady-state k (cat) and K (m) values occurred with phenol/4-aminoantipyrine as reducing substrate which might be due to conformational changes. No significant alteration in flexibility was detected by acrylamide quenching analyses, but ANS binding experiments purposed lesser binding of ANS to hydrophobic patches in mutated HRPs. Double mutation was non-additive and non-synergistic.

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Section: Resultsmentioning

confidence: 98%

Investigating the Structural and Functional Effects of Mutating Asn Glycosylation Sites of Horseradish Peroxidase to Asp

Asad

Khajeh

Ghaemi

2010

Appl Biochem Biotechnol

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“…As alluded to above, Phe41 makes a pstacking interaction with the heme porphyrin ring and thus limits the peroxygenase activity of the enzyme (Colas and De Montellano, 2004). Phe41 acts as hydrophobic barrier between residues Arg38 and His42, which are key residues in the enzymatic catalysis (Ryan et al, 2008;Gazaryan et al, 1994). Mutations of HRP-C Phe41 have shown that the hydrophobicity of the active site region is important for the enzymatic catalysis (Ryan et al, 2006;Feng et al, 2008).…”

Section: Sca Of the Plant Peroxidase Superfamilymentioning

confidence: 97%

“…Surrounding the heme plane, there are several invariant or highly conserved amino acid residues that are essential for the catalytic properties of peroxidases. A number of studies involving mutagenesis have explored the presence of these structurally and catalytically important residues in the active site in order to enhance the applicability and stability of peroxidases (Ryan et al, 2008;Feng et al, 2008).…”

Section: Introductionmentioning

confidence: 99%

Crystal structure and statistical coupling analysis of highly glycosylated peroxidase from royal palm tree (Roystonea regia)

Watanabe

Moura

Bleicher

et al. 2010

Journal of Structural Biology

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Royal palm tree peroxidase (RPTP) is a very stable enzyme in regards to acidity, temperature, H(2)O(2), and organic solvents. Thus, RPTP is a promising candidate for developing H(2)O(2)-sensitive biosensors for diverse applications in industry and analytical chemistry. RPTP belongs to the family of class III secretory plant peroxidases, which include horseradish peroxidase isozyme C, soybean and peanut peroxidases. Here we report the X-ray structure of native RPTP isolated from royal palm tree (Roystonea regia) refined to a resolution of 1.85A. RPTP has the same overall folding pattern of the plant peroxidase superfamily, and it contains one heme group and two calcium-binding sites in similar locations. The three-dimensional structure of RPTP was solved for a hydroperoxide complex state, and it revealed a bound 2-(N-morpholino) ethanesulfonic acid molecule (MES) positioned at a putative substrate-binding secondary site. Nine N-glycosylation sites are clearly defined in the RPTP electron-density maps, revealing for the first time conformations of the glycan chains of this highly glycosylated enzyme. Furthermore, statistical coupling analysis (SCA) of the plant peroxidase superfamily was performed. This sequence-based method identified a set of evolutionarily conserved sites that mapped to regions surrounding the heme prosthetic group. The SCA matrix also predicted a set of energetically coupled residues that are involved in the maintenance of the structural folding of plant peroxidases. The combination of crystallographic data and SCA analysis provides information about the key structural elements that could contribute to explaining the unique stability of RPTP.

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“…Although HRP A2, HRP C and SBP are closely related [8], their enzymatic stabilities differ considerably. Of these three proteins, SBP is the most thermostable, and HRP A2 the least thermostable [9][10][11][12]. Their stabilities towards their primary substrate, H 2 O 2 , vary in an apparently inverse pattern with their thermal stabilities.…”

Section: Introductionmentioning

confidence: 99%

Stability properties of an ancient plant peroxidase

et al. 2014

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Consensus mutagenesis reveals that non-helical regions influence thermal stability of horseradish peroxidase

Cited by 13 publications

References 51 publications

Investigating the Structural and Functional Effects of Mutating Asn Glycosylation Sites of Horseradish Peroxidase to Asp

Investigating the Structural and Functional Effects of Mutating Asn Glycosylation Sites of Horseradish Peroxidase to Asp

Crystal structure and statistical coupling analysis of highly glycosylated peroxidase from royal palm tree (Roystonea regia)

Stability properties of an ancient plant peroxidase

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