Effects of mutations in the helix G region of horseradish peroxidase

Abstract

Summary.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 usefulness. Based on prior art, we substituted solvent-exposed lysine and glutamic acid residues near the proximal helix G (Lys 232, 241; Glu 238, 239) and between helices F and F' (Lys 174). Three single mutants (K232N, K232F, K241N) demonstrated increased stabilities against heat (up to two-fold) and solvents (up to four-fold). Stability gains are likely due to improved hydrogen bonding and space-fill characteristics introduced by the relevant substitution. Two double mutants showed stability gains but most double mutations were non-additive and non-synergistic.Substitutions of Lys 174 or Glu 238 were destabilising. Unexpectedly, notable alterations in steady-state V m /E values occurred with reducing substrate ABTS (2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid)), despite the distance of the mutated positions from the active site.

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

“…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].…”

“…Little information is available regarding the effects of rational, sitedirected amino acid substitution on the HRP stability toward critical inactivating factors such as temperature and hydrogen peroxide. Till now, the sole reports concerning genetic manipulation of HRP in order to improve its thermal and peroxide stability are performed by Ryan and Fagain [12,14]. In this research, Asn 13 and Asn 268 residues of recombinant HRP substituted with Asp, and the effect of these substitutions on the activity and stability of the enzyme was investigated.…”

How Modification of Accessible Lysines to Phenylalanine Modulates the Structural and Functional Properties of Horseradish Peroxidase: A Simulation Study

“…In another study, they analyzed the effects of 13 single and 3 double point mutations on the stability of HRP. Three single mutants (K232N, K232F, K241N) demonstrated increased stability against heat (up to 2-fold) and solvents (up to 4-fold) [27] . These studies indicate that single substitution of either K232 or K241 by Phe residues is beneficial for the stability of HRP structure, although it cannot be inferred that simultaneous mutation of these two is additive or synergistic.…”

“…(4) The hydrogen bonding strength between some residues involved in proximal Ca 2+ coordination shows considerable improvements upon substitutions ( Table 4 ). Such changes could enhance the stability of proximal Ca 2+ binding pocket which is assumed to be essential for the structural and functional integrity of the enzyme [27] , [60] . (5) Formation of two persistent stitching hydrogen bonds between the connecting loops GH and F′F″ could enhance the integrity of this part of p-HRP structure.…”

Enhancing thermostability of the biocatalysts beyond their natural function via protein engineering

“…Some of the studies carried out previously reflect that most of these mutations are present in the loop and random coils, e.g. mutation G195E in galactose oxidase (Sun et al, 2001) lipase (Acharya et al, 2004;Ahmad et al, 2008) subtilisin E (Zhao, 1999) horse radish peroxidase (Ryan and O'Fágáin, 2008), α glucosidase, (Zhou et al, 2010), L-asparaginase (Kotzia and Labrou, 2009), N-carbamyl-D-amino acid amidohydrolase (Yu et al, 2009). 2.…”