Elsevier

Enzyme and Microbial Technology

Volume 118, November 2018, Pages 13-19
Enzyme and Microbial Technology

Leu169Trp substitution in MnSOD from Staphylococcus equorum created an active new form of similar resistance to UVC irradiation

https://doi.org/10.1016/j.enzmictec.2018.06.015Get rights and content

Highlights

  • Leu169Trp substitution has little effect on the activity upon exposure to the UVC.

  • The mutant appears a dimer in solution.

  • The mutant has similar resistance towards inactivation by UVC.

  • The substitution results in generation of a new molecular form.

  • The presence of the new form of altered structure explains the enzyme resistance towards the UVC.

Abstract

Manganese superoxide dismutase from Staphylococcus equorum (MnSODSeq) maintains its activity after up to 45 minutes of UVC radiation. The enzyme occurs in a dimeric form that likely contributes to its activity and stability. Therefore, maintaining the dimeric form could be a way to improve the enzyme’s stability. One of the main interactions for dimer formation occurs between Tyr168 and His31, of which the latter is also involved in the enzymatic reaction. UVC radiation may cause alterations in the electronic structure of the phenolic ring in the Tyr168 side chain: this may disrupt the Tyr168-His31 pairing and lead to enzyme instability and/or activity loss. In this report, a Leu169Trp substitution was carried out to protect the Tyr168 residue by introducing an amino acid with an aromatic side chain for better photon absorption of the UV light. Interestingly, although the substitution appeared to have a minor effect on enzyme stability and activity upon UVC irradiation, the melting temperature (TM) of the Leu169Trp mutant was different. Unlike the native protein, the TM of the mutant had not changed after UV irradiation. Thus, our effort to extend the resistance to UVC radiation was not successful, but we have discovered a biologically active new form. The present finding provides evidence that MnSODSeq maintains most of its activity and resistance to UVC irradiation as long as the dimer and its glutamate-bridge are intact, despite an alteration that destabilizes its monomeric structure. The present finding further unravels the relationship between the structure of the enzyme and its activity. Furthermore, the results may provide further insight in how to modify the enzyme to improve its characteristics for application in medicine or cosmetics.

Introduction

The presence of superoxide (SO) species in living organisms is often associated with various diseases or biological processes that are related to oxidative stress, cancer, diabetes, cell death, or aging [1]. In the body, superoxides are countered by endogenous antioxidants including proteins, one of which is superoxide dismutase (SOD, E.C. 1.15.1.1). SOD is ubiquitous and can be found in various organisms that make use of oxygen [2]. Its antioxidant property is much stronger than that of vitamin C, making the enzyme an effective first line of defense against SO species [3]. However, SOD levels in the body decrease with aging, therefore increasing the risk of diseases. SOD eliminates the SO species by facilitating its conversion into hydrogen peroxide (H2O2) and oxygen (O2) [2]. SOD is also reported to protect cells and tissues from ultraviolet (UV) radiation [4], which is known to cause or trigger damage, and endangering health. Therefore, SOD has also been developed as a component in sun block cream [2].

UV radiation alters the reactive sulfur side chain of cysteine (Cys) and the aromatic side chain of certain bulky amino acids i.e. tryptophan (Trp), tyrosine (Tyr), and phenylalanine (Phe) by photon absorption. In some cases, other reactive amino acids such as serine (Ser), histidine (His), methionine (Met), glycine (Gly), arginine (Arg) and lysine (Lys) can also be affected [5]. Exposure to UV radiation results in a conversion of Trp to its kynurenine derivatives [6]. The quantum yield at longer wavelength is lower than at shorter wavelength, in agreement with their respective excitation energies: Thus, a stronger damaging effect is anticipated at shorter wavelengths. Solar UV radiation is divided into long (UVA, 315–400 nm), medium (UVB, 280–315 nm), and short (UVC, 100–280 nm) wavelengths, each range having its own damaging effect to biological systems [7]. Although UVA cause the least cellular damage, UVA represents 95% of the solar UV radiation and poses a similar risk as UVB (5%). UVC is screened off by the ozone layer in the atmosphere [7,8]. Here, the stability of a recombinant MnSOD from Staphylococcus equorum (rMnSODSeq) against UV irradiation was studied at molecular level. UVC radiation was employed as it has the highest energy and most severe radiation effect [5].

Previously, we have reported the unique properties of rMnSODSeq [9]. The enzyme displays thermal stability, which may be due to its dimeric form. The dimer is formed through interactions between Glu164 (rMnSODSeq amino acid numbering) of one monomer (monomer A) to His165 from the other monomer (monomer B). Glu164 (A) forms a glutamate-bridge with Glu164 (B) and stabilizes the orientation of His165 [10,11]. Since His165 is also involved in coordinating the manganese ion in the active site, disruption of the glutamate-bridge affects the activity of the enzyme. The dimeric form appears to be related to enzyme activity. In the structure of bacterial E. coli MnSOD, the manganese ion is located near the dimer interface [11]. Another significant dimeric interaction occurs between Tyr168 (A) and His31 (B) [12]. The Tyr168-His31 pair is proposed to be involved in the reaction by orienting the substrate into the active site [11]. The Glu164-His165 and Tyr168-His31 interactions stabilize the dimeric form of rMnSODSeq. In E. coli MnSOD, a mutation of Glu164Ala results in the dissociation of the dimer, which consequently also weakens the Tyr168-His31 pair. In addition, the activity of the Glu164Ala mutant dimer was very low, suggesting the importance of this pair to the enzymatic catalysis. Furthermore, the disruption of the Tyr168-His31 pair may also have an effect on the enzymatic reaction because it affects the chemistry of His31, which in turns prevents the histidine to perform its proper function. Possibly, alteration of Tyr168 by e.g. UVC could lead to a decrease in enzyme activity, although the dimer form remains intact because of the conservation of Glu164-His165 pair. Recently, the crystal structure of rMnSODSeq has become available [13]. The rMnSODSeq structure shows its high structural resemblance to the other bacterial MnSODs, including MnSOD from E. coli. Most importantly, all aforementioned interactions and the amino acid targeted in the mutagenesis in this study are conserved. Therefore the present works remains relevant. Moreover, the findings may provide also structural information about other bacterial MnSODs.

rMnSODSeq resistance towards inactivation by UVC [14] is likely due to amino acids with an aromatic side chain, as the enzyme contains no cysteine residue. A relation between resistance towards inactivation by UV and the dimeric form of rMnSODSeq is unknown. Nevertheless, its resistance to UV is important for future application of the enzyme.

A Leu169Trp mutant was produced to evaluate whether the introduction of an amino acid with aromatic side chain could extend the enzyme resistance towards inactivation by UVC. The results suggest that both the rMnSODSeq and the Leu169Trp mutant retained 75–80% of their activity after 15 min exposure to UVC. The decrease in activity upon prolonged exposure to UVC of the mutant was similar to that of the native protein. However, the melting temperature (TM) of the mutant was different, suggesting that the structure of the enzyme had been altered by the mutation. More intriguingly, the TM and activity of the mutant were similar to those of a partially unfolded form [15] and the structure resembles a dimer with weakened monomers. Thus, we discovered an alternative monomeric form of the enzyme that as a dimer has similar activity and resistance towards inactivation by UVC. The present results further dissect the relation between the structure, the activity, and UV resistance of rMnSODSeq. In addition, the recently published crystal structure of rMnSODSeq shows the compactness of the enzyme globular structure. This may explain that the minor substitution in this study slightly affects the enzyme activity, but significantly impacts its structure. This finding indicates that enzyme modification for improvement of its characteristics could be a challenge.

Section snippets

Material and methods

All chemicals were purchased from Sigma (St. Louis, MO-USA) or Merck AG (Darmstadt, Germany) except when stated differently.

Structural model of rMnSODSeq and site directed mutagenesis

The in silico rMnSODSeq structural models from the employed protein modeling programs were highly similar, with variations present only at stretches 45–47 and 89–95 (rMnSODSeq numbering). These stretches are regions containing a loop that may adopt different orientations depending on the structure templates. These stretches were manually inspected and then finalized according to the most proposed orientation in the output models from the modeling programs and the highest likelihood from

Conflict of interests

The authors declare that there are no conflicts of interest.

Acknowledgement

This work is supported by Program Riset Inovasi ITB (2016). We thank Dr. H. J. Doddema for editing the manuscript.

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    1

    Present address: Molecular Biology and Proteomics Core Facilities, Indonesia Medical Education and Research Institute (IMERI), Faculty of Medicine, University of Indonesia, Salemba Raya No. 6, Jakarta 10430, Indonesia.

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