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Introduction
Uric acid is the end product
of purine metabolism in the body.
Overproduction and obstruction of uric acid production and excretion can lead to hyperuricemia
.
In recent years, the prevalence of hyperuricemia has been on
the rise due to increased intake of high-purine foods.
Uric acid nephropathy has also been reported to be associated
with hyperuricemia.
Reducing the intake of high-purine foods, inhibiting the synthesis of uric acid or promoting the excretion of uric acid can effectively control the level of uric acid in
the blood in the body.
Xanthine oxidase is a liver enzyme that catalyzes the oxidation of hypoxanthines to xanthines and xanthines to uric acid
.
Therefore, xanthine oxidase is a key molecular target for the development of drugs for the treatment of
hyperuricemia.
Xanthine oxidase inhibitors (allopurinol, febuxostat) are widely used in the treatment of hyperuricemia
.
But allopurinol and febuxostat have certain side effects
.
Therefore, the identification of xanthine oxidase inhibitors from food has become the mainstay of the
treatment of gout.
Some xanthine oxidase inhibitory peptides
have been isolated from seafood such as bonito, tuna, and shark cartilage.
Egg whites are an excellent source
of bioactive peptides.
Previous studies have successfully identified bioactive peptides such as antioxidant peptides, immunoactive peptides, and antimicrobial peptides
from ovalbumin, oomucin, and lysozyme.
Thus, ovalbumin, ooslime, and lysozyme can be used to screen for potential xanthine oxidase inhibitory peptides
.
However, traditional bioactive peptide separation and purification methods are time-consuming and
costly.
Many studies have demonstrated the effectiveness of virtual screening, so virtual screening is an effective alternative to traditional enzymatic digestion
.
Associate Professor Yu Zhipeng and Associate Professor Zhao Wenzhu of the School of Food Science and Engineering of Hainan University identified the new xanthine oxidase inhibitory peptide by combining virtual screening, molecular docking and in vitro activity verification using egg protein as raw material, and revealed the molecular mechanism
of xanthine oxidase and xanthine oxidase inhibitory peptide interaction.
This work may provide new ideas
for the application of egg protein active peptides in the treatment of hyperuricemia.
Results and Discussion
Virtual screening results of active peptides
Virtual screening is a fast, reliable, and efficient bioactive peptide screening method
.
A total of 62 tripeptides, tetrapeptides and pentapeptides
were obtained from the hydrolysates of egg albumin, ootic mucin, and lysozyme.
Among the 36 active peptides with good water solubility and non-toxicity, 28 peptides successfully docked with xanthine oxidase, and their CDOCKER-Energy scores are shown in Table 1
.
Lower CDOCKER-Energy scores indicate that the active peptide binds tightly to xanthine oxidases, resulting in a more favorable conformation
.
The CDOCKER-Energy scores of active peptides EEK, TNDC, EGK, EER and DNEC were lower, -96.
88, -86.
44, -86.
30, -84.
04 and -82.
55 kcal/mol, respectively, which were lower than the positive control allopurinol (5.
91 kcal/mol) and febuxostat (31.
62 kcal/mol).
Therefore, in vitro activity studies
were carried out on active peptides EEK, TNDC, EGK, EER and DNEC.
.
At a concentration of 0.
50 mg/mL, the xanthine oxidase inhibition rate of active peptide EEK was the highest (73.
67%), and the xanthine oxidase inhibition rate of active peptide TNDC, EGK, EER and DNEC was 4.
38%, 10.
49%, 5.
41% and 13.
24%,
respectively.
Therefore, we chose the active peptide EEK for follow-up research
.
The active peptide EEK was located in residue 289 ~ 291 of ovalbumin and exhibited the best xanthine oxidase inhibitory activity with an IC50 value of 141 μmol/L (0.
40 mg/mL).
。 The inhibitory activity of xanthine oxidase was lower than that of the inhibitor allopurinol (IC50 = 10.
10 μg/mL), but significantly better than the previously reported xanthine oxidase inhibitory peptide WPPKN (IC50 = 17.
75 mg/mL), ADIYTE (IC50 = 19.
01 mg/mL), and EEAK (IC50 = 0.
58 mg/mL).
。 Table 1 Water-solubility, toxicity prediction of polypeptide and docking results with xanthine oxidase Study on the interaction mechanism of tripeptide EEK and xanthine oxidase Xanthine oxidase is a dimer, each monomer has an active site, Glu802, Glu1261 and Arg880 residues in the molybdenum center play a key role in catalyzing xanthine oxidation, and Leu648, Phe649, Phe914, Phe1009, Val1011, Residues such as Phe1013 and Leu1014 play a key role
in catalyzing xanthine oxidation.
The optimal docking posture for EEK with xanthine oxidase is shown
in Figure 1.
EEK and xanthine oxidases are stabilized into bonds
by three carbon-hydrogen bond interactions (the carbon-hydrogen bond interaction is a weaker hydrogen bond where the donor is a polarized carbon atom), two salt bridges, five conventional hydrogen bonds, and four charge attraction.
Two carbon-hydrogen bond interactions are formed between the hydrogen atoms (HB1 and HB2) of the xanthine oxidase residue Ser876 and the oxygen atom of EEK (O17), both with a length of 2.
82 å
.
Carbon-hydrogen bonding
is also formed between the hydrogen atom (HA) of the amino acid residue Phe1009 and the oxygen atom (O14) of EEK (2.
45 Å).
The oxygen atoms (OE2) of xanthine oxidase residue Glu879 and the oxygen atoms of Glu802 (OE1) interact with EEK via salt bridges at distances of 1.
82 Å and 1.
87 Å
, respectively.
The H52, O15, O14, O15, and O30 of EEK interact with the nitrogen atom (NE2) of His875, the hydrogen atom of Arg880 (HE, HH21), the hydrogen atom of Thr1010 (HG1), and the hydrogen atom of Asn768 (HD22) on xanthine oxidase
, respectively.
Four charge attractiones
were found in the EEK-xanthine oxidase complex.
The first is composed of oxygen atoms (OE2) of Glu879 residues on xanthine oxidase and hydrogen atoms (H51) of EEK with a length of 1.
82 Å; The second is the nitrogen atom (NH2) of the residue Arg880 on the xanthine oxidase and the oxygen atom (O15) of EEK with a length of 3.
35 Å; The third is the oxygen atom (OE1) of the residue Glu802 on the xanthine oxidase and the hydrogen atom (H3) of EEK with a length of 1.
87 Å; The fourth is the nitrogen atom (NZ) of residue Lys771 and the oxygen atom (O30) of EEK with a length of 4.
70 å
.
Analysis of previous studies of the structure of xanthine oxidase suggests that Glu802, Phe1009 and Arg880 on xanthine oxidase may play a major role
in xanthine oxidase binding.
Tripeptide EEK has advantages
over quercetin, the proligand in xanthine oxidase, in binding to amino acid residues predicted in xanthine oxidase (Glu802, Phe1009, Arg880).
Quercetin forms two unfavorable chemical bonds with Glu802 of xanthine oxidase, while tripeptide EEK binds
stably to Glu802 through salt bridges and charge attraction.
Quercetin is linked
to Phe1009 of xanthine oxidase through two Pi-Pi-T type interactions.
In contrast, tripeptide EEK binds
to the Phe1009 bond of xanthine oxidase by hydrocarbon bonding.
Quercetin binds to Arg880 of xanthine oxidase through traditional hydrogen bond interactions, and the tripeptide EEK forms a charge attraction
with Arg880.
The number of carbon-hydrogen bond interactions and charge attraction formed by the ligation of tripeptide EEK with xanthine oxidase were higher than those of allopurinol, febuxostat, EEAK, FH, DN and YLD (Figure 2).
Therefore, carbon-hydrogen bond interactions and charge attraction may be key
to screening xanthine oxidase inhibitors.
(a) is the three-dimensional structure of the EEK-xanthine oxidase complex, red is the xanthine oxidase residue amino acid, black represents the atomic number in the xanthine oxidase, and blue represents the atomic number
in the peptide EEK.
(b) The figure shows a 2D schematic of the EEK-xanthine oxidase molecular interaction
.
Light blue represents carbon-hydrogen bonds, green represents regular hydrogen bonds, orange represents salt bridges and electrostatic interactions, and red represents adverse negative-negative interactions
.
Fig.
1 Interaction between EEK and xanthine oxidase docking
(a) The docking of the original ligand quercetin to xanthine oxidase
.
Green represents regular hydrogen bonds
.
Light blue represents pi-donor hydrogen bonds
.
Bright pink indicates the interaction between Pi-Pi stacking and Pi-Pi T-shape
.
Pink represents π-alkyl interactions
.
Purple represents interactions
.
Red represents adverse collisions and adverse receptor-receptor interactions
.
(b), (c), (d), (e), (f) (g) are molecular interactions
of allopurinol, febuxostat, polypeptides EEAK, FH, DN and YLD with xanthine oxidase, respectively.
Fig.
2 Interaction of active peptide with xanthine oxidase
Conclusion
In this study, a tripeptide EEK with xanthine oxidase inhibitory activity was identified from egg protein with an IC50 value of 141 μmol/L
.
Tripeptide EEK binds to xanthine oxidase active centers by three carbon-hydrogen bonds, two salt bridges, five conventional hydrogen bonds
, and four attraction charge forces.
Glu802, Phe1009 and Arg880 are key amino acids
in the interaction of tripeptide EEK with xanthine oxidase.
Carbon-hydrogen bond interactions and charge attraction play a major role
in EEK interactions with key amino acid residues (Glu802, Phe1009 and Arg880) of xanthine oxidases.
Tripeptide EEK is a promising natural xanthine oxidase inhibitor for the control of hyperuricemia
.
First author bio
D.
, Associate Professor, Master Supervisor
.
The research mainly focuses on the enzymatic hydrolysis preparation and structure identification of ACE inhibitory peptide (hypotensive active peptide), hypoglycemic active peptide and anti-Alzheimer's active peptide.
Research on the mechanism of action and homeostatic protection mechanism based on molecular simulation; To explore the mechanism
of action of active peptides in vivo based on food omics technology.
At present, 45 SCI search papers have been published as the first author/corresponding author (including 21 papers in the first division of the JCR category of the Chinese Academy of Sciences); He has applied for 50 national invention patents related to functional factors such as active peptides, of which 16 patents have been authorized by the first inventor and 1 has completed the transformation of
achievements.
About the corresponding author
Wenzhu Zhao, Ph.D.
, associate professor, master supervisor
.
At present, the main research direction is the identification of active factors and glycoprotein structures of fruit-aided targeted screening of fruits and vegetables and the development of functional products of fruits and vegetables with the help of computer-aided targeting.
He presided over the projects of the National Natural Science Foundation of China and the Provincial Department of Education, participated in the special projects of the National Key R&D Program and the Provincial Department of Science and Technology, and won a number of provincial natural academic achievement awards and municipal natural academic achievement awards
from 2014 to 2019.
He has published more than 50 scientific papers as the first or corresponding author, including more than 20 papers retrieved by SCI; Authorized 5 national invention patents related to fruit and vegetable glycoprotein, active polypeptides, etc.
, and completed 1 transformation of
achievements.
He is currently a reviewer
for SCI-searched journals such as Food Chemistry, Food Hydrocolloids and Journal of the Science of Food and Agriculture.
Identification of egg protein-derived peptides as xanthine oxidase inhibitors: virtual hydrolysis, molecular docking, and in vitro activity evaluation
Zhipeng Yua, Yaxin Caob, Ruotong Kanb, Huizhuo Jib, Wenzhu Zhaoa,*, Sijia Wuc, Jingbo Liuc, David Shiuand
a School of Food Science and Engineering, Hainan University, Haikou 570228, China
b College of Food Science and Engineering, Bohai University, Jinzhou 121013, China
c Lab of Nutrition and Functional Food, Jilin University, Changchun 130062, China
d Institute of Drug Discovery Technology, Ningbo University, Ningbo 315211, China
*Corresponding author.
E-mail address: zhaowenzhu777@163.
com
The purpose of this study was to screen the xanthine oxidase (XO) inhibitory peptides from egg white proteins through virtual hydrolysis, in vitro activity validation, and molecular docking.
The results demonstrated that tripeptide EEK from ovalbumin exhibited potent XO inhibitory activity with an IC50 value of 141 µmol/L.
The molecular docking results showed that tripeptide EEK bound with the active center of XO via 3 carbon hydrogen bond interactions, 2 salt bridges, 5 conventional hydrogen bond interactions, and 4 attractive charge interactions.
The residues Glu802, Phe1009, and Arg880 may play key roles in the XO catalytic reaction.
Especially, the key intermolecular forces of inhibiting XO activity may be special type of hydrogen bonds including carbon hydrogen bond interactions and attraction charge interactions.
The novel tripeptide EEK is potential candidates for controlling hyperuricemia.
Reference:
YU Z P, CAO Y X, KAN R T, et al.
Identification of egg protein-derived peptides as xanthine oxidase inhibitors: virtual hydrolysis, molecular docking, and in vitro activity evaluation[J].
Food Science and Human Wellness, 2022, 11(6): 1591-1597.
DOI:10.
1016/j.
fshw.
2022.
06.
017.
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