/
results.html
272 lines (253 loc) · 13.6 KB
/
results.html
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
61
62
63
64
65
66
67
68
69
70
71
72
73
74
75
76
77
78
79
80
81
82
83
84
85
86
87
88
89
90
91
92
93
94
95
96
97
98
99
100
101
102
103
104
105
106
107
108
109
110
111
112
113
114
115
116
117
118
119
120
121
122
123
124
125
126
127
128
129
130
131
132
133
134
135
136
137
138
139
140
141
142
143
144
145
146
147
148
149
150
151
152
153
154
155
156
157
158
159
160
161
162
163
164
165
166
167
168
169
170
171
172
173
174
175
176
177
178
179
180
181
182
183
184
185
186
187
188
189
190
191
192
193
194
195
196
197
198
199
200
201
202
203
204
205
206
207
208
209
210
211
212
213
214
215
216
217
218
219
220
221
222
223
224
225
226
227
228
229
230
231
232
233
234
235
236
237
238
239
240
241
242
243
244
245
246
247
248
249
250
251
252
253
254
255
256
257
258
259
260
261
262
263
264
265
266
267
268
269
270
271
272
<!DOCTYPE html>
<html>
<head>
<!-- Basic -->
<meta charset="utf-8" />
<meta http-equiv="X-UA-Compatible" content="IE=edge" />
<!-- Mobile Metas -->
<meta name="viewport" content="width=device-width, initial-scale=1, shrink-to-fit=no" />
<!-- Site Metas -->
<meta name="keywords" content="" />
<meta name="description" content="" />
<meta name="author" content="" />
<title>Results</title>
<!-- slider stylesheet -->
<link rel="stylesheet" type="text/css" href="https://cdnjs.cloudflare.com/ajax/libs/OwlCarousel2/2.1.3/assets/owl.carousel.min.css" />
<!-- bootstrap core css -->
<link rel="stylesheet" type="text/css" href="css/bootstrap.css" />
<!-- fonts style -->
<link href="https://fonts.googleapis.com/css?family=Baloo+Chettan|Dosis:400,600,700|Poppins:400,600,700&display=swap" rel="stylesheet" />
<!-- Custom styles for this template -->
<link href="css/style.css" rel="stylesheet" />
<!-- responsive style -->
<link href="css/responsive.css" rel="stylesheet" />
</head>
<body>
<div class="hero_area">
<!-- <!– header section strats –>-->
<!-- <div class="brand_box">-->
<!-- <a class="navbar-brand" href="index.html">-->
<!-- <span>-->
<!-- Ninom-->
<!-- </span>-->
<!-- </a>-->
<!-- </div>-->
<!-- <!– end header section –>-->
</div>
<!-- slider section -->
<section class=" slider_section position-relative">
<div id="carouselExampleControls" class="carousel slide " data-ride="carousel">
<div class="carousel-inner">
<div class="carousel-item active">
<div class="img-box">
<img src="images/bgpic1.png" alt="">
</div>
</div>
<!-- <div class="carousel-item">-->
<!-- <div class="img-box">-->
<!-- <img src="images/bgpic1.png" alt="">-->
<!-- </div>-->
<!-- </div>-->
<!-- <div class="carousel-item">-->
<!-- <div class="img-box">-->
<!-- <img src="images/bgpic1.png" alt="">-->
<!-- </div>-->
<!-- </div>-->
</div>
<!-- <a class="carousel-control-prev" href="#carouselExampleControls" role="button" data-slide="prev">-->
<!-- <span class="sr-only">Previous</span>-->
<!-- </a>-->
<!-- <a class="carousel-control-next" href="#carouselExampleControls" role="button" data-slide="next">-->
<!-- <span class="sr-only">Next</span>-->
<!-- </a>-->
</div>
</section>
<!-- end slider section -->
<!-- nav section -->
<section class="nav_section">
<div class="container">
<div class="custom_nav2">
<nav class="navbar navbar-expand custom_nav-container ">
<button class="navbar-toggler" type="button" data-toggle="collapse" data-target="#navbarSupportedContent" aria-controls="navbarSupportedContent" aria-expanded="false" aria-label="Toggle navigation">
<span class="navbar-toggler-icon"></span>
</button>
<div class="collapse navbar-collapse" id="navbarSupportedContent">
<div class="d-flex flex-column flex-lg-row align-items-center">
<ul class="navbar-nav ">
<li class="nav-item active" style="font-size: 20px">
<a class="nav-link" href="project.html">Project </a>
</li>
<li class="nav-item active" style="font-size: 20px">
<a class="nav-link" href="introduction.html">Introduction <span class="sr-only">(current)</span></a>
</li>
<li class="nav-item" style="font-size: 20px">
<a class="nav-link" href="results.html">Results </a>
</li>
<li class="nav-item" style="font-size: 20px">
<a class="nav-link" href="poster.html">Poster </a>
</li>
<li class="nav-item" style="font-size: 20px">
<a class="nav-link" href="index.html">Home </a>
</li>
</ul>
</div>
</div>
</nav>
</div>
</div>
</section>
<!-- end nav section -->
<!-- about section -->
<section class="about_section layout_padding">
<div>
<h1 align="center" style="font-size: 60px">Results and Discussions </h1>
</div>
<div class="container-fluid">
<div align="center">
<hr>
<h2 id="result1">
Cytotoxic activity evaluation of malonylated GPA
</h2>
<img src="images/result1.jpg" alt="">
<h5 align="center">Figure 3. Cytotoxic activity evaluation of malonylated GPA.</h5>
</div>
<div class="detail-box">
<p align="left" style="font-size: 25px">
Previously, we identified BmmI could transfer malonyl group onto the anti-tumor compound GPA (Fig 1, 2). We optimized the reaction conditions, and found the convention rate could be ~50% in the presence of 20 μM enzyme (Fig 3A, 3B). To evaluate whether malonyl group could influence the cytotoxic activity of GPA, we thought to use fluorescent tumor cells to measure the cytotoxic activity by observing their density and morphology. We chose human fibrosarcoma cells HT1080 expressing the enhanced green fluorescent protein (EGFP), and respectively incubated it with a negative control without GPA, GPA without BmmI, and GPA with 20 μM of BmmI. After 24 hours, the density and morphology of the fluorescent cells were observed using fluorescence microscope. As shown in Fig 3C, compared to that of the negative control, the densities of the fluorescent tumor cells have reduced dramatically in the presence of GPA with or without BmmI, indicating both GPA and malonylated GPA have cytotoxic activity against HT1080. Furthermore, the density of fluorescent tumor cells in the presence of GPA and 20 μM BmmI (generating ~50% malonylated GPA) is much lower than that with the addition of GPA only. These results indicated that malonylation could improve the cytotoxic activity of GPA, prompting us to engineer BmmI by directed evolution for higher catalytic efficiency toward GPA.
</p>
</div>
</div>
</section>
<hr>
<section class="about_section layout_padding">
<div class="container-fluid">
<div align="center">
<h2 id="result2">
Identifying the candidate residues of BmmI for directed evolution
</h2>
<img src="images/result2.jpg" alt="">
<h5 align="center">Figure 4. The binding mode of GPA with BmmI (PDB id: 6KJH).</h5>
</div>
<div class="detail-box">
<p align="left" style="font-size: 25px">
Previously we elucidated the crystal structure of BmmI (PDB id: 6KJH), which belongs to the structures of BLRE family enzymes [7]. To find out the possible binding residues of BmmI with GPA (2), we built the models for BmmI/GPA (2) through computer-aided molecular docking. As shown in Fig 4, GPA is located at the catalytic cavity of BmmI; the sugar moiety forms hydrogen bonds with Glu70, Ser238, His219 and His229, and the oxygen at the C3' of pyridine moiety hydrogen bonds with Arg292; in addition, the aliphatic chain of GPA is stabilized by nonpolar interactions with Phe346, Trp324, Phe287, Phe288 and Phe127. Based on the substrate binding mode, these residues were deduced to be potentially related to the catalytic activity of BmmI toward GPA. Therefore, we selected Glu70, Ser238, His219, His229, Arg292, Phe346, Trp324, Phe287, Phe288 and Phe127 for further directed evolution study.
</p>
</div>
</div>
</section>
<hr>
<section class="about_section layout_padding">
<div class="container-fluid">
<div align="center">
<h2 id="result3">
Structure-based virtual screening of Bmml
</h2>
<img src="images/result3.png" alt="">
<h5 align="center">Table 1. Mutation sites of the top-ranking 9 mutants with high binding affinities toward GPA</h5>
</div>
<div class="detail-box">
<p align="left" style="font-size: 25px">
We constructed a virtual enzyme library of 190 saturated mutants of Glu70, Ser238, His219, His229, Arg292, Phe346, Trp324, Phe287, Phe288 and Phe127 using Molecular Operating Environment (MOE). To select the possible hints with higher catalytic efficiency, we performed docking-based virtual screening. We measured the binding affinities of these mutants with GPA, and their protein stabilities. As shown in Table 1, the top-ranking 9 mutants with high binding affinities toward GPA, and good stabilities were selected for further study. </p>
</div>
</div>
</section>
<hr>
<section class="about_section layout_padding">
<div class="container-fluid">
<div align="center">
<h2 id="result4">
Construction and activity tests of mutants
</h2>
<img src="images/result4.png" alt="">
<h5 align="center">Figure 5. The relative activities of the crude enzymes of BmmI and its variants.</h5>
</div>
<div class="detail-box">
<p align="left" style="font-size: 25px">
We then constructed the nine mutants W324A, F127G, F288A, H229A, F287P, H219A, H219T, R292D and H346A. Reverse complementary primers with mutation sites were designed to amplify the linear expression plasmids (pET28a/bmmI) with mutated sequences (Table S2). The resulting linear plasmids were ligated to circular molecules by seamless cloning, and then introduced into E. coli Rossetta (DE3). The expression strains were individually cultured, followed by addition of 0.2 mM IPTG to induce protein expression (Fig S1). Crude enzymes from each expression strain were incubated with GPA and malonyl-CoA for 2 h, and the generation of malonylated GPA was detected by HPLC. As shown in Fig 5, compared to that of the wild-type BmmI, the catalytic activities of W324A and F288A toward malonyl-CoA and GPA increased by ~15% and 10%. </div>
</div>
</section>
<hr>
<section class="about_section layout_padding">
<div class="container-fluid">
<div align="center">
<h2 id="result5">
In vitro characterization of the BmmI variant W324A with other acyl donors
</h2>
<!-- <img src="images/result5.jpg" alt="">-->
<img src="images/result5.png" alt="">
<h5 align="center">Figure 6. In vitro characterization of the BmmI variant W324A with different acyl donors.</h5>
</div>
<div class="detail-box">
<p align="left" style="font-size: 25px">
Furthermore, we tested the catalytic activities of W324A toward different acyl donors. We chose carboxyl-terminated methylmalonyl-CoA and succinyl-CoA as acyl donors. As shown in Fig 6, W324A could completely transform GPA (2) to methylmalonylated GPA (4, Fig 6A, 6B); and exhibited ~ 50% conversion rate to succinylated GPA (5, Fig 6C, 6D). These results indicated that the W324A mutant serves as an efficient enzyme for the malonylation/methylmalonylation/succinylation of GPA.
</div>
</div>
</section>
<hr>
<section class="about_section layout_padding">
<div class="container-fluid">
<div align="center">
<h2 id="conclusion">
Conclusion
</h2>
<!-- <img src="images/result4.jpg" alt="">-->
<!-- <h5 align="center">Figure 5. The relative activities of the crude enzymes of BmmI and its variants.</h5>-->
</div>
<div class="detail-box">
<p align="left" style="font-size: 25px">
Acyltransfer is of crucial importance for the modification of many secondary metabolites. The Bacillus-derived acyltransferase BmmI could catalyze the malonylation of the anti-tumor agent GPA. In this project, we identified the malonyl group could improve the cytotoxic activity of GPA. By using MOE, we constructed a virtual enzyme library of 190 saturated mutants of the binding residues, and selected top9 mutants with highest binding affinities by structure-based virtual screening. The mutant W324A exhibited ~15% increased malonyltransfer activity compared to that of the wild-type BmmI, and exhibited high catalytic efficiency with methylmalonyl-CoA and succinyl-CoA. Given that malonylation could improve the cytotoxic activity of GPA, BmmI could be exploited as a tool enzyme in expanding the chemical diversity of these compounds with better bioactivities.
</div>
</div>
</section>
<!-- end about section -->
<!-- info section -->
<section class="info_section layout_padding">
<div class="container">
<div class="info_logo">
<h2>
OUC-Marine Drugs
</h2>
</div>
<div class="info_contact">
<div class="row">
<div class="col-md-4">
<a href="">
<img src="images/location.png" alt="">
<span>
Ocean University of China, Qingdao 266003, China
</span>
</a>
</div>
<div class="col-md-4">
<a href="">
<span>
</span>
</a>
</div>
<div class="col-md-4">
<a href="">
<img src="images/mail.png" alt="">
<span>
xiaofei3450@ouc.edu.cn
</span>
</a>
</div>
</div>
</div>
<div class="row">
</div>
</div>
</section>
<!-- end info section -->
<!-- footer section -->
<section class="container-fluid footer_section">
<p>
© <span id="displayYear"></span> All Rights Reserved. Design by Ocean University of China
</p>
</section>
<!-- footer section -->
<script type="text/javascript" src="js/jquery-3.4.1.min.js"></script>
<script type="text/javascript" src="js/bootstrap.js"></script>
<script type="text/javascript" src="js/custom.js"></script>
</body>
</html>