Fucoidan Sulfatases from Marine Bacterium Wenyingzhuangia fucanilytica CZ1127T
Abstract
:1. Introduction
2. Materials and Methods
2.1. Reagents
2.2. General Methods
2.3. Identification and Amino Acid Sequences Analysis of Sulfatases
2.4. Homology Modeling of SWF1 and SWF4
2.5. Cloning of Fucoidan Sulfatases SWF1 and SWF4
2.6. Production of Recombinant Fucoidan Sulfatases SWF1 and SWF4
2.7. Purification of Sulfatases SWF1 and SWF4
2.8. Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis of Proteins
2.9. Fucoidan Sulfatase Activity Assay
2.10. Determination of Substrate Specificity of Sulfatases SWF1 and SWF4
2.11. Determination of the pH Optimum for Sulfatases SWF1 and SWF4 Activity
2.12. Determination of the Optimal Temperature for Sulfatases SWF1 and SWF4 Activity
2.13. Influence of Different Compounds on SWF1 and SWF4 Activity
2.14. Preparation of Reaction Products for Nuclear Magnetic Resonance Analysis
2.15. Nuclear Magnetic Resonance Spectroscopy
3. Results
3.1. Amino Acid Sequence Analysis of SWF1 and SWF4
3.2. Expression and Purification of Fucoidan Sulfatases SWF1 and SWF4
3.3. Optimal Conditions for Catalytic Activity of SWF1 and SWF4
3.4. Substrate Specificity and Mode of Action of SWF1 and SWF4
4. Discussion
4.1. Identification of Putative Fucoidan Sulfatases in Marine Bacteria Wenyingzhuangia fucanilytica CZ1127T
4.2. Analysis of Amino Acid Sequences of Putative Fucoidan Sulfatases SWF1 and SWF4
4.3. Conservativeness of Sulfate-Binding S-Subsites of SWF1 and SWF4
4.4. Role of a Calcium Cation in Enzyme Activities of SWF1 and SWF4
4.5. Substrate Specificity of Sulfatases SWF1 and SWF4
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Conflicts of Interest
References
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Residue * | PDB: 5g2v | PDB: 4ug4 | PDB: 1hdh | SWF1 | SWF4 | Proposed Function |
---|---|---|---|---|---|---|
FGly/(modified residue) | Ser 77 | Cys 57 | Cys 51 | Cys 79 | Cys 78 | Catalytic nucleophile. Formation of sulfoenzyme intermediate complex. |
M | Ca2+ | Mn2+ | Ca2 | Ca2 | Ca2 | Coordination and stabilization of sulfate group of substrate. |
AsnA | Gln 350 | His 300 | Asn 318 | Asn 283 | Asn 311 | Coordination of metal ion. |
AspA | Asp 349 | Asp 299 | Asp 317 | Asp 282 | Asp 310 | Coordination of metal ion and activation of FGly. |
AspB | Asp 37 | Asp 17 | Asp 13 | Asp 40 | Asp 38 | Coordination of metal ion. |
AspC | Asp 38 | Gln 18 | Asp14 | Asp 41 | Asp 39 | Coordination of metal ion. |
LysA | Lys 125 | Lys 105 | Lys 113 | Lys 130 | Lys 128 | Sulfate binding and stabilisation of FGly. |
LysB | Lys 362 | Lys 312 | Lys 375 | Lys 308 | Lys 323 | Sulfate binding and protonation of ester group of substrate. |
ArgA | Arg 81 | Arg 61 | Arg 55 | Arg 83 | Arg 82 | Stabilization of FGly. |
HisA | His 127 | His 107 | His 115 | His 132 | (Gly 130) | Deprotonation of FGly, elimination of sulfoenzyme intermediate complex. |
HisB | His 199 | His 204 | His 211 | His 223 | His 213 | Sulfate binding and protonation of ester group of substrate. |
Position | δC, Type | δH (J in Hz) | ROESY | HMBCα |
---|---|---|---|---|
1 | 96.8, CH | 5.06, d (3.9) | 2, 9, 10 | 3, 5, 9 |
2 | 69.4, CH | 3.79, dd (9.9, 3.8) | 1 | 3 |
3 | 70.7, CH | 3.96, dd (10.3, 3.4) | 4, 5 | 1, 2, 4 |
4 | 73.3, CH | 3.83, d (3.4) | 3, 5, 6 | 3, 5 |
5 | 68.1, CH | 4.46, m | 3, 4, 6 | 1, 4, 6 |
6 | 16.5, CH3 | 1.21, d (6.6) | 4, 5 | 5 |
7 | 100.3, CH | 5.27, d (3.3) | 8, 16, 18 | 9, 11, 16 |
8 | 74.6, CH | 4.57, dd (10.3, 3.6) | 7 | 9 |
9 | 72.9, CH | 4.18, dd (10.8, 3.1) | 1, 10, 11 | 1, 7, 8, 10 |
10 | 70.1, CH | 4.11, d (2.2) | 1, 9, 11, 12 | 9, 11 |
11 | 68.6, CH | 4.44, m | 9, 10, 12 | 7, 10, 12 |
12 | 16.5, CH3 | 1.24, d (6.6) | 10, 11 | 11 |
13 | 95.4, CH | 5.34, d (3.9) | 14, 21, 22 | 15, 17, 21 |
14 | 76.6, CH | 4.49, dd (10.5, 3.8) | 13 | 15, 16 |
15 | 68.6, CH | 4.16, dd (10.9, 2.9) | 16, 17 | 13, 14, 16 |
16 | 83.7, CH | 3.99, d (2.6) | 7, 15, 17, 18 | 7, 14, 15, 16 |
17 | 68.9, CH | 4.52, m | 15, 16, 18 | 13, 17, 18 |
18 | 16.8, CH3 | 1.38, d (6.8) | 7, 16, 17 | 17 |
19 | 91.7, CH | 5.48, d (3.9) | 20 | 21, 23 |
20 | 74.7, CH | 4.51, dd (10.2, 3.2) | 19 | 21 |
21 | 73.9, CH | 4.05, dd (9.9, 3.1) | 13, 23 | 13, 19, 20 |
22 | 69.9, CH | 4.08, d (3.2) | 13, 23, 24 | 23 |
23 | 67.2, CH | 4.23, q (13.2, 6.6) | 21, 22, 24 | 19, 22, 24 |
24 | 16.7, CH3 | 1.24, d (6.6) | 22, 23 | 23 |
Position | δH (J in Hz) | ROESY |
---|---|---|
1 | 5.36, d (4.6) | 2, 9, 10 |
2 | 4.46, dd (9.5, 4.1) | 1 |
3 | 4.11, m | 5 |
4 | 3.90, d (4.2) | 5 |
5 | 4.51, m | 3, 4 |
6 | 1.23, d (6.0) | |
7 | 5.28, d (3.7) | 8, 16, 18 |
8 | 4.57, m | 7 |
9 | 4.19, dd (10.4, 3.2) | 11 |
10 | 4.11, m | 11 |
11 | 4.43, m | 9, 10 |
12 | 1.29, d (6.5) | |
13 | 5.37, d (4.4) | 21, 22 |
14 | 4.65, m | |
15 | 4.76, dd (11.2, 2.8) | 16, 17 |
16 | 4.28, d (2.9) | 7, 15, 17 |
17 | 4.55, m | 15, 16 |
18 | 1.40, d (6.8) | 7 |
19 | 5.50, d (3.9) | 20 |
20 | 4.54, m | 19 |
21 | 4.06, dd (10.2, 3.2) | 13, 23 |
22 | 4.09, d (4.8) | 13, 23 |
23 | 4.23, m | 21, 22 |
24 | 1.24, d (6.4) |
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Silchenko, A.S.; Rasin, A.B.; Zueva, A.O.; Kusaykin, M.I.; Zvyagintseva, T.N.; Kalinovsky, A.I.; Kurilenko, V.V.; Ermakova, S.P. Fucoidan Sulfatases from Marine Bacterium Wenyingzhuangia fucanilytica CZ1127T. Biomolecules 2018, 8, 98. https://doi.org/10.3390/biom8040098
Silchenko AS, Rasin AB, Zueva AO, Kusaykin MI, Zvyagintseva TN, Kalinovsky AI, Kurilenko VV, Ermakova SP. Fucoidan Sulfatases from Marine Bacterium Wenyingzhuangia fucanilytica CZ1127T. Biomolecules. 2018; 8(4):98. https://doi.org/10.3390/biom8040098
Chicago/Turabian StyleSilchenko, Artem S., Anton B. Rasin, Anastasiya O. Zueva, Mikhail I. Kusaykin, Tatiana N. Zvyagintseva, Anatoly I. Kalinovsky, Valeriya V. Kurilenko, and Svetlana P. Ermakova. 2018. "Fucoidan Sulfatases from Marine Bacterium Wenyingzhuangia fucanilytica CZ1127T" Biomolecules 8, no. 4: 98. https://doi.org/10.3390/biom8040098