Enzymatic Activity Analysis and Catalytic Essential Residues Identification of Brucella abortus Malate Dehydrogenase
Malate dehydrogenase (MDH) plays important metabolic roles in bacteria. In this study, the recombinant MDH protein (His-MDH) of Brucella abortus was purified and its ability to catalyze the conversion of oxaloacetate (OAA) to L-malate (hereon referred to as MDH activity) was analyzed. Michaelis Cons...
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| Format: | Article |
| Language: | English |
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Wiley
2014-01-01
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| Series: | The Scientific World Journal |
| Online Access: | http://dx.doi.org/10.1155/2014/973751 |
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| author | Xiangan Han Yongliang Tong Mingxing Tian Yuxi Zhang Xiaoqing Sun Shaohui Wang Xusheng Qiu Chan Ding Shengqing Yu |
| author_facet | Xiangan Han Yongliang Tong Mingxing Tian Yuxi Zhang Xiaoqing Sun Shaohui Wang Xusheng Qiu Chan Ding Shengqing Yu |
| author_sort | Xiangan Han |
| collection | DOAJ |
| description | Malate dehydrogenase (MDH) plays important metabolic roles in bacteria. In this study, the recombinant MDH protein (His-MDH) of Brucella abortus was purified and its ability to catalyze the conversion of oxaloacetate (OAA) to L-malate (hereon referred to as MDH activity) was analyzed. Michaelis Constant (Km) and Maximum Reaction Velocity (Vmax) of the reaction were determined to be 6.45×10−3 M and 0.87 mM L−1 min−1, respectively. In vitro studies showed that His-MDH exhibited maximal MDH activity in pH 6.0 reaction buffer at 40°C. The enzymatic activity was 100%, 60%, and 40% inhibited by Cu2+, Zn2+, and Pb2+, respectively. In addition, six amino acids in the MDH were mutated to investigate their roles in the enzymatic activity. The results showed that the substitutions of amino acids Arg 89, Asp 149, Arg 152, His 176, or Thr 231 almost abolished the activity of His-MDH. The present study will help to understand MDH’s roles in B. abortus metabolism. |
| format | Article |
| id | doaj-art-ff1ba6930df8490baf4935dcfd479d76 |
| institution | Kabale University |
| issn | 2356-6140 1537-744X |
| language | English |
| publishDate | 2014-01-01 |
| publisher | Wiley |
| record_format | Article |
| series | The Scientific World Journal |
| spelling | doaj-art-ff1ba6930df8490baf4935dcfd479d762025-02-03T01:31:46ZengWileyThe Scientific World Journal2356-61401537-744X2014-01-01201410.1155/2014/973751973751Enzymatic Activity Analysis and Catalytic Essential Residues Identification of Brucella abortus Malate DehydrogenaseXiangan Han0Yongliang Tong1Mingxing Tian2Yuxi Zhang3Xiaoqing Sun4Shaohui Wang5Xusheng Qiu6Chan Ding7Shengqing Yu8Shanghai Veterinary Research Institute, The Chinese Academy of Agricultural Sciences (CAAS), 518 Ziyue Road, Shanghai 200241, ChinaShanghai Veterinary Research Institute, The Chinese Academy of Agricultural Sciences (CAAS), 518 Ziyue Road, Shanghai 200241, ChinaShanghai Veterinary Research Institute, The Chinese Academy of Agricultural Sciences (CAAS), 518 Ziyue Road, Shanghai 200241, ChinaShanghai Veterinary Research Institute, The Chinese Academy of Agricultural Sciences (CAAS), 518 Ziyue Road, Shanghai 200241, ChinaShanghai Veterinary Research Institute, The Chinese Academy of Agricultural Sciences (CAAS), 518 Ziyue Road, Shanghai 200241, ChinaShanghai Veterinary Research Institute, The Chinese Academy of Agricultural Sciences (CAAS), 518 Ziyue Road, Shanghai 200241, ChinaShanghai Veterinary Research Institute, The Chinese Academy of Agricultural Sciences (CAAS), 518 Ziyue Road, Shanghai 200241, ChinaShanghai Veterinary Research Institute, The Chinese Academy of Agricultural Sciences (CAAS), 518 Ziyue Road, Shanghai 200241, ChinaShanghai Veterinary Research Institute, The Chinese Academy of Agricultural Sciences (CAAS), 518 Ziyue Road, Shanghai 200241, ChinaMalate dehydrogenase (MDH) plays important metabolic roles in bacteria. In this study, the recombinant MDH protein (His-MDH) of Brucella abortus was purified and its ability to catalyze the conversion of oxaloacetate (OAA) to L-malate (hereon referred to as MDH activity) was analyzed. Michaelis Constant (Km) and Maximum Reaction Velocity (Vmax) of the reaction were determined to be 6.45×10−3 M and 0.87 mM L−1 min−1, respectively. In vitro studies showed that His-MDH exhibited maximal MDH activity in pH 6.0 reaction buffer at 40°C. The enzymatic activity was 100%, 60%, and 40% inhibited by Cu2+, Zn2+, and Pb2+, respectively. In addition, six amino acids in the MDH were mutated to investigate their roles in the enzymatic activity. The results showed that the substitutions of amino acids Arg 89, Asp 149, Arg 152, His 176, or Thr 231 almost abolished the activity of His-MDH. The present study will help to understand MDH’s roles in B. abortus metabolism.http://dx.doi.org/10.1155/2014/973751 |
| spellingShingle | Xiangan Han Yongliang Tong Mingxing Tian Yuxi Zhang Xiaoqing Sun Shaohui Wang Xusheng Qiu Chan Ding Shengqing Yu Enzymatic Activity Analysis and Catalytic Essential Residues Identification of Brucella abortus Malate Dehydrogenase The Scientific World Journal |
| title | Enzymatic Activity Analysis and Catalytic Essential Residues Identification of Brucella abortus Malate Dehydrogenase |
| title_full | Enzymatic Activity Analysis and Catalytic Essential Residues Identification of Brucella abortus Malate Dehydrogenase |
| title_fullStr | Enzymatic Activity Analysis and Catalytic Essential Residues Identification of Brucella abortus Malate Dehydrogenase |
| title_full_unstemmed | Enzymatic Activity Analysis and Catalytic Essential Residues Identification of Brucella abortus Malate Dehydrogenase |
| title_short | Enzymatic Activity Analysis and Catalytic Essential Residues Identification of Brucella abortus Malate Dehydrogenase |
| title_sort | enzymatic activity analysis and catalytic essential residues identification of brucella abortus malate dehydrogenase |
| url | http://dx.doi.org/10.1155/2014/973751 |
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