α-Lys424 Participates in Insertion of FeMoco to MoFe Protein and Maintains Nitrogenase Activity in Klebsiella oxytoca M5al
Our previous investigation of substrates reduction catalyzed by nitrogenase suggested that α-Ile423 of MoFe protein possibly functions as an electron transfer gate to Mo site of active center-“FeMoco”. Amino acid residue α-Lys424 connects directly to α-Ile423, and they are located in the same α-heli...
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Frontiers Media S.A.
2019-04-01
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| Series: | Frontiers in Microbiology |
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| Online Access: | https://www.frontiersin.org/article/10.3389/fmicb.2019.00802/full |
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| author | Lina Song Pengxi Liu Wei Jiang Qingjuan Guo Chunxi Zhang Abdul Basit Ying Li Jilun Li |
| author_facet | Lina Song Pengxi Liu Wei Jiang Qingjuan Guo Chunxi Zhang Abdul Basit Ying Li Jilun Li |
| author_sort | Lina Song |
| collection | DOAJ |
| description | Our previous investigation of substrates reduction catalyzed by nitrogenase suggested that α-Ile423 of MoFe protein possibly functions as an electron transfer gate to Mo site of active center-“FeMoco”. Amino acid residue α-Lys424 connects directly to α-Ile423, and they are located in the same α-helix (α423-431). In the present study, function of α-Lys424 was investigated by replacing it with Arg (alkaline, like Lys), Gln (neutral), Glu (acidic), and Ala (neutral) through site-directed mutagenesis and homologous recombination. The mutants were, respectively, termed 424R, 424Q, 424E, and 424A. Studies of diazotrophic cell growth, cytological, and enzymatic properties indicated that none of the substitutions altered the secondary structure of MoFe protein, or normal expression of nifA, nifL, and nifD. Substitution of alkaline amino acid (i.e., 424R) maintained acetylene (C2H2) and proton (H+) reduction activities at normal levels similar to that of wild-type (WT), because its FeMoco content did not reduce. In contrast, substitution of acidic or neutral amino acid (i.e., 424Q, 424E, 424A) impaired the catalytic activity of nitrogenase to varying degrees. Combination of MoFe protein structural simulation and the results of a series of experiments, the function of α-Lys424 in ensuring insertion of FeMoco to MoFe protein was further confirmed, and the contribution of α-Lys424 in maintaining low potential of the microenvironment causing efficient catalytic activity of nitrogenase was demonstrated. |
| first_indexed | 2024-12-23T10:33:14Z |
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| id | doaj.art-56df7a40be574038aeec2e6900ca5bad |
| institution | Directory Open Access Journal |
| issn | 1664-302X |
| language | English |
| last_indexed | 2024-12-23T10:33:14Z |
| publishDate | 2019-04-01 |
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| series | Frontiers in Microbiology |
| spelling | doaj.art-56df7a40be574038aeec2e6900ca5bad2022-12-21T17:50:22ZengFrontiers Media S.A.Frontiers in Microbiology1664-302X2019-04-011010.3389/fmicb.2019.00802446517α-Lys424 Participates in Insertion of FeMoco to MoFe Protein and Maintains Nitrogenase Activity in Klebsiella oxytoca M5alLina Song0Pengxi Liu1Wei Jiang2Qingjuan Guo3Chunxi Zhang4Abdul Basit5Ying Li6Jilun Li7State Key Laboratory of Agrobiotechnology, College of Biological Sciences, China Agricultural University, Beijing, ChinaState Key Laboratory of Agrobiotechnology, College of Biological Sciences, China Agricultural University, Beijing, ChinaState Key Laboratory of Agrobiotechnology, College of Biological Sciences, China Agricultural University, Beijing, ChinaState Key Laboratory of Agrobiotechnology, College of Biological Sciences, China Agricultural University, Beijing, ChinaLaboratory of Photochemistry, Institute of Chemistry, Chinese Academy of Sciences, Beijing, ChinaState Key Laboratory of Agrobiotechnology, College of Biological Sciences, China Agricultural University, Beijing, ChinaState Key Laboratory of Agrobiotechnology, College of Biological Sciences, China Agricultural University, Beijing, ChinaState Key Laboratory of Agrobiotechnology, College of Biological Sciences, China Agricultural University, Beijing, ChinaOur previous investigation of substrates reduction catalyzed by nitrogenase suggested that α-Ile423 of MoFe protein possibly functions as an electron transfer gate to Mo site of active center-“FeMoco”. Amino acid residue α-Lys424 connects directly to α-Ile423, and they are located in the same α-helix (α423-431). In the present study, function of α-Lys424 was investigated by replacing it with Arg (alkaline, like Lys), Gln (neutral), Glu (acidic), and Ala (neutral) through site-directed mutagenesis and homologous recombination. The mutants were, respectively, termed 424R, 424Q, 424E, and 424A. Studies of diazotrophic cell growth, cytological, and enzymatic properties indicated that none of the substitutions altered the secondary structure of MoFe protein, or normal expression of nifA, nifL, and nifD. Substitution of alkaline amino acid (i.e., 424R) maintained acetylene (C2H2) and proton (H+) reduction activities at normal levels similar to that of wild-type (WT), because its FeMoco content did not reduce. In contrast, substitution of acidic or neutral amino acid (i.e., 424Q, 424E, 424A) impaired the catalytic activity of nitrogenase to varying degrees. Combination of MoFe protein structural simulation and the results of a series of experiments, the function of α-Lys424 in ensuring insertion of FeMoco to MoFe protein was further confirmed, and the contribution of α-Lys424 in maintaining low potential of the microenvironment causing efficient catalytic activity of nitrogenase was demonstrated.https://www.frontiersin.org/article/10.3389/fmicb.2019.00802/fullKlebsiella oxytocanitrogenaseMoFe proteinα-Lys424FeMoco |
| spellingShingle | Lina Song Pengxi Liu Wei Jiang Qingjuan Guo Chunxi Zhang Abdul Basit Ying Li Jilun Li α-Lys424 Participates in Insertion of FeMoco to MoFe Protein and Maintains Nitrogenase Activity in Klebsiella oxytoca M5al Frontiers in Microbiology Klebsiella oxytoca nitrogenase MoFe protein α-Lys424 FeMoco |
| title | α-Lys424 Participates in Insertion of FeMoco to MoFe Protein and Maintains Nitrogenase Activity in Klebsiella oxytoca M5al |
| title_full | α-Lys424 Participates in Insertion of FeMoco to MoFe Protein and Maintains Nitrogenase Activity in Klebsiella oxytoca M5al |
| title_fullStr | α-Lys424 Participates in Insertion of FeMoco to MoFe Protein and Maintains Nitrogenase Activity in Klebsiella oxytoca M5al |
| title_full_unstemmed | α-Lys424 Participates in Insertion of FeMoco to MoFe Protein and Maintains Nitrogenase Activity in Klebsiella oxytoca M5al |
| title_short | α-Lys424 Participates in Insertion of FeMoco to MoFe Protein and Maintains Nitrogenase Activity in Klebsiella oxytoca M5al |
| title_sort | α lys424 participates in insertion of femoco to mofe protein and maintains nitrogenase activity in klebsiella oxytoca m5al |
| topic | Klebsiella oxytoca nitrogenase MoFe protein α-Lys424 FeMoco |
| url | https://www.frontiersin.org/article/10.3389/fmicb.2019.00802/full |
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