Bacillus Subtilis Class Ib Ribonucleotide Reductase: High Activity and Dynamic Subunit Interactions
The class Ib ribonucleotide reductase (RNR) isolated from Bacillus subtilis was recently purified as a 1:1 ratio of NrdE (α) and NrdF (β) subunits and determined to have a dimanganic-tyrosyl radical (Mn[superscript III][subscript 2]-Y·) cofactor. The activity of this RNR and the one reconstituted fr...
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| Format: | Article |
| Language: | en_US |
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American Chemical Society (ACS)
2015
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| Online Access: | http://hdl.handle.net/1721.1/95673 https://orcid.org/0000-0001-7174-0485 https://orcid.org/0000-0001-8076-4489 |
| _version_ | 1811072171483594752 |
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| author | Zhu, Xuling Stubbe, JoAnne Parker, Mackenzie James |
| author2 | Massachusetts Institute of Technology. Department of Biology |
| author_facet | Massachusetts Institute of Technology. Department of Biology Zhu, Xuling Stubbe, JoAnne Parker, Mackenzie James |
| author_sort | Zhu, Xuling |
| collection | MIT |
| description | The class Ib ribonucleotide reductase (RNR) isolated from Bacillus subtilis was recently purified as a 1:1 ratio of NrdE (α) and NrdF (β) subunits and determined to have a dimanganic-tyrosyl radical (Mn[superscript III][subscript 2]-Y·) cofactor. The activity of this RNR and the one reconstituted from recombinantly expressed NrdE and reconstituted Mn[superscript III][subscript 2]-Y· NrdF using dithiothreitol as the reductant, however, was low (160 nmol min[superscript –1] mg[superscript –1]). The apparent tight affinity between the two subunits, distinct from all class Ia RNRs, suggested that B. subtilis RNR might be the protein that yields to the elusive X-ray crystallographic characterization of an “active” RNR complex. We now report our efforts to optimize the activity of B. subtilis RNR by (1) isolation of NrdF with a homogeneous cofactor, and (2) identification and purification of the endogenous reductant(s). Goal one was achieved using anion exchange chromatography to separate apo-/mismetalated-NrdFs from Mn[superscript III][subscript 2]-Y· NrdF, yielding enzyme containing 4 Mn and 1 Y·[over β [subscript 2]]. Goal two was achieved by cloning, expressing, and purifying TrxA (thioredoxin), YosR (a glutaredoxin-like thioredoxin), and TrxB (thioredoxin reductase). The success of both goals increased the specific activity to ~1250 nmol min[superscript –1] mg[superscript –1] using a 1:1 mixture of NrdE:Mn[superscript III][subscript 2]-Y· NrdF and either TrxA or YosR and TrxB. The quaternary structures of NrdE, NrdF, and NrdE:NrdF (1:1) were characterized by size exclusion chromatography and analytical ultracentrifugation. At physiological concentrations (~1 μM), NrdE is a monomer (α) and Mn[superscript III][subscript 2]-Y· NrdF is a dimer (β[subscript 2]). A 1:1 mixture of NrdE:NrdF, however, is composed of a complex mixture of structures in contrast to expectations. |
| first_indexed | 2024-09-23T09:01:45Z |
| format | Article |
| id | mit-1721.1/95673 |
| institution | Massachusetts Institute of Technology |
| language | en_US |
| last_indexed | 2024-09-23T09:01:45Z |
| publishDate | 2015 |
| publisher | American Chemical Society (ACS) |
| record_format | dspace |
| spelling | mit-1721.1/956732022-09-30T12:54:34Z Bacillus Subtilis Class Ib Ribonucleotide Reductase: High Activity and Dynamic Subunit Interactions Zhu, Xuling Stubbe, JoAnne Parker, Mackenzie James Massachusetts Institute of Technology. Department of Biology Massachusetts Institute of Technology. Department of Chemistry Parker, Mackenzie James Zhu, Xuling Stubbe, JoAnne The class Ib ribonucleotide reductase (RNR) isolated from Bacillus subtilis was recently purified as a 1:1 ratio of NrdE (α) and NrdF (β) subunits and determined to have a dimanganic-tyrosyl radical (Mn[superscript III][subscript 2]-Y·) cofactor. The activity of this RNR and the one reconstituted from recombinantly expressed NrdE and reconstituted Mn[superscript III][subscript 2]-Y· NrdF using dithiothreitol as the reductant, however, was low (160 nmol min[superscript –1] mg[superscript –1]). The apparent tight affinity between the two subunits, distinct from all class Ia RNRs, suggested that B. subtilis RNR might be the protein that yields to the elusive X-ray crystallographic characterization of an “active” RNR complex. We now report our efforts to optimize the activity of B. subtilis RNR by (1) isolation of NrdF with a homogeneous cofactor, and (2) identification and purification of the endogenous reductant(s). Goal one was achieved using anion exchange chromatography to separate apo-/mismetalated-NrdFs from Mn[superscript III][subscript 2]-Y· NrdF, yielding enzyme containing 4 Mn and 1 Y·[over β [subscript 2]]. Goal two was achieved by cloning, expressing, and purifying TrxA (thioredoxin), YosR (a glutaredoxin-like thioredoxin), and TrxB (thioredoxin reductase). The success of both goals increased the specific activity to ~1250 nmol min[superscript –1] mg[superscript –1] using a 1:1 mixture of NrdE:Mn[superscript III][subscript 2]-Y· NrdF and either TrxA or YosR and TrxB. The quaternary structures of NrdE, NrdF, and NrdE:NrdF (1:1) were characterized by size exclusion chromatography and analytical ultracentrifugation. At physiological concentrations (~1 μM), NrdE is a monomer (α) and Mn[superscript III][subscript 2]-Y· NrdF is a dimer (β[subscript 2]). A 1:1 mixture of NrdE:NrdF, however, is composed of a complex mixture of structures in contrast to expectations. Massachusetts Institute of Technology. Biophysical Instrumentation Facility (NSF-007031) 2015-02-26T18:59:02Z 2015-02-26T18:59:02Z 2014-01 2013-11 Article http://purl.org/eprint/type/JournalArticle 0006-2960 1520-4995 http://hdl.handle.net/1721.1/95673 Parker, Mackenzie J., Xuling Zhu, and JoAnne Stubbe. “ Bacillus Subtilis Class Ib Ribonucleotide Reductase: High Activity and Dynamic Subunit Interactions .” Biochemistry 53, no. 4 (February 4, 2014): 766–776. https://orcid.org/0000-0001-7174-0485 https://orcid.org/0000-0001-8076-4489 en_US http://dx.doi.org/10.1021/bi401056e Biochemistry Article is made available in accordance with the publisher's policy and may be subject to US copyright law. Please refer to the publisher's site for terms of use. application/pdf American Chemical Society (ACS) ACS |
| spellingShingle | Zhu, Xuling Stubbe, JoAnne Parker, Mackenzie James Bacillus Subtilis Class Ib Ribonucleotide Reductase: High Activity and Dynamic Subunit Interactions |
| title | Bacillus Subtilis Class Ib Ribonucleotide Reductase: High Activity and Dynamic Subunit Interactions |
| title_full | Bacillus Subtilis Class Ib Ribonucleotide Reductase: High Activity and Dynamic Subunit Interactions |
| title_fullStr | Bacillus Subtilis Class Ib Ribonucleotide Reductase: High Activity and Dynamic Subunit Interactions |
| title_full_unstemmed | Bacillus Subtilis Class Ib Ribonucleotide Reductase: High Activity and Dynamic Subunit Interactions |
| title_short | Bacillus Subtilis Class Ib Ribonucleotide Reductase: High Activity and Dynamic Subunit Interactions |
| title_sort | bacillus subtilis class ib ribonucleotide reductase high activity and dynamic subunit interactions |
| url | http://hdl.handle.net/1721.1/95673 https://orcid.org/0000-0001-7174-0485 https://orcid.org/0000-0001-8076-4489 |
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