Chemistry with an Artificial Primer of Polyhydroxybutyrate Synthase Suggests a Mechanism for Chain Termination
Polyhydroxybutyrate (PHB) synthases (PhaCs) catalyze the conversion of 3-(R)-hydroxybutyryl CoA (HBCoA) to PHB, which is deposited as granules in the cytoplasm of microorganisms. The class I PhaC from Caulobacter crescentus (PhaC[subscript Cc]) is a highly soluble protein with a turnover number of 7...
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| Format: | Article |
| Language: | en_US |
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American Chemical Society (ACS)
2016
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| Online Access: | http://hdl.handle.net/1721.1/103958 https://orcid.org/0000-0001-8076-4489 |
| _version_ | 1811071273359376384 |
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| author | Stubbe, JoAnne Buckley, Rachael M. |
| author2 | Massachusetts Institute of Technology. Department of Biology |
| author_facet | Massachusetts Institute of Technology. Department of Biology Stubbe, JoAnne Buckley, Rachael M. |
| author_sort | Stubbe, JoAnne |
| collection | MIT |
| description | Polyhydroxybutyrate (PHB) synthases (PhaCs) catalyze the conversion of 3-(R)-hydroxybutyryl CoA (HBCoA) to PHB, which is deposited as granules in the cytoplasm of microorganisms. The class I PhaC from Caulobacter crescentus (PhaC[subscript Cc]) is a highly soluble protein with a turnover number of 75 s[superscript –1] and no lag phase in coenzyme A (CoA) release. Studies with [1-[superscript 14]C]HBCoA and PhaC[subscript Cc] monitored by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) and autoradiography reveal that the rate of elongation is much faster than the rate of initiation. Priming with the artificial primer [[superscript 3]H]sTCoA and monitoring for CoA release reveal a single CoA/PhaC, suggesting that the protein is uniformly loaded and that the elongation process could be studied. Reaction of sT-PhaC[subscript Cc] with [1-[superscript 14]C]HBCoA revealed that priming with sTCoA increased the uniformity of elongation, allowing distinct polymerization species to be observed by SDS–PAGE and autoradiography. However, in the absence of HBCoA, [3H]sT-PhaC unexpectedly generates [3H]sDCoA with a rate constant of 0.017 s[superscript –1]. We propose that the [[superscript 3]H]sDCoA forms via attack of CoA on the oxoester of the [[superscript 3]H]sT-PhaC chain, leaving the synthase attached to a single HB unit. Comparison of the relative rate constants of thiolysis by CoA and elongation by PhaC[subscript Cc], and the size of the PHB polymer generated in vivo, suggests a mechanism for chain termination and reinitiation. |
| first_indexed | 2024-09-23T08:48:37Z |
| format | Article |
| id | mit-1721.1/103958 |
| institution | Massachusetts Institute of Technology |
| language | en_US |
| last_indexed | 2024-09-23T08:48:37Z |
| publishDate | 2016 |
| publisher | American Chemical Society (ACS) |
| record_format | dspace |
| spelling | mit-1721.1/1039582022-09-23T14:43:10Z Chemistry with an Artificial Primer of Polyhydroxybutyrate Synthase Suggests a Mechanism for Chain Termination Stubbe, JoAnne Buckley, Rachael M. Massachusetts Institute of Technology. Department of Biology Massachusetts Institute of Technology. Department of Chemistry Buckley, Rachael Marie Stubbe, JoAnne Polyhydroxybutyrate (PHB) synthases (PhaCs) catalyze the conversion of 3-(R)-hydroxybutyryl CoA (HBCoA) to PHB, which is deposited as granules in the cytoplasm of microorganisms. The class I PhaC from Caulobacter crescentus (PhaC[subscript Cc]) is a highly soluble protein with a turnover number of 75 s[superscript –1] and no lag phase in coenzyme A (CoA) release. Studies with [1-[superscript 14]C]HBCoA and PhaC[subscript Cc] monitored by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) and autoradiography reveal that the rate of elongation is much faster than the rate of initiation. Priming with the artificial primer [[superscript 3]H]sTCoA and monitoring for CoA release reveal a single CoA/PhaC, suggesting that the protein is uniformly loaded and that the elongation process could be studied. Reaction of sT-PhaC[subscript Cc] with [1-[superscript 14]C]HBCoA revealed that priming with sTCoA increased the uniformity of elongation, allowing distinct polymerization species to be observed by SDS–PAGE and autoradiography. However, in the absence of HBCoA, [3H]sT-PhaC unexpectedly generates [3H]sDCoA with a rate constant of 0.017 s[superscript –1]. We propose that the [[superscript 3]H]sDCoA forms via attack of CoA on the oxoester of the [[superscript 3]H]sT-PhaC chain, leaving the synthase attached to a single HB unit. Comparison of the relative rate constants of thiolysis by CoA and elongation by PhaC[subscript Cc], and the size of the PHB polymer generated in vivo, suggests a mechanism for chain termination and reinitiation. National Institutes of Health (U.S.) (NIH Grant GM49171) 2016-08-18T20:40:38Z 2016-08-18T20:40:38Z 2015-03 2015-02 Article http://purl.org/eprint/type/JournalArticle 0006-2960 1520-4995 http://hdl.handle.net/1721.1/103958 Rachael M. Buckley, and JoAnne Stubbe. "Chemistry with an Artificial Primer of Polyhydroxybutyrate Synthase Suggests a Mechanism for Chain Termination." Biochemistry 54:12 (2015), pp. 2117-2125. © 2015 American Chemical Society. https://orcid.org/0000-0001-8076-4489 en_US http://dx.doi.org/10.1021/bi501405b 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 | Stubbe, JoAnne Buckley, Rachael M. Chemistry with an Artificial Primer of Polyhydroxybutyrate Synthase Suggests a Mechanism for Chain Termination |
| title | Chemistry with an Artificial Primer of Polyhydroxybutyrate Synthase Suggests a Mechanism for Chain Termination |
| title_full | Chemistry with an Artificial Primer of Polyhydroxybutyrate Synthase Suggests a Mechanism for Chain Termination |
| title_fullStr | Chemistry with an Artificial Primer of Polyhydroxybutyrate Synthase Suggests a Mechanism for Chain Termination |
| title_full_unstemmed | Chemistry with an Artificial Primer of Polyhydroxybutyrate Synthase Suggests a Mechanism for Chain Termination |
| title_short | Chemistry with an Artificial Primer of Polyhydroxybutyrate Synthase Suggests a Mechanism for Chain Termination |
| title_sort | chemistry with an artificial primer of polyhydroxybutyrate synthase suggests a mechanism for chain termination |
| url | http://hdl.handle.net/1721.1/103958 https://orcid.org/0000-0001-8076-4489 |
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