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The Discovery of a β‑Lactone Synthetase Frank M. Raushel* Department of Chemistry, Texas A&M University, College Station, Texas 77844, United States
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reaction requires MgATP, but the full set of the reaction products was apparently not measured. However, OleC is related to a superfamily of enzymes that have been shown to catalyze the ATP-dependent ligation of substrates via the formation of an adenylate intermediate.4 It was suggested by the authors that MgATP is likely required to activate the β-hydroxyl group or the carboxylate of the substrate prior to β-lactone formation.1 It is relatively unlikely that β-lactone formation would occur via the formation of an adenylate intermediate with the β-hydroxy group, and thus, the most probable intermediate would occur by the initial attack of the substrate carboxylate on the α-phosphoryl group of ATP as shown in Scheme 2.
n a recent paper that appeared as a Rapid Report in Biochemistry, the laboratories of Carrie Wilmot and Larry Wackett from the University of Minnesota reported the identification and characterization of the first enzyme capable of catalyzing the formation of a β-lactone.1 This is a significant milestone in the discovery of new enzyme-catalyzed reactions because a number of important natural products such as lipstatin, a precursor of the anti-obesity drug Orlistat, have been shown to contain a β-lactone functional group.2 However, no other enzyme reported to date that actually catalyzes the formation of this unstable entity has been identified. The inherent chemical instability of the β-lactone functional group initially hampered the discovery and characterization of this enzyme. The Minnesota group was investigating a cluster of four genes from Stenotrophomonas maltophilia that was apparently responsible for the biosynthesis of long-chain cis-olefins starting from two CoA-activated carboxylic acids. One of the intermediates in this proposed biosynthetic pathway was a β-hydroxy acid (1) that was formed by the reduction of the corresponding β-keto acid. Incubation of the β-hydroxy acid (1) with MgATP in the presence of the enzyme OleC was initially thought to directly produce the cis-olefin (3) when the products of this transformation were analyzed by gas chromatography.3 However, when the structures of the reaction products were analyzed by nuclear magnetic resonance spectroscopy, the data clearly demonstrated that the actual product was the β-lactone (2) as illustrated in Scheme 1.1 The high
Scheme 2. Proposed Reaction Mechanism for the Formation of β-Lactone 2 from β-Hydroxy Acid 1 via the Formation of Adenylate Intermediate 4
Scheme 1. Biosynthesis of a cis-Olefin from a β-Hydroxy Acid via a β-Lactone Intermediate The proposed reaction mechanism for β-lactone synthesis is in stark contrast with the biosynthetic routes for the formation of β-lactam functional groups found within the structures of many antibiotics. For example, during the biosynthesis of the antibiotic isopenicillin N (6), the amide bond is formed in a prior step via the ATP-dependent condensation of a dipeptide with D-valine to form tripeptide ACV (5). Formation of the β-lactam is ultimately catalyzed by isopenicillin N synthase (IPNS), a mononuclear iron protein that requires molecular oxygen (Scheme 3).5 How easily will other enzymes be identified that catalyze the formation of β-lactone functional groups? In a survey of the nonredundant protein sequence database, Wackett and co-workers have identified more than 900 sequences with an amino acid sequence identity of >35% with the enzyme OleC, and more than 16000 sequences with an amino acid sequence identity of >25%. Therefore, it would appear that the formation
temperatures needed to separate the substrates from the products during the gas chromatographic procedures apparently caused the dehydration and decarboxylation of the β-lactone to the cis-olefin product. It is likely that the enzyme OleB catalyzes the formation of the cis-olefin from the β-lactone. How is the β-lactone product actually synthesized by the action of OleC? The Wackett laboratory has shown that the © XXXX American Chemical Society
Received: January 25, 2017 Published: January 31, 2017 A
DOI: 10.1021/acs.biochem.7b00061 Biochemistry XXXX, XXX, XXX−XXX
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Biochemistry Scheme 3. Formation of the β-Lactam Functional Group in Isopenicillin N by IPNS
of natural products with β-lactone functional groups is rather widespread, and other enzymes are likely to be soon identified and structurally characterized. This is a remarkable time for genomic enzymology.
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AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. Telephone: (979) 845-3373. ORCID
Frank M. Raushel: 0000-0002-5918-3089 Notes
The author declares no competing financial interest.
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REFERENCES
(1) Christenson, J. K., Richman, J. E., Jensen, M. R., Neufeld, J. Y., Wilmot, C. M., and Wackett, L. P. (2017) β-Lactone synthetase found in the olefin biosynthesis pathway. Biochemistry 56, 348−351. (2) Weibel, E. K., Hadvary, P., Hochuli, E., Kupfer, E., and Lengsfeld, H. (1987) Lipstatin, an inhibitor of pancreatic lipase, produced by Streptomyces toxytricini. I. Producing organism, fermentation, isolation and biological activity. J. Antibiot. 40, 1081−1085. (3) Kancharla, P., Bonnett, S. A., and Reynolds, K. A. (2016) Stenotrophomonas maltophilia OleC-catalyzed ATP-dependent formation of long-chain Z-Olefins from 2-alkyl-3-hydroxyalkanoic acids. ChemBioChem 17, 1426−1429. (4) Conti, E., Franks, H. P., and Brick, P. (1996) Crystal structure of firefly luciferase throws light on a superfamily of adenylate-forming enzymes. Structure 4, 287−298. (5) Elkins, J. M., Rutledge, P. J., Burzlaff, N. I., Clifton, I. J., Adlington, R. M., Roach, P. L., and Baldwin, J. E. (2003) Crystallographic stidies on the reaction of isopenicillin N synthase with an unsaturated substrated substrate analog. Org. Biomol. Chem. 1, 1455−1466.
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DOI: 10.1021/acs.biochem.7b00061 Biochemistry XXXX, XXX, XXX−XXX
