Crystal structure of albaflavenone monooxygenase containing a moonlighting terpene synthase active site.
Albaflavenone synthase (CYP170A1) is a monooxygenase catalyzing the final two steps in the biosynthesis of this antibiotic in the soil bacterium, Streptomyces coelicolor A3(2). Interestingly, CYP170A1 shows no stereo selection forming equal amounts of two albaflavenol epimers, each of which is oxidized in turn to albaflavenone. To explore the structural basis of the reaction mechanism, we have studied the crystal structures of both ligand-free CYP170A1 (2.6 A) and complex of endogenous substrate (epi-isozizaene) with CYP170A1 (3.3 A). The structure of the complex suggests that the proximal epi-isozizaene molecules may bind to the heme iron in two orientations. In addition, much to our surprise, we have found that albaflavenone synthase also has a second, completely distinct catalytic activity corresponding to the synthesis of farnesene isomers from farnesyl diphosphate. Within the cytochrome P450 alpha-helical domain both the primary sequence and x-ray structure indicate the presence of a novel terpene synthase active site that is moonlighting on the P450 structure. This includes signature sequences for divalent cation binding and an alpha-helical barrel. This barrel is unusual because it consists of only four helices rather than six found in all other terpene synthases. Mutagenesis establishes that this barrel is essential for the terpene synthase activity of CYP170A1 but not for the monooxygenase activity. This is the first bifunctional P450 discovered to have another active site moonlighting on it and the first time a terpene synthase active site is found moonlighting on another protein.
Department of Biochemistry, Center for Structural Biology, and Center in Molecular Toxicology, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-0146. Electronic address: bin.zhao@vanderbilt.edu.
Bin
B
Zhao
Department of Biochemistry, Center for Structural Biology, and Center in Molecular Toxicology, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-0146.
Li
L
Lei
Department of Biochemistry and Molecular Genetics, Schools of Medicine and Dentistry, University of Alabama at Birmingham, Birmingham, Alabama 35294.
Dmitry G
DG
Vassylyev
Department of Chemistry, Brown University, Providence, Rhode Island 02912-9108.
Xin
X
Lin
Department of Chemistry, Brown University, Providence, Rhode Island 02912-9108.
David E
DE
Cane
Institute of Life Science, Medical School, Swansea University, Swansea SA2 8PP, United Kingdom.
Steven L
SL
Kelly
Department of Biochemistry, Center for Structural Biology, and Center in Molecular Toxicology, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-0146.
Hang
H
Yuan
Institute of Life Science, Medical School, Swansea University, Swansea SA2 8PP, United Kingdom.
David C
DC
Lamb
Department of Biochemistry, Center for Structural Biology, and Center in Molecular Toxicology, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-0146.
Michael R
MR
Waterman
D019883:Alkyl and Aryl Transferases / Q000737:chemistry* / Q000378:metabolism; D001426:Bacterial Proteins / Q000737:chemistry* / Q000378:metabolism; D020134:Catalytic Domain / Q000502:physiology; D018360:Crystallography, X-Ray; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry* / Q000378:metabolism; D006418:Heme / Q000737:chemistry / Q000378:metabolism; D007501:Iron / Q000737:chemistry / Q000378:metabolism; D011106:Polyisoprenyl Phosphates / Q000096:biosynthesis / Q000737:chemistry; D011485:Protein Binding / Q000502:physiology; D017433:Protein Structure, Secondary / Q000502:physiology; D017434:Protein Structure, Tertiary / Q000502:physiology; D012717:Sesquiterpenes / Q000737:chemistry / Q000378:metabolism; D048372:Streptomyces coelicolor / Q000201:enzymology*
D001426:Bacterial Proteins; D011106:Polyisoprenyl Phosphates; D012717:Sesquiterpenes; C087466:albaflavenone; D006418:Heme; C004808:farnesyl pyrophosphate; D003577:Cytochrome P-450 Enzyme System; D007501:Iron; D019883:Alkyl and Aryl Transferases; C108877:terpene synthase