Biosynthesis of the immunosuppressants FK506, FK520, and rapamycin involves a previously undescribed family of enzymes acting on chorismate

被引:96
作者
Andexer, Jennifer N. [1 ]
Kendrew, Steven G. [2 ]
Nur-e-Alam, Mohammad [2 ]
Lazos, Orestis [1 ]
Foster, Teresa A. [2 ]
Zimmermann, Anna-Sophie [1 ]
Warneck, Tony D. [2 ]
Suthar, Dipen [2 ]
Coates, Nigel J. [2 ]
Koehn, Frank E. [3 ]
Skotnicki, Jerauld S. [3 ]
Carter, Guy T. [3 ]
Gregory, Matthew A. [2 ]
Martin, Christine J. [2 ]
Moss, Steven J. [2 ]
Leadlay, Peter F. [1 ]
Wilkinson, Barrie [2 ]
机构
[1] Univ Cambridge, Dept Biochem, Cambridge CB2 1GA, England
[2] Biotica, Cambridge CB10 1XL, England
[3] Pfizer Inc, Nat Prod Unit, Groton, CT 06340 USA
基金
英国生物技术与生命科学研究理事会;
关键词
calcineurin; mTOR; GENETICALLY HETEROGENEOUS MICE; CAMPESTRIS PV. CAMPESTRIS; EXTENDS LIFE-SPAN; STREPTOMYCES-HYGROSCOPICUS; GENE-CLUSTER; POLYKETIDE SYNTHASE; STARTER UNIT; XANTHOMONAS-CAMPESTRIS; ORGANIZATION; PATHWAY;
D O I
10.1073/pnas.1015773108
中图分类号
O [数理科学和化学]; P [天文学、地球科学]; Q [生物科学]; N [自然科学总论];
学科分类号
07 ; 0710 ; 09 ;
摘要
The macrocyclic polyketides FK506, FK520, and rapamycin are potent immunosuppressants that prevent T-cell proliferation through initial binding to the immunophilin FKBP12. Analogs of these molecules are of considerable interest as therapeutics in both metastatic and inflammatory disease. For these polyketides the starter unit for chain assembly is (4R,5R)-4,5-dihydroxycyclohex-1-enecarboxylic acid derived from the shikimate pathway. We show here that the first committed step in its formation is hydrolysis of chorismate to form (4R,5R)-4,5-dihydroxycyclohexa1,5-dienecarboxylic acid. This chorismatase activity is encoded by fkbO in the FK506 and FK520 biosynthetic gene clusters, and by rapK in the rapamycin gene cluster of Streptomyces hygroscopicus. Purified recombinant FkbO (from FK520) efficiently catalyzed the chorismatase reaction in vitro, as judged by HPLC-MS and NMR analysis. Complementation using fkbO from either the FK506 or the FK520 gene cluster of a strain of S. hygroscopicus specifically deleted in rapK (BIOT-4010) restored rapamycin production, as did supplementation with (4R,5R)-4,5-dihydroxycyclohexa-1,5-dienecarboxylic acid. Although BIOT-4010 produced no rapamycin, it did produce low levels of BC325, a rapamycin analog containing a 3-hydroxybenzoate starter unit. This led us to identify the rapK homolog hyg5 as encoding a chorismatase/3-hydroxybenzoate synthase. Similar enzymes in other bacteria include the product of the bra8 gene from the pathway to the terpenoid natural product brasilicardin. Expression of either hyg5 or bra8 in BIOT-4010 led to increased levels of BC325. Also, purified Hyg5 catalyzed the predicted conversion of chorismate into 3-hydroxybenzoate. FkbO, RapK, Hyg5, and Bra8 are thus founder members of a previously unrecognized family of enzymes acting on chorismate.
引用
收藏
页码:4776 / 4781
页数:6
相关论文
共 54 条
[1]   Gapped BLAST and PSI-BLAST: a new generation of protein database search programs [J].
Altschul, SF ;
Madden, TL ;
Schaffer, AA ;
Zhang, JH ;
Zhang, Z ;
Miller, W ;
Lipman, DJ .
NUCLEIC ACIDS RESEARCH, 1997, 25 (17) :3389-3402
[2]   Organization of the biosynthetic gene cluster for rapamycin in Streptomyces hygroscopicus: Analysis of the enzymatic domains in the modular polyketide synthase [J].
Aparicio, JF ;
Molnar, I ;
Schwecke, T ;
Konig, A ;
Haydock, SF ;
Khaw, LE ;
Staunton, J ;
Leadlay, PF .
GENE, 1996, 169 (01) :9-16
[3]   The crystal structure of Escherichia coli TdcF, a member of the highly conserved YjgF/YER057c/UK114 family [J].
Burman, Julia D. ;
Stevenson, Clare E. M. ;
Sawers, R. Gary ;
Lawson, David M. .
BMC STRUCTURAL BIOLOGY, 2007, 7
[4]   Molecular Interplay between Mammalian Target of Rapamycin (mTOR), Amyloid-β, and Tau EFFECTS ON COGNITIVE IMPAIRMENTS [J].
Caccamo, Antonella ;
Majumder, Smita ;
Richardson, Arlan ;
Strong, Randy ;
Oddo, Salvatore .
JOURNAL OF BIOLOGICAL CHEMISTRY, 2010, 285 (17) :13107-13120
[5]   Biosynthesis of ansatrienin (mycotrienin) and naphthomycin -: Identification and analysis of two separate biosynthetic gene clusters in Streptomyces collinus Tu 1892 [J].
Chen, S ;
von Bamberg, D ;
Hale, V ;
Breuer, M ;
Hardt, B ;
Müller, R ;
Floss, HG ;
Reynolds, KA ;
Leistner, E .
EUROPEAN JOURNAL OF BIOCHEMISTRY, 1999, 261 (01) :98-107
[6]   Identification of a cyclohexylcarbonyl CoA biosynthetic gene cluster and application in the production of doramectin [J].
Cropp, TA ;
Wilson, DJ ;
Reynolds, KA .
NATURE BIOTECHNOLOGY, 2000, 18 (09) :980-983
[7]   A metabolic node in action: Chorismate-utilizing enzymes in microorganisms [J].
Dosselaere, F ;
Vanderleyden, J .
CRITICAL REVIEWS IN MICROBIOLOGY, 2001, 27 (02) :75-131
[8]  
Drummond AJ., 2010, GENEIOUS V51
[9]   Antascomicins A, B, C, D and E - Novel FKBP12 binding compounds from a micromonospora a strain [J].
Fehr, T ;
Sanglier, JJ ;
Schuler, W ;
Gschwind, L ;
Ponelle, M ;
Schilling, W ;
Wioland, C .
JOURNAL OF ANTIBIOTICS, 1996, 49 (03) :230-233
[10]   Natural products derived from unusual variants of the shikimate pathway [J].
Floss, HG .
NATURAL PRODUCT REPORTS, 1997, 14 (05) :433-452