==Novobiocin Biosynthesis==

This aminocoumarin antibiotic consists of three major substituents. The 3-dimethylallyl-4-hydroxybenzoic acid moiety, known as ring A, is derived from prephenate and dimethylallyl pyrophosphate. The aminocoumarin moiety, known as ring B, is derived from L-Tyrosine. The final component of novobiocin is the sugar derivative L-noviose, known as ring C, which is derived from glucose-1-phosphate. The biosynthetic gene cluster for novobiocin was identified by Heide and coworkers in 1999 (published 2000) from Streptomyces spheroides NCIB 11891<ref name="pmid10770754">{{cite journal |author=Steffensky M, Mühlenweg A, Wang ZX, Li SM, Heide L |title=Identification of the novobiocin biosynthetic gene cluster of Streptomyces spheroides NCIB 11891 |journal=Antimicrob. Agents Chemother. |volume=44 |issue=5 |pages=1214–22 |year=2000 |month=May |pmid=10770754 |pmc=89847 |doi= |url=http://aac.asm.org/cgi/pmidlookup?view=long&pmid=10770754}}</ref>. They identified 23 putative open reading frames (ORFs) and more than 11 other ORFs that may play a role in novobiocin biosynthesis.

The biosynthesis of ring A (see Fig. 1) begins with prephenate which is a derived from the shikimic acid biosynthetic pathway. The enzyme NovF catalyzes the decarboxylation of prephenate while simultaneously reducing nicotinamide adenine dinucleotide phosphate (NADP^+) to produce NADPH. Following this NovQ catalyzes the electrophilic substitution of the phenyl ring with dimethylallyl pyrophosphate (DMAPP) otherwise known as prenylation<ref name="pmid12618544">{{cite journal |author=Pojer F, Wemakor E, Kammerer B, ''et al'' |title=CloQ, a prenyltransferase involved in clorobiocin biosynthesis |journal=Proc. Natl. Acad. Sci. U.S.A. |volume=100 |issue=5 |pages=2316–21 |year=2003 |month=March |pmid=12618544 |pmc=151338 |doi=10.1073/pnas.0337708100 |url=http://www.pnas.org/cgi/pmidlookup?view=long&pmid=12618544}}</ref>. DMAPP can come from either the mevalonic acid pathway or the deoxyxylulose biosynthetic pathway. Next the 3-dimethylallyl-4-hydroxybenzoate molecule is subjected to two oxidative decarboxylations by NovR and molecular oxygen <ref name="pmid12777382">{{cite journal |author=Pojer F, Kahlich R, Kammerer B, Li SM, Heide L |title=CloR, a bifunctional non-heme iron oxygenase involved in clorobiocin biosynthesis |journal=J. Biol. Chem. |volume=278 |issue=33 |pages=30661–8 |year=2003 |month=August |pmid=12777382 |doi=10.1074/jbc.M303190200 |url=http://www.jbc.org/cgi/pmidlookup?view=long&pmid=12777382}}</ref>. NovR is a non-heme iron oxygenase with a unique bifunctional catalysis. In the first stage both oxygens are incorporated from the molecular oxygen while in the second step only one is incorporated as determined by isotope labeling studies. This completes the formation of ring A.

Figure 1. Biosynthesis of 3-dimethylallyl-4-hydroxybenzoic acid component of novobiocin (ring A)

The biosynthesis of ring B (see Fig. 2) begins with the natural amino acid L-Tyrosine. This is then adenylated and thioesterified onto the peptidyl carrier protein (PCP) of NovH by ATP and NovH itself<ref name="pmid11325587">{{cite journal |author=Chen H, Walsh CT |title=Coumarin formation in novobiocin biosynthesis: beta-hydroxylation of the aminoacyl enzyme tyrosyl-S-NovH by a cytochrome P450 NovI |journal=Chem. Biol. |volume=8 |issue=4 |pages=301–12 |year=2001 |month=April |pmid=11325587 |doi= |url=http://linkinghub.elsevier.com/retrieve/pii/S1074-5521(01)00009-6}}</ref>. NovI then further modifies this PCP bound molecule by oxidizing the beta-position using NADPH and molecular oxygen. NovJ and NovK form a heterodimer of J2K2 which is the active form of this benzylic oxygenase<ref name="pmid16171397">{{cite journal |author=Pacholec M, Hillson NJ, Walsh CT |title=NovJ/NovK catalyze benzylic oxidation of a beta-hydroxyl tyrosyl-S-pantetheinyl enzyme during aminocoumarin ring formation in novobiocin biosynthesis |journal=Biochemistry |volume=44 |issue=38 |pages=12819–26 |year=2005 |month=September |pmid=16171397 |doi=10.1021/bi051297m |url=http://dx.doi.org/10.1021/bi051297m}}</ref>. This process uses NADP+ as a hydride acceptor in the oxidation of the beta-alcohol to the ketone. This ketone will prefer to exist in its enol tautomer in solution. Next a still unidentified protein catalyzes the selective oxidation of the benzene (as shown in Fig. 2). Upon oxidation this intermediate will spontaneously lactonize to form the aromatic ring B and lose NovH in the process.

Figure 2. Biosynthesis of 3-amino-4,7-dihydroxy-2H-chromen-2-one component of novobiocin (ring B)

The biosynthesis of L-noviose (ring C) is shown in Fig. 3. This process starts from glucose-1-phosphate where NovV takes dTTP and replaces the phosphate group with a dTDP group. NovT then oxidizes the 4-hydroxy group using NAD+. NovT also accomplishes a dehydroxylation of the 6 position of the sugar. NovW then epimerizes the 3 position of the sugar<ref name="pmid15752721">{{cite journal |author=Thuy TT, Lee HC, Kim CG, Heide L, Sohng JK |title=Functional characterizations of novWUS involved in novobiocin biosynthesis from Streptomyces spheroides |journal=Arch. Biochem. Biophys. |volume=436 |issue=1 |pages=161–7 |year=2005 |month=April |pmid=15752721 |doi=10.1016/j.abb.2005.01.012 |url=http://linkinghub.elsevier.com/retrieve/pii/S0003-9861(05)00027-5}}</ref>. The methylation of the 5 position is accomplished by NovU and S-adenosyl methionine (SAM). Finally NovS reduces the 4 position again to achieve epimerization of that position from the starting glucose-1-phosphate using NADH.

Figure 3. Biosynthesis of L-noviose component of novobiocin (ring C)

Rings A, B, and C are coupled together and modified to give the finished novobiocin molecule. Rings A and B are coupled together by the enzyme NovL using ATP to diphosphorylate the carboxylate group of ring A so that the carbonyl can be attacked by the amine group on ring B. The resulting compound is methylated by NovO and SAM prior to glycosylation<ref name="pmid16274243">{{cite journal |author=Pacholec M, Tao J, Walsh CT |title=CouO and NovO: C-methyltransferases for tailoring the aminocoumarin scaffold in coumermycin and novobiocin antibiotic biosynthesis |journal=Biochemistry |volume=44 |issue=45 |pages=14969–76 |year=2005 |month=November |pmid=16274243 |doi=10.1021/bi051599o |url=http://dx.doi.org/10.1021/bi051599o}}</ref>. NovM adds ring C (L-noviose) to the hydroxyl group derived from tyrosine with the loss of dTDP. Another methylation is accomplished by NovP and SAM at the 4 position of the L-noviose sugar<ref name="pmid14694473">{{cite journal |author=Freel Meyers CL, Oberthür M, Xu H, Heide L, Kahne D, Walsh CT |title=Characterization of NovP and NovN: completion of novobiocin biosynthesis by sequential tailoring of the noviosyl ring |journal=Angew. Chem. Int. Ed. Engl. |volume=43 |issue=1 |pages=67–70 |year=2004 |month=January |pmid=14694473 |doi=10.1002/anie.200352626 |url=http://dx.doi.org/10.1002/anie.200352626}}</ref>. This methylation allows NovN to carbamylate the 3 position of the sugar as shown in Fig. 4 completing the biosynthesis of novobiocin.

Figure 4. Completed biosynthesis of novobiocin from ring systems A, B, and C