Tailoring Enzymes Acting on Carrier Protein-Tethered Substrates in Natural Product Biosynthesis

CHAPTER SIXTEEN

Shuangjun Lin*, Tingting Huang{, Ben Shen{,{,},1 *The State Key Laboratory of Microbial Metabolism, School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, Shanghai, PR China {Department of Chemistry, The Scripps Research Institute, Jupiter, Florida, USA { Department of Molecular Therapeutics, The Scripps Research Institute, Jupiter, Florida, USA }Natural Products Library Initiative at TSRI, The Scripps Research Institute, Jupiter, Florida, USA 1Corresponding author: e-mail address: [email protected]

Contents 1. Introduction 322 2. Methods 331 2.1 In vitro characterization of SgcC3-catalyzed chlorination of (S)-b-tyrosyl-SgcC2 331 2.2 In vitro characterization of SgcC-catalyzed hydroxylation of (S)-b-3-chloro-tyrosinyl-SgcC2 336 2.3 Exploitation of SgcC2-tethered (S)-b-tyrosine analogues for structural diversification 338 3. Conclusion 339 Acknowledgment 340 References 340

Abstract Carrier proteins (CPs) are integral components of fatty acid synthases, polyketide synthases, and nonribosomal peptide synthetases and play critical roles in the biosynthesis of fatty acids, polyketides, and nonribosomal peptides. An emerging role CPs play in natural product biosynthesis involves tailoring enzymes that act on CP-tethered substrates. These enzymes provide a new opportunity to engineer natural product diversity by exploiting CPs to increase substrate promiscuity for the tailoring steps. This chapter describes protocols for in vitro biochemical characterization of SgcC3 and SgcC that catalyze chlorination and hydroxylation of SgcC2-tethered (S)-b-tyrosine and analogues in the biosynthesis of the enediyne chromophore of the chromoprotein C-1027. These protocols are applicable to mechanistic characterization and engineered exploitation of other tailoring enzymes that act on CP-tethered substrates in natural product

biosynthesis and structural diversification. The ultimate goal is to use the in vitro findings to guide in vivo engineering of designer natural products.

1. INTRODUCTION

Acyl carrier proteins (ACPs) and peptidyl carrier proteins (PCPs) are small (10 kDa) proteins, existing as either a discrete protein in a type II multienzyme complex or a distinct domain interspersed among the catalytic domains of a type I multifunctional megasynthase (Marahiel & Essen, 2009; Mercer & Burkart, 2007; Shen, 2000; Staunton & Weissman, 2001; Weissman, 2009). While the overall amino acid sequence identity among the carrier proteins (CPs) is modest, they are characterized by a highly conserved signature motif of GxxSL/I. The serine residue in this motif is the site for 40-phosphopantetheinylation, a posttranslational modification catalyzed by 40-phosphopantetheinyl transferases (PPTases) (Lambalot et al., 1996; Sanchez, Du, Edwards, Toney, & Shen, 2001). PPTases convert the apo-CPs into the functional holo-CPs by installing the 20 A˚ -long 40- phosphopantetheine prosthetic group with a free terminal thiol (Fig. 16.1A). At this thiol, both substrates and the growing intermediates are tethered as thioesters. While the 40-phosphopantetheinyl arm facilitates the delivery of substrates into each of the active sites and channels the growing intermediates between each of the elongation cycles, the CPs provide necessary protein–protein recognition among the various enzymatic partners. CPs that carry short carboxylic acids or other acyl intermediates are known as ACPs, which were first characterized from fatty acid synthases (FASs) (Chan & Vogel, 2010; Gago, Diacovich, Arabolaza, Tsai, & Gramajo, 2011; Mercer & Burkart, 2007). Type I FASs are multifunctional proteins consisting of domains for individual activities, while type II FASs are multienzyme complexes consisting of discrete, monofunctional proteins. ACPs, either as a domain in type I FASs or a discrete protein in type II FASs, play a pivotal role in fatty acid biosynthesis by tethering the starter and extender units for condensation and by channeling the growing acyl intermediates for complete b-ketoreduction (i.e., b-ketoreduction, dehydration, and enoylreduction) during each cycle of chain elongation to afford the fully reduced fatty acid as the final product (Fig. 16.1B). Tailoring Enzymes Acting on Carry Protein-Tethered Substrates 323

A 4Ј-Phosphopantetheine

O O PPTase O OH OO SH P N N OOHH H apo-ACP CoA ADP SH apo-PCP holo-ACP holo-PCP

B b-Keto reduction AT Elongation (complete) ACP ACP ACP ACP ACP

SH S O S S S O O O O R S-Enz O O n O R RR Fatty acids C b-Keto reduction AT Elongation (selective) ACP ACP ACP ACP ACP

SH S O S S S R O O O R S-Enz OOOH O O OH Polyketides O R R D A Elongation PCP PCP PCP PCP R O R 1 H n+1 SH S SSN PCP N NH O O H n 2 R R OORn 1 S 1 NH2 NH O O Peptides R2 R2 NH2 H2N E AT/A Tailoring enzymes Natural products A/P-CP A/P-CP A/P-CP Cyclization (see Fig. 16.2 for examples) SH S Halogenation S O Methylation O Oxidation Reduction (see Table 16.1 for examples) Rs Groups introduced Rs: by tailoring enzymes Figure 16.1 Carrier proteins and their roles in fatty acid, polyketide, and nonribosomal peptide biosynthesis: (A) posttranslational modification of an apo-ACP or apo-PCP into a holo-ACP or holo-PCP by a PPTase; (B) ACP-mediated substrate activation and intermediate channeling in fatty acid biosynthesis; (C) ACP-mediated substrate activation and intermediate channeling in polyketide biosynthesis; (D) PCP-mediated substrate activation and intermediate channeling in nonribosomal peptide biosynthesis; and (E) tailoring enzymes acting on ACP- or PCP-tethered substrates in natural product biosynthesis. See Table 16.1 for specific tailoring enzymes, ACP- or PCP-tethered substrates and their corresponding products, and the types of modification and Fig. 16.2 for structures of natural products with moieties modified by tailoring enzymes highlighted in gray. A, adenylation enzyme; ACP, acyl carrier protein; AT, acyltransferase; PCP, peptidyl carrier protein; PPTase, 40-phosphopantetheinyl transferase. 324 Shuangjun Lin et al.

ACPs were subsequently characterized from polyketide synthases (PKSs), which catalyze the biosynthesis of polyketides, a large family of natural products with profound biological activities (Mercer & Burkart, 2007; Shen, 2000; Staunton & Weissman, 2001; Weissman, 2009). Following the convention of FASs, PKSs have also been classified into types I and II according to their enzyme architectures (Shen, 2003). Thus, similar to FASs, ACPs in type I PKSs are domains, ACPs in type II PKSs are discrete proteins, and regardless of their architectural difference, both ACP domains and proteins tether the acyl CoA substrates for condensation and channel the growing acyl intermediates during each cycle of chain elongation. However, in contrast to FASs, the b-ketone groups of the ACP-tethered growing acyl intermediates in PKSs can undergo no, partial, or full reduction, depending on the given cycle of elongation, thereby providing a mechanistic basis to account for the vast structural diversity of polyketide natural products (Fig. 16.1C). CPs from nonribosomal peptide synthetases (NRPSs) are known as PCPs, carrying amino acids or peptidyl intermediates. NRPSs catalyze the biosynthesis of nonribosomal peptides, another major family of natural products including many clinically important drugs (Marahiel & Essen, 2009; Mercer & Burkart, 2007). Although PKSs and NRPSs catalyze the biosynthesis of two distinct classes of natural products from two different pools of substrates, they apparently use a very similar molecular logic for substrate activation and intermediate channeling. While the type I and II nomenclature for FASs and PKSs has not been widely accepted to classify NRPSs, both multifunctional NRPSs with distinct domains and discrete NRPSs with largely monofunctions are known. In a mechanism analogous to FASs and PKSs, NRPSs use PCPs to tether the amino acid substrates for condensation and channel the growing peptidyl intermediates during each cycle of chain elongation (Fig. 16.1D). These striking structural and mechanistic similarities between PKSs and NRPSs have inspired the discovery and characterization of natural NRPS–PKS megasynthases for the biosynthesis of hybrid peptide–polyketide natural products and the construction of engineered hybrid NRPS–PKS systems to further expand the size and diversity of natural product libraries (Du et al., 2001; Fischbach & Walsh, 2006, 2010). CP-dependent PKSs and NRPSs catalyze the assembly of a myriad of polyketide, peptide, and hybrid polyketide–peptide backbones from a vast array of short carboxylic acids and amino acids. The nascent scaffolds are often heavily modified by the coordinated action of specialized enzymes, Tailoring Enzymes Acting on Carry Protein-Tethered Substrates 325 known as tailoring enzymes, to further imbue structural and functional diversity. While tailoring enzymes that act during chain elongation, that is, with the growing intermediates still tethered to specific ACPs or PCPs, are known, most tailoring enzymes act on the peptide, polyketide, or hybrid peptide–polyketide intermediates after they are released from the PKS or NRPS megasynthases as free substrates (Fischbach & Walsh, 2010; Walsh et al., 2001). A subset of tailoring enzymes is emerging that specifically act on CP- tethered substrates; the corresponding free substrates are not recognized. This strategy is most commonly associated with biosynthesis of unusual building blocks incorporated into many polyketide and nonribosomal peptide natural products. Modifications catalyzed by tailoring enzymes acting on both ACP- and PCP-tethered substrates are known, including cyclization, halogenation, methylation, oxidation (dehydrogenation, epoxidation, and hydroxylation), and reduction (Fig. 16.1E). Table 16.1 summarizes the tailoring enzymes known to date that have been biochemically characterized and act on CP-tethered substrates in natural product biosynthesis (Fig. 16.2). Tailoring enzymes that act on CP-tethered substrates therefore represent a new molecular logic for natural product biosynthesis. The tethering of pre- cursors to CPs ensures that the resultant building blocks will be sequestered from endogenous metabolite pools and efficiently incorporated into the final natural products. The enediyne chromophore of the C-1027 chromoprotein, one of the most potent antitumor antibiotics known to date, features a highly modified b-amino acid moiety (Fig. 16.3; Van Lanen & Shen, 2008). The gene cluster for C-1027 biosynthesis was cloned and sequenced from Streptomyces globisporus (Liu, Christenson, Standage, & Shen, 2002). Bioinformatics analysis of the genes within the C-1027 biosynthetic gene cluster predicted, and biochemical characterizations subsequently confirmed, that the biosynthesis of the b-amino acid moiety from the a-tyrosine precursor involved tailoring enzymes that act on PCP-tethered substrates (Van Lanen et al., 2005). Thus, a-tyrosine is first converted by the SgcC4 aminomutase to (S)-b-tyrosine (Christenson, Liu, Toney, & Shen, 2003; Christenson, Wu, Spies, Shen, & Toney, 2003), which is then tethered by the SgcC1 adenylation enzyme to the SgcC2 PCP (Van Lanen, Lin, Dorrestein, Kelleher, & Shen, 2006). Sequential chlorination and hydroxylation of the SgcC2-tethered (S)-b- tyrosine by the SgcC3 halogenase (Lin et al., 2007) and SgcC monooxygenase (Lin et al., 2008), respectively, affords the fully modified b-tyrosine building block, which, still tethered to the SgcC2 PCP, is Table 16.1 Tailoring enzymes acting on carrier protein-tethered substrates that have been biochemically characterized from natural product biosynthetic pathways. Tailoring Carrier Natural productsa enzyme protein Type of reaction Substrates Products Reference

Armentomycin CytC3 CytC2 Chlorination L-aminobutanoic g,g-Dichloro-L- Ueki et al. (2006) acid aminobutanoic acid

Barbamide BarB1 PCPBarA Chlorination L-leucine d-trichloro-L-leucine Galonic´, BarB2 Vaillancourt, and Walsh (2006) Flatt et al. (2006) C-1027 SgcC3 SgcC2 Chlorination (S)-b-tyrosine (S)-3-chloro-b-tyrosine Lin, Van Lanen, and Shen (2007) C-1027 SgcC SgcC2 Hydroxylation (S)-3-chloro-b- (S)-3-chloro-5-hydroxy-b- Lin, Van Lanen, and tyrosine tyrosine Shen (2008) CDA HxcO ACP Dehydrogenation hexanoic acid hex-2-enoic acid Kopp, Linne, Oberthu¨r, and Marahiel (2008) CDA HcmO ACP Oxidation hex-2-enoic acid 2,3-Epoxyhexanoic acid Kopp et al. (2008)

Chloramphenicol CmlA PCPCmlP Hydroxylation L-p- b-Hydroxy-L-p- Makris, Chakrabarti, aminophenylalanine aminophenylalanine Mu¨nck, and Lipscomb (2010)

Chlorobiocin CloN3 CloN5 Dehydrogenation L-proline Pyrrole-2-carboxylic acid Garneau-Tsodikova, Dorrestein, Kelleher, and Walsh (2005) Coronatine CmaB CmaD Chlorination L-allo-isoleucine g-Chloro-L-allo-isoleucine Vaillancourt, Yeh, Vosburg, O’Connor, and Walsh (2005)

Coronatine CmaC CmaD Cyclization g-Chloro-L-allo- (1S,2S)-1-amino-2- Vaillancourt, et al. isoleucine ethylcyclopropanecarboxylic (2005) acid

Dapdiamide DdaC PCPDpaD Epoxidation Nb-fumaramoyl-L- Nb-epoxysuccinamoyl-L- Hollenhorst et al. 2,3- 2,3-diaminopropionate (2010) diaminopropionate

FK506 TcsC ACPTcsA Reduction/ E-pent-2-enoic 2-Propylmalonic acid Mo et al. (2011) carboxylation acid

Kutzneride KtzD KtzC Chlorination L-isoleucine g-Chloro-L-isoleucine Neumann and Walsh (2008)

Kutzneride KtzA KtzC Cyclization g-Chloro-L- (1S,2R)-1-amino-2- Neumann and Walsh isoleucine ethylcyclopropanecarboxylic (2008) acid Kutzneride KthP KtzC Chlorination Piperazate (3S,5S)-5-chloropiperazate Jiang et al. (2011)

Kutzneride KtzO PCPKtzH Hydroxylation L-glutamic acid L-threo-b-hydroxy-glutamic Strieker, Nolan, acid Walsh, and Marahiel (2009)

Continued Table 16.1 Tailoring enzymes acting on carrier protein-tethered substrates that have been biochemically characterized from natural product biosynthetic pathways.—cont'd Tailoring Carrier Natural products enzyme protein Type of reaction Substrates Products Reference

Kutzneride KtzP PCPKtzH Hydroxylation L-glutamic acid L-erythro-b-hydroxy- Strieker et al. (2009) glutamic acid

Nikkomycin NikQ PCPNikP1 Hydroxylation Histidine b-Hydroxy-histidine Chen, Hubbard, O’Connor, and Walsh (2002)

Novobiocin NovI PCPNovH Hydroxylation L-tyrosine b-Hydroxy-L-tyrosine Chen and Walsh (2001)

Novobiocin NovJ/K PCPNovH Oxidation b-OH-L-tyrosine b-Ketotyrosine Pacholec, Hillson, and Walsh (2005)

Pacidamycin PacV PCPPacP Methylation L-2,3- L-3-N-methyl-2,3- Zhang et al. (2011) diaminobutyrate diamniobutyrate

Pyochelin PchG PCPPchF Reduction Hydroxyphenyl- Des-N-methyl-pyochelinic Reimmann et al. bisthiazolinic acid acid (2001) Pyoluteorin PltA PltL Chlorination Pyrrole-2- 4,5-Dichloropyrrole-2- Dorrestein, Yeh, carboxylic acid carboxylic acid Garneau-Tsodikova, Kelleher, and Walsh (2005)

Pyoluteorin PltE PltL Dehydrogenation L-proline Pyrrole-2-carboxylic acid Thomas, Burkart, and Walsh (2002) Sibiromycin SibG PCPSibE Hydroxylation 3-hydroxy-4- 3,5-dihydroxy-4- Giessen, Kraas, and methylanthranilic methylanthranilic acid. Marahiel (2011) acid

Syringomycin E SyrB2 PCPSyrB1 Chlorination L-threonine g-Chloro-L-threonine Vaillancourt, Yin, and Walsh (2005), Blasiak, Vaillancourt, Walsh, and Drennan (2006)

Syringomycin E SyrP PCP8SyrE Hydroxylation L-aspartic acid L-threo-b-hydroxy-aspartic Singh, Fortin, acid Koglin, and Walsh (2008)

Undecylprodigiosin RedW ORF9 Dehydrogenation L-proline pyrrole-2-carboxylic acid Thomas et al. (2002)

Vancomycin OxyD PCPBpsD Hydroxylation (R)-tyrosine (R)-b-hydroxy-tyrosine Cryle, Meinhart, and Schlichting (2010) aSee Fig. 16.2 for structures of the natural products with moieties (highlighted in gray) that were modified by the tailoring enzymes acting on carrier protein-tethered substrates. 330 Shuangjun Lin et al.

O O O OH H Cl N OMe N OH O N CO H O CCl 2 Cl NH 3 H O O 2 NS H OO N N N Armentomycin Barbamide H H O (dichloroaminobutanoic acid) O NH O OH O HO C HN O 2 HO C O O N 2 O OH H N O O O NH O O H NOC OH NH 2 NO OH O 2 O OH O H N Cl N N O O H H N O O NH O H 2 Cl O C-1027 H HO CO H N OH N 2 R H HO Cl CDA O O O HO Chlorobiocin (R = Cl) NH Chloramphenicol Novobiocin (R = CH ) O H 3 O NH OO2 H H CO H O H OH N 2 H N N HO 2 N OH H O O O O Coronatine Dapdiiamide E O

O OH N OH O O HO Cl HN HN O O O N HO O O O N O Cl N N H OH O O OH HO O H O O O O NNH H FK506 3 HN N O H N OHH HO NH 2 O H O OH Cl OR N O Kutzneride 2 (3S) R O N Kutzneride 8 (3 ) Nikkomycin (I, R = Glu) O Nikkomycin (X, R = OH) OH OH S S HN O H H O O N Cl N OH N N N NH CO H N 2 2 N Cl O H H Pyochelin N HO O O N H H Pacidamycin 1 OH O HO Pyoluteoin O NH H 2 N O HO HN N NH NH H H 2 H C N O HN OH 19 9 O N NH HO O OH H 2 OH N H OOH HO COH NH O O 2 O 2 HN O O OH H H O Cl N N O N O N O O H H O O O O HO OH N OH Cl HO Sibiromycin HO Cl OOO E H H H Syringomycin O N N N N N N H H H OMe HN O O H N O HO C 2 N N C H 2 H N 11 23 H Vancomycin OH Undecylprodigiosin HO OH Figure 16.2 Structures of natural products whose biosynthetic pathways feature tailoring enzymes that have been biochemically characterized to act on carrier protein-tethered substrates. Moieties resulted from tailoring enzymes acting on carrier protein-tethered substrates are highlighted in gray. See Table 16.1 for specific tailoring enzymes, ACP- or PCP-tethered substrates and their corresponding products, and the types of modification. incorporated directly into the C-1027 enediyne chromophore by the SgcC5 condensation enzyme (Lin, Huang, Horsman, Huang, Guo, & Shen, 2012; Lin, Van Lanen, & Shen, 2009; Fig. 16.3). In this chapter, we describe protocols for in vitro biochemical characterization of SgcC3 and SgcC that catalyze chlorination and hydroxylation of SgcC2-tethered (S)-b-tyrosine and analogues. They include: (i) preparation Tailoring Enzymes Acting on Carry Protein-Tethered Substrates 331

SgcC2 SgcC2 SgcC2 O O H H H S S S O N H N H N H N H 2 O SgcC3 2 O SgcC 2 SgcC5 O O O

Cl Cl OH Enediyne core OH OH OH Benzoxazolinate O Deoxy aminosugar O O R N 1 SgcC1 O O Å OH OH H N H 3 R ÅH O O 2 NH H N 2 3 O SgcC4 O C-1027 (R = OH, R = Cl) 1 2 20-Deschloro-C-1027 (R = OH, R = H) 1 2 22-Deshydroxy-C-1027 (R = H, R = Cl) 1 2 OH OH 20-Deschloro-22-deshydroxy-C-1027 (R = R = H) 1 2 (S)-b-Tyr L-Tyr Figure 16.3 Biosynthesis of the (S)-3-chloro-5-hydroxy-b-tyrosine moiety of C-1027 and engineered biosynthesis of C-1027 analogues. (S)-b-Tyrosine was first activated and tethered to the SgcC2 PCP by the SgcC1 adenylation enzyme. (S)-b-Tyrosyl-SgcC2 was sequentially chlorinated by SgcC3 and hydroxylated by SgcC to afford (S)-3- chloro-5-hydroxy-b-tyrosyl-SgcC2, which was directly incorporated into C-1027 by SgcC5. Manipulation of SgcC3 or SgcC in C-1027 biosynthesis resulted in the production of three C-1027 analogues, 20-deschloro-C-1027, 22-deshydroxy-C-1027, and 20-deschloro-22-deshydroxy-C-1027. of the holo-SgcC2 PCP; (ii) preparation of SgcC2-tethered (S)-b-tyrosine substrates; (iii) SgcC3-catalyzed chlorination of (S)-b-tyrosyl-SgcC2; (iv) SgcC-catalyzed hydroxylation of (S)-3-chloro-b-tyrosyl-SgcC2; and (v) exploitation of SgcC2-tethered (S)-b-tyrosine substrates for structural diversification.

2. METHODS

2.1. In vitro characterization of SgcC3-catalyzed chlorination of (S)-b-tyrosyl-SgcC2 SgcC3 is a FAD-dependent halogenase, acting only on SgcC2-tethered substrates and accepting both (S)- and (R)-b-tyrosyl-SgcC2. SgcC3 catalyzes preferentially chlorination but also bromination, and it does not catalyze fluorination or iodination. SgcC3 requires Cl (or Br ), O2, and reduced FAD. The latter can be supplied by the C-1027 pathway-specific flavin reductase SgcE6 or Escherichia coli flavin reductase Fre from FAD and NADH (Fig. 16.4B; Lin et al., 2007).

2.1.1 Expression in E. coli and overproduction and purification of apo-SgcC2 1. Most ACPs or PCPs of Streptomyces origin, upon expression in E. coli, are overproduced in apo-form. Follow the protocols provided in Methods Enzymology, volume 459 (Cheng, Coughlin, Lim, & Shen, 2009; 332 Shuangjun Lin et al.

A Svp SgcC2 SgcC2 OH SH CoA ADP apo-ACP holo-ACP

B SgcC2 SgcC2 ÅHHO SgcC1 S SgcC3 H S KOH Å H O H NHN H N H N 3 2 2 3 O O O H O O ATP PPi HO-X 2 + + X X X AMP OH SgcC2 OH OH OH S b (S)-b-Tyr SH FAD-OH FAD-OOH ( )-3-chloro- -Tyr (X = Cl) (S)-3-bromo-b-Tyr (X = Br)

H O O 2 2 FAD FADH 2

SgcE6 NADH NAD

C SgcC2 SgcC2 ÅH O SgcC1 H S SgcC H S KOH ÅH O H N H N H N H N 3 O 2 O 2 O 3 O ATP PPi FAD-OOH FAD-OH X + + X X OH X OH SgcC2 AMP OH OH O H O OH OH 2 2 (S)-b-Tyr SH FADH (S)-3-hydroxy-b-Tyr (X = H) 2 FAD (S)-3-fluoro-5-hydroxy-b-Tyr (X = F) (S)-3-chloro-5-hydroxy-b-Tyr (X = Cl) SgcE6 (S)-3-bromo-5-hydroxy-b-Tyr (X = Br) NAD NADH (S)-3-iodo-5-hydroxy-b-Tyr (X = I) (S)-3-methyl-5-hydroxy-b-Tyr (X = CH ) 3 Figure 16.4 In vitro characterization of SgcC3 as a FAD-dependent halogenase and SgcC as a FAD-dependent hydroxylase that act on SgcC2-tethered (S)-b-tyrosine and analogues: (A) Svp PPTase-catalyzed in vitro conversion of apo-SgcC2 into holo-SgcC2; (B) SgcC1-catalyzed preparation of (S)-b-tyrosyl-SgcC2, SgcC3-catalyzed chlorination or bromination of (S)-b-tyrosyl-SgcC2, and hydrolytic release from SgcC2 of the haloge- nated products (S)-3-chloro-b-tyrosine and (S)-3-bromo-b-tyrosine; and (C) SgcC1- catalyzed preparation of SgcC2-tethered (S)-b-tyrosine and analogues, SgcC-catalyzed hydroxylation of SgcC2-tethered (S)-b-tyrosine and analogues, and hydrolytic release from SgcC2 of the hydroxylated products (S)-3-hydroxy-b-tyrosine, (S)-3-fluoro-5- hydroxy-b-tyrosine, (S)-3-chloro-5-hydroxy-b-tyrosine, (S)-3-bromo-5-hydroxy-b-tyrosine, (S)-3-iodo-5-hydroxy-b-tyrosine, and (S)-3-methyl-5-hydroxy-b-tyrosine.

Horsman, Van Lanen, & Shen, 2009; Jiang, Rajski, & Shen, 2009)to express sgcC2 in E. coli BL21 (DE3) and to purify the overproduced apo-SgcC2 as an N-terminal His6-tagged fusion protein. 2. Dialyze the purified SgcC2 into 50 mM Tris–HCl (pH 7.5), containing 50 mM NaCl and 1 mM dithiothreitol (DTT), and concentrate using an Amicon Ultra-4 (3K, GE Healthcare, Piscataway, NJ). 3. Check the purity of the isolated protein by SDS-PAGE on a 15% gel (Fig. 16.5A), determine the concentration by Bradford assay (Bio- Rad, Hercules, CA), and store in 40% glycerol at 20 C until use. Tailoring Enzymes Acting on Carry Protein-Tethered Substrates 333

SgcE6 SgcC3 SgcC2 MW Stds SgcC1 MW Stds kD MW StdsSgcC 97 66

21 14

BCD mAU at 280 nm mAU at 280 nm mAU at 280 nm

I I I

II II II

III III III

15.0 20.0 18.0 20.0 22.0 24.0 15.0 20.0 25.0 Time (min) Time (min) Time (min) Figure 16.5 Representative data from in vitro characterization of SgcC3 and SgcC with SgcC2-tethered (S)-b-tyrosine and analogues as substrates. (A) SDS-PAGE analysis of SgcC2, SgcC3, and SgcE6 on a 15% gel and SgcC1 and SgcC on a 12% gel. (B) HPLC chromatograms of SgcC3-catalyzed chlorination of (S)-b-tyrosyl-SgcC2: (I) authentic (S)-b-tyrosine standard (●), (II) assay solution, and (III) authentic (S)-3-chloro-b-tyrosine standard (ç). (C) HPLC chromatograms of SgcC-catalyzed hydroxylation of (S)-b-tyrosyl- SgcC2: (I) authentic (S)-3-chloro-b-tyrosine standard (ç), (II) assay solution, (III) authentic (S)-3-chloro-5-hydroxy-b-tyrosine standard (r), and 4,5-dihydroxy-1,2-dithiane (*) presented in the assay. (D) HPLC chromatograms of SgcC3-catalyzed bromination of (S)-b-tyrosyl-SgcC2: (I) authentic (S)-b-tyrosine standard (●), (II) assay solution, and (III) authentic (S)-3-bromo-b-tyrosine standard (◊).

2.1.2 In vitro preparation of holo-SgcC2 by Svp and of (S)-b-tyrosyl-SgcC2 by SgcC1 1. Follow the protocols provided in Methods Enzymology, volume 459 (Cheng et al., 2009; Horsman et al., 2009; Jiang et al., 2009) to convert apo-SgcC2 into holo-SgcC2 using the Svp PPTase (Sanchez et al., 2001; Fig. 16.4A). Mix 0.8 mL of solution containing 160 mM apo-SgcC2, 0.8 mM CoA, 12.5 mM MgCl2,and2mM tris(2-carboxyethyl)phosphine hydrochloride (TCEP) in 100 mM Tris–HCl (pH 7.5), initiate the reaction by adding 5 mM Svp, and incubate at 25 C for 45 min. 334 Shuangjun Lin et al.

2. Express sgcC1 in E. coli BL21 (DE3) and purify the overproduced SgcC1 adenylation enzyme as an N-terminal His6-tagged fusion protein according to the literature procedure (Fig. 16.5A; Van Lanen et al., 2006); use SgcC1 to catalyze the tethering of (S)-b-tyrosine to holo-SgcC2 (Fig. 16.4B). Add 0.8 mL of solution, containing 4 mM (S)-b-tyrosine, 8 mM ATP, 2 mM TCEP, and 12.5 mM MgCl2,to the holo-SgcC2 solution from step 1. Initiate the reaction by adding 2 mM SgcC1, and incubate at 25 C for 1 h. 3. Purify (S)-b-tyrosyl-SgcC2 by ion exchange chromatography on a 5-mL HiTrap Q column (GE Healthcare). Preequilibrate the column with 50 mM Bis–Tris–HCl (pH 7.0), load the reaction mixture from step 2 to the column, and wash it with five column volumes of the same buffer. Elute the column with a linear gradient from 0% to 100% 1 M NaCl in 50 mM Bis–Tris–HCl (pH 7.0), in 25 column volumes at a flow rate of 3 mL/min. (S)-b-Tyrosinyl-SgcC2 is typically eluted between 0.35 and 0.4 M NaCl. 4. Desalt b-tyrosyl-S-SgcC2 from step 3 using a Superose 12 column (GE Healthcare) in 20 mM sodium phosphate, pH 7.0, and concentrate using an Amicon Ultra-4 (3K, GE Healthcare) prior to use in SgcC3 assay.

2.1.3 Expression in E. coli and overproduction and purification of SgcC3 1. Prepare PCR primers for amplification of sgcC3 from cosmid pBS1005 (Liu et al., 2002), clone the PCR product into the pET-30Xa/LIC vector (Novagen, Madison, WI) using a ligation-independent cloning procedure to yield the expression plasmid pBS1041, and sequence the construct to confirm PCR fidelity. With this construct, SgcC3 will be overproduced as an N-terminal His6-tagged fusion protein (Lin et al., 2007). 2. Introduce pBS1041 into E. coli BL21 (DE3) by transformation, and select transformants on LB agar plates containing 50 mg/mL kanamycin. 3. Pick a single colony to grow in 3 mL of LB containing 50 mg/mL kanamycin overnight at 37 C, and transfer 0.5 mL into 50 mL of LB containing 50 mg/mL kanamycin to grow again overnight at 37 Cto prepare the seed culture. Inoculate 500 mL of LB containing 50 mg/ mL kanamycin with 5 mL of the seed culture, and incubate at 18 C until it reaches an OD600 of 0.6. 4. Induce sgcC3 expression by adding IPTG to 0.1 mM and continue incu- bation at 18 C for 15–20 h. 5. Harvest the cells by centrifugation at 4 C, resuspend the cells in buffer A (100 mM sodium phosphate, pH 7.5, 300 mM NaCl) supplemented Tailoring Enzymes Acting on Carry Protein-Tethered Substrates 335

with a complete protease inhibitor tablet, EDTA-free (Roche Applied Science, Indianapolis, IN), lyse the cells by sonication (430 s pulse cycle), and centrifuge the lysate at 4 C and 15,000 rpm for 30 min to collect the clear supernatant. 6. Load the supernatant to a preequilibrated Ni-NTA agarose column (Qiagen, Valencia, CA) with buffer B (buffer A plus 10% glycerol), and wash the column sequentially with five column volumes of buffer B and five column volumes of buffer B containing 20 mM imidazole. Elute the column with five column volumes of buffer B containing 250 mM imidazole, and pool fractions containing SgcC3. 7. Desalt the purified SgcC3 using a PD-10 column (GE Healthcare) into 50 mM Tris–HCl (pH 7.5), containing 50 mM NaCl and 1 mM DTT, and concentrate using an Amicon Ultra-4 (10K, GE Healthcare). 8. Check the purity of the isolated protein by SDS-PAGE on a 15% gel (Fig. 16.5A), determine the concentration by Bradford assay (Bio-Rad), and store in 40% glycerol at 20 C until use.

2.1.4 Expression in E. coli and overproduction and purification of SgcE6 1. Prepare PCR primers for amplification of sgcE6 from cosmid pBS1006 (Liu et al., 2002) and clone the PCR product into the pET-30Xa/LIC vector (Novagen) using a ligation-independent cloning procedure to yield the expression plasmid pBS1042, in which SgcE6 will be overproduced as an N-terminal His6-tagged fusion protein. Sequence the construct to confirm PCR fidelity. 2. Follow steps 2–8, Section 2.1.3, to afford pure SgcE6 (Fig. 16.5A), and store in 40% glycerol at 20 C until use.

2.1.5 In vitro assay of SgcC3-catalyzed chlorination of (S)-b-tyrosyl-SgcC2 1. Set up the SgcC3-catalyzed halogenation of (S)-b-tyrosinyl-SgcC2 in 200 mL of reaction solution, containing 50 mM (S)-b-tyrosyl-SgcC2, 5mM NADH, 0.10 mM FAD, 100 mM NaCl, 1 mM TCEP, and 5 mM SgcE6 in 50 mM sodium phosphate buffer (pH 6.0), at 37 C(Fig. 16.4B). 2. Initiate the reaction by adding 20 mM SgcC3, and incubate at 37 C for 1 h. 3. Terminate the reaction by adding 35 mL of 70% trichloroacetic acid (TCA), and incubate on ice for 15 min to precipitate all proteins. 336 Shuangjun Lin et al.

4. Pellet the proteins by centrifugation at 4 C and 14,000 rpm for 15 min, wash the protein pellet twice with 200 mL of 5% cold TCA and once with 200 mL of ice-cold ethanol, and dry the pellet in a speed-vac for 10 min. 5. Redissolve the protein pellet in 150 mL of 0.1 N KOH solution, and incubate at 70 C for 15 min to hydrolyze the SgcC2-tethered substrate (S)-b-tyrosine and product (S)-3-chloro-b-tyrosine (Fig. 16.4B). 6. Adjust the solution with 2 N HCl to pH 6, cool on ice for 10 min, and remove the precipitated proteins by centrifugation at 4 C and 14,000 rpm for 15 min. Collect the supernatant, concentrate to dryness in a speed-vac, and redissolve the residue in 50 mLofH2O. 7. Subject 20 mL of the sample from step 6 to HPLC analysis on an Apollo C18 column (5 mM, 2504.6 mm, Alltech Associates Inc., Deerfield, IL) with UV detection at 280 nm. Elute the column at a flow rate of 1 mL/min with a 24-min linear gradient from 0% to 40% acetonitrile in 0.1% TFA. 8. Determine the peaks corresponding to (S)-b-tyrosine and (S)-3-chloro- b-tyrosine by comparison to authentic standards (see Fig. 16.5B for a representative HPLC chromatogram) and confirm their identity by ESI-MS analysis.

2.2. In vitro characterization of SgcC-catalyzed hydroxylation of (S)-b-3-chloro-tyrosinyl-SgcC2 SgcC is a FAD-dependent monooxygenase, acting only on SgcC2-tethered substrates, and requiring O2 and reduced FAD. The latter can be generated by the C-1027 pathway-specific flavin reductase SgcE6 or E. coli flavin reductase Fre from FAD and NADH. While (S)-3-chloro-b-tyrosyl-SgcC2 is the natural substrate for SgcC in C-1027 biosynthesis (Fig. 16.3), both (S)-3-bromo-b-tyrosyl-SgcC2 and (S)-3-iodo-b-tyrosyl-SgcC2 are better substrates, with (S)-3-fluoro-b-tyrosyl-SgcC2, (S)-3-methyl-b-tyrosyl- SgcC2, and (S)-b-tyrosyl-SgcC2 also serving as substrates albeit significantly poorer ones (Fig. 16.4C; Lin et al., 2008).

2.2.1 Expression in E. coli and overproduction and purification of SgcC 1. Follow steps 1–8, Section 2.1.3, to clone sgcC from pBS1005 (Liu et al., 2002), construct expression plasmid pBS1092, overproduce SgcC in E. coli BL21 (DE3), and purify SgcC as an N-terminal His6-tagged fusion protein (Lin et al., 2008). Tailoring Enzymes Acting on Carry Protein-Tethered Substrates 337

2. Check the purity of the isolated SgcC protein by SDS-PAGE on a 12% gel (Fig. 16.5A), determine the concentration by Bradford assay (Bio-Rad), and store in 40% glycerol at 25 C until use.

2.2.2 In vitro assay of SgcC-catalyzed hydroxylation of (S)-3-chloro-b-tyrosyl-SgcC2 1. Prepare (S)-3-chloro-b-tyrosyl-SgcC2 from (S)-3-chloro-b-tyrosine and holo-SgcC2 by taking advantage of the substrate promiscuity of SgcC1 (Van Lanen et al., 2006; Fig. 16.4C). Steps 2–4, Section 2.1.2, provide a protocol to prepare (S)-b-tyrosyl-SgcC2 from purified holo-SgcC2 using SgcC1. An alternative protocol is provided in this section for the preparation of (S)-3-chloro-b-tyrosyl-SgcC2 from apo- SgcC2 directly by coupled assay using both Svp and SgcC1. The two protocols afford comparative yields with >90% of the free (S)-b-tyrosine or analogues tethered to SgcC2 (Fig. 16.4). 2. Set up the in vitro 40-phosphopantetheinylation of apo-SgcC2 in 1.8 mL of reaction solution containing 200 mM apo-SgcC2, 1.0 mM CoA, 12.5 mM MgCl2, and 2.0 mM TCEP in 100 mM Tris–HCl (pH 7.5), at 25 C. Initiate the reaction by adding 10 mM Svp, and incubate at 25 C for 45 min. 3. Prepare a loading solution containing 7.0 mM (S)-3-chloro-b-tyrosine, 8mM ATP, 2.0 mM TCEP, and 12.5 mM MgCl2 in 100 mM Tris–HCl (pH 7.5), and mix it with an equal volume of the holo-SgcC2 reaction solution from step 2. Initiate the loading reaction by adding 5 mM SgcC1, and incubate at 25 C for 1 h. Follow steps 3 and 4, Section 2.1.2,to purify (S)-3-chloro-b-tyrosyl-SgcC2. 4. Set up the SgcC-catalyzed hydroxylation of (S)-3-chloro-b-tyrosyl- SgcC2 in 200 mL of reaction solution containing 250 mM (S)-3- chloro-b-tyrosyl-SgcC2, 5 mM NADH, 10 mM FAD, 1 mM TCEP, 50 mM NaCl, and 5 mM SgcC in 50 mM sodium phosphate (pH 6.0), at 25 C. 5. Initiate thereactions byadding1.5 mM SgcE6and incubate at 25 C for 1 h. 6. Terminate the reaction and recover (S)-3-chloro-b-tyrosyl-SgcC2 and its hydroxylated product (S)-3-chloro-5-hydroxy-b-tyrosyl-SgcC2 by following the steps 3 and 4, Section 2.1.5. 7. Redissolve the protein pellet from step 6 by adding first 5 mL of 1.5 M DTT and then 150 mL of 0.1N KOH, and incubate at 50 C for 15 min to hydrolyze the SgcC2-tethered substrate (S)-3-chloro-b- tyrosine and product (S)-3-chloro-5-hydroxy-b-tyrosine (Fig. 16.4C). 338 Shuangjun Lin et al.

8. Follow steps 6–8, Section 2.1.5, for sample preparation and HPLC analysis. Determine the peaks corresponding to (S)-3-chloro-b-tyrosine and (S)-3-chloro-5-hydroxy-b-tyrosine by comparison to authentic standards (see Fig. 16.5C for a representative HPLC chromatogram), and confirm their identity by ESI-MS analysis.

2.3. Exploitation of SgcC2-tethered (S)-b-tyrosine analogues for structural diversification 2.3.1 SgcC3-catalyzed bromination of (S)-b-tyrosyl-SgcC2 1. Prepare SgcC3 according to Section 2.1.3 and SgcE6 according to Sec- tion 2.1.4 with the exception of excluding NaCl in all buffers used for their purification. 2. Prepare the (S)-b-tyrosyl-SgcC2 according to Section 2.1.2. 3. Desalt the (S)-b-tyrosyl-SgcC2 sample from step 2 using a Superose 12 column (GE Healthcare) in 20 mM sodium phosphate (pH 7.0), and run the sample twice to ensure the complete removal of residual NaCl. 4. Set up the SgcC3-catalyzed bromination of (S)-b-tyrosyl-SgcC2 reaction in an identical condition to that of chlorination with the exception of replacing NaCl with 0.1 M NaBr and excluding TCEP from the assay solution, and follow the steps in Section 2.1.5 to carry out the reaction and analyze the product (Fig. 16.4B). Determine the formation of (S)-3- bromo-b-tyrosine by HPLC analysis and comparison with authentic standard (see Fig. 16.5D for a representative HPLC chromatogram), and confirm its identity by ESI-MS analysis.

2.3.2 SgcC-catalyzed hydroxylation of SgcC2-tethered (S)-b-tyrosine analogues 1. Prepare SgcE6 according to Section 2.1.4 and SgcC according to Section 2.2.2. 2. Prepare SgcC2-tethered b-tyrosine analogues of (S)-3-fluoro-b-tyrosyl- SgcC2, (S)-3-bromo-b-tyrosyl-SgcC2, (S)-3-iodo-b-tyrosyl-SgcC2, and (S)-3-methyl-b-tyrosyl-SgcC2 according to Section 2.1.2 with the exception of replacing (S)-b-tyrosine with corresponding analogues (Fig. 16.4C). 3. Since SgcC hydroxylates SgcC2-tethered (S)-b-tyrosine analogues with varying rates, the assay condition described for (S)-3-chloro-b-tyrosyl- SgcC2 in Section 2.2.2 needs optimization for each of the analogues to ensure efficient formation of the hydroxylated products. Tailoring Enzymes Acting on Carry Protein-Tethered Substrates 339

4. Set up the SgcC-catalyzed hydroxylation reaction in 200 mL of solution containing 250 mM SgcC2-tethered (S)-b-tyrosine or analogues, 5 mM NADH, 10 mM FAD, 1 mM TCEP, and 50 mM NaCl, in 50 mM sodium phosphate (pH 6.0) at 25 C. For (S)-3-bromo-b-tyrosyl-SgcC2 and (S)-3-iodo-b-tyrosyl-SgcC2, add 1.5 mM SgcC and 2 mM SgcE6 and incubate the reaction at 25 C for 20 min, while for (S)-3- methyl-b-tyrosyl-SgcC2, (S)-3-fluoro-b-tyrosyl-SgcC2, and (S)-b- tyrosyl-SgcC2, add 6 mM SgcC and 2 mM SgcE6 and incubate the reaction at 25 C for 1 h. 5. Terminate the reaction, recover SgcC2-tethered substrates and their hydroxylated products, release them from SgcC2 by hydrolysis, and determine their identities by HPLC and ESI-MS analyses by following the steps 6–8, Section 2.2.2 (Fig. 16.4C). For maximal sensitivity, use varying wavelengths to detect the formation of each of the hydroxylated products: (S)-3-fluoro-b-5-hydroxy-tyrosine from (S)-3-fluoro-b- tyrosyl-SgcC2 at UV 272 nm, (S)-3-bromo-5-hydroxy-b-tyrosine from (S)-3-bromo-b-tyrosyl-SgcC2 at UV 282 nm, (S)-3-iodo-5-hydroxy- b-tyrosine from (S)-3-iodo-b-tyrosyl-SgcC2 at UV 284 nm, (S)-3- methyl-5-hydroxy-b-tyrosine from (S)-3-methyl-b-tyrosyl-SgcC2 at UV 278 nm, and (S)-3-hydroxy-b-tyrosine from (S)-b-tyrosyl-SgcC2 at UV 277 nm.

3. CONCLUSION

We highlighted in this chapter the emerging roles CPs play in precursor biosynthesis and post-PKS or post-NRPS modifications and summarized tailoring enzymes that are known to act on CP-tethered substrates (Figs. 16.1 and 16.2; Table 16.1). By covalently tethering, CPs sequester the substrates from endogenous metabolite pools, thereby increasing their concentration at the active sites for catalysis. CPs also provide the critical protein–protein recognitions among the various enzymatic partners, and this feature provides a new opportunity to engineer natural product diversity by exploiting CPs to increase substrate promiscuity for the tailoring steps. Realization of the full potential of tailoring enzymes that act on CP- tethered substrates in engineered biosynthesis of natural product structural diversity depends on continued discovery of new members of this family of enzymes, further expansion of the catalytic portfolio, fundamental characterization of their reaction mechanisms, and exploitation of their 340 Shuangjun Lin et al. portability in the broad context of natural product biosynthetic machinery. The protocols provided here were developed from our current effort to characterize the SgcC3 halogenase and SgcC hydroxylase, acting exclusively on SgcC2-tethered b-tyrosine and analogues, in the biosynthesis of the (S)- 3-chloro-5-hydroxy-b-tyrosine moiety of the antitumor antibiotic C-1027 (Van Lanen & Shen, 2008), but should be applicable to mechanistic characterization and engineered exploitation of other tailoring enzymes that act on CP-tethered substrates in natural product biosynthesis and structural diversification. The ultimate goal would be to use the in vitro findings to guide in vivo engineering to produce designer natural product analogues. For example, it has already been demonstrated that variants of the b-tyrosine moiety can be tolerated by the C-1027 biosynthetic machinery, resulting in the production of several C-1027 analogues (Fig. 16.3; Kennedy et al., 2007; Van Lanen et al., 2005). It would be fascinating to investigate if the sets of b-tyrosine analogues that can be readily generated by SgcC3 and SgcC in vitro (Fig. 16.4) can be recapitulated in vivo to produce a focused library of C-1027 analogues, some of which could be developed into novel anticancer drugs.

ACKNOWLEDGMENT This work was supported in part by National Institute of Health (NIH) grant CA078747.

REFERENCES Blasiak, L. C., Vaillancourt, F. H., Walsh, C. T., & Drennan, C. L. (2006). Crystal structure of the non-haem iron halogenase in syringomycin biosynthesis. Nature, 440, 368–371. Chan, D. I., & Vogel, H. J. (2010). Current understanding of fatty acid biosynthesis and the acyl carrier protein. The Biochemical Journal, 430, 1–19. Chen, H., Hubbard, B. K., O’Connor, S. E., & Walsh, C. T. (2002). Formation of b-hydroxy histidine in the biosynthesis of nikkomycin antibiotics. Chemistry & Biology, 9, 103–112. Chen, H., & Walsh, C. T. (2001). Coumarin formation in novobiocin biosynthesis: b-Hydroxylation of the aminoacyl enzyme tyrosyl-S-NovH by a cytochrome P450 NovI. Chemistry & Biology, 8, 301–312. Cheng, Y.-Q., Coughlin, J. M., Lim, S.-K., & Shen, B. (2009). Type I polyketide synthases that require discrete acyltransferases. Methods in Enzymology, 459, 165–186. Christenson, S. D., Liu, W., Toney, M. D., & Shen, B. (2003). A novel 4-methylideneimidazole-5-one-containing tyrosine aminomutase in enediyne antitumor antibiotic C-1027 biosynthesis. Journal of the American Chemical Society, 125, 6062–6063. Christenson, S. D., Wu, W., Spies, M. A., Shen, B., & Toney, M. D. (2003). Kinetic analysis of the 4-methylideneimidazole-5-one-containing tyrosine aminomutase in enediyne antitumor antibiotic C-1027 biosynthesis. Biochemistry, 42, 12708–12728. Cryle, M. J., Meinhart, A., & Schlichting, I. (2010). Structural characterization of OxyD, a cytochrome P450 involved in b-hydroxytyrosine formation in vancomycin biosynthesis. The Journal of Biological Chemistry, 285, 24562–24574. Tailoring Enzymes Acting on Carry Protein-Tethered Substrates 341

Dorrestein, P. C., Yeh, E., Garneau-Tsodikova, S., Kelleher, N. L., & Walsh, C. T. (2005). Dichlorination of a pyrrolyl-S-carrier protein by FADH2-dependent halogenase PltA during pyoluterin biosynthesis. Proceedings of the National Academy of Sciences of the United States of America, 102, 13843–13848. Du, L., Sanchez, C., & Shen, B. (2001). Biosynthesis of hybrid peptide and polyketide metabolites: Prospects towards engineering novel molecules. Metabolic Engineering, 3, 78–95. Fischbach, M. A., & Walsh, C. T. (2006). Assembly-line enzymology for polyketide and nonribosomal peptide antibiotics: Logic, machinery, and mechanisms. Chemistry Review, 106, 3468–3496. Fischbach, M. A., & Walsh, C. T. (2010). Natural products version 2.0: Connecting genes to molecules. Journal of the American Chemical Society, 132, 2469–2493. Flatt, P. M., O’Connell, S. J., McPhail, K. L., Zeller, G., Willis, C. L., Sherman, D. H., et al. (2006). Characterization of the initial enzymatic steps of barbamide biosynthesis. Journal of Natural Products, 69, 938–944. Gago, G., Diacovich, L., Arabolaza, A., Tsai, S.-C., & Gramajo, H. (2011). Fatty acid biosynthesis in actinomycetes. FEMS Microbiology Reviews, 35, 475–497. Galonic´, D. P., Vaillancourt, F. H., & Walsh, C. T. (2006). Halogenation of unactivated car- bon centers in natural product biosynthesis: Trichlorination of leucine during barbamide biosynthesis. Journal of the American Chemical Society, 128, 3900–3901. Garneau-Tsodikova, S., Dorrestein, P. C., Kelleher, N. L., & Walsh, C. T. (2005). Charac- terization of the formation of the pyrrole moiety during clorobiocin and coumermycin A1 biosynthesis. Biochemistry, 44, 2770–2780. Giessen, T. W., Kraas, F. I., & Marahiel, M. A. (2011). A four-enzyme pathway for 3,5-dihydroxy-4-methylanthranilic acid formation and incorporation into the antitumor antibiotic sibiromycin. Biochemistry, 50, 5680–5692. Hollenhorst, M. A., Bumpus, S. B., Matthews, M. L., Jr., Bollinger, J. M., Kelleher, N. L., & Walsh, C. T. (2010). The nonribosomal peptide synthetase enzyme DdaD tethers Nb- fumaramoyl-L-2,3-diaminopropionate for Fe(II)/a-ketoglutarate-dependent epoxidation by DdaC during dapdiamide antibiotic biosynthesis. Journal of the American Chemical Society, 132, 15773–15781. Horsman, G. P., Van Lanen, S. G., & Shen, B. (2009). Iterative type I polyketide synthases for enediyne core biosynthesis. Methods in Enzymology, 459, 97–112. Jiang, W., Heemstra, J. R., Jr., Forseth, R. R., Neumann, C. S., Manaviazar, S., Schroeder, F. C., et al. (2011). Biosynthetic chlorination of the piperazate residue in kutzneride biosynthesis by KthP. Biochemistry, 50, 6063–6072. Jiang, H., Rajski, S. R., & Shen, B. (2009). Tandem acyl carrier protein domains in poly- unsaturated fatty acid synthases. Methods in Enzymology, 459, 79–96. Kennedy, D. R., Gawron, L. S., Ju, J., Liu, W., Shen, B., & Beerman, T. A. (2007). Single chemical modifications of the C-1027 enediyne core, a radiomimetic antitumor drug, affect both drug potency and the role of ataxia-telangiectasia mutated in cellular responses to DNA double-strand breaks. Cancer Research, 67, 773–781. Kopp, F., Linne, U., Oberthu¨r, M., & Marahiel, M. A. (2008). Harnessing the chemical activation inherent to carrier protein-bound thioesters for the characterization of lipopeptide fatty acid tailoring enzymes. Journal of the American Chemical Society, 130, 2656–2666. Lambalot, R. H., Gehring, A. M., Flugel, R. S., Zuber, P., LaCelle, M., Marahiel, M. A., et al. (1996). A new enzyme superfamily—The phosphopantetheinyl transferase. Chem- istry & Biology, 3, 923–936. Lin, S., Huang, T., Horsman, G. P., Huang, S.-X., Guo, X., & Shen, B. (2012). Specificity of the ester bond forming condensation enzyme SgcC5 in C-1027 biosynthesis. Organic Let- ters, 14, 2300–2303. 342 Shuangjun Lin et al.

Lin, S., Van Lanen, S. G., & Shen, B. (2007). Regiospecific chlorination of (S)-beta-tyrosyl- S-carrier protein catalyzed by SgcC3 in the biosynthesis of the enediyne antitumor antibiotic C-1027. Journal of the American Chemical Society, 129, 12432–12438. Lin, S., Van Lanen, S. G., & Shen, B. (2008). Characterization of the two-component, FAD- dependent monooxygenase SgcC that requires carrier protein-tethered substrates for the biosynthesis of the enediyne antitumor antibiotic C-1027. Journal of the American Chemical Society, 130, 6616–6623. Lin, S., Van Lanen, S. G., & Shen, B. (2009). A free-standing condensation enzyme catalyzing ester bond formation in C-1027 biosynthesis. Proceedings of the National Academy of Sciences of the United States of America, 106, 4183–4188. Liu, W., Christenson, S. D., Standage, S., & Shen, B. (2002). Biosynthesis of the enediyne antitumor antibiotic C-1027. Science, 297, 1170–1173. Makris, T. M., Chakrabarti, M., Mu¨nck, E., & Lipscomb, J. D. (2010). A family of diiron monooxygenase catalyzing amino acid beta-hydroxylation in antibiotic biosynthersis. Proceedings of the National Academy of Sciences of the United States of America, 107, 15391–15396. Marahiel, M. A., & Essen, L.-O. (2009). Nonribosomal peptide synthetases: Mechanistic and structural aspects of essential domains. Methods in Enzymology, 458, 337–351. Mercer, A. C., & Burkart, M. D. (2007). The ubiquitous carrier protein—A window to metabolite biosynthesis. Natural Product Reports, 24, 750–773. Mo, S. J., Kim, D. H., Lee, J. H., Park, J. W., Basnet, D. B., Ban, Y. H., et al. (2011). Biosynthesis of the allylmalonyl-CoA extender unit for the FK506 polyketide synthase proceeds through a dedicated polyketide synthase and facilitates the mutasynthesis of analogues. Journal of the American Chemical Society, 133, 976–985. Neumann, C. S., & Walsh, C. T. (2008). Biosynthesis of ()-(1S,2R)-allocoronamic acyl thioester by an FeII-dependent halogenase and a cyclopropane-forming flavoprotein. Journal of the American Chemical Society, 130, 14022–14023. Pacholec, M., Hillson, N. J., & Walsh, C. T. (2005). NovJ/NovK catalyze benzylic oxidation of a b-hydroxyl tyrosyl-S-pantetheinyl enzyme during aminocoumarin ring formation in novobiocin biosynthesis. Biochemistry, 44, 2819–2826. Reimmann, C., Patel, H. M., Serino, L., Barone, M., Walsh, C. T., & Haas, D. (2001). Essential PchG-dependent reduction in pyochelin biosynthesis of Pseudomonas aeruginosa. Journal of Bacteriology, 183, 813–820. Sanchez, C., Du, L., Edwards, D. J., Toney, M. D., & Shen, B. (2001). Cloning and characterization of a phosphopantetheinyl transferase from Streptomyces verticillus ATCC15003, the producer of the hybrid peptide-polyketide antitumor drug bleomycin. Chemistry & Biology, 8, 725–738. Shen, B. (2000). Aromatic polyketide biosynthesis. Topics in Current Chemistry, 209, 1–51. Shen, B. (2003). Polyketide biosynthesis beyond the type I, II, and III polyketide synthase paradigms. Current Opinion in Chemical Biology, 7, 285–295. Singh, G. M., Fortin, P., Koglin, A., & Walsh, C. T. (2008). Beta hydroxylation of the aspartyl residue in the phytotoxin syringomycin E: Characterization of two candidate hydroxylases AspH and SyrP in Pseudomonas syringae. Biochemistry, 47, 11310–11320. Staunton, J., & Weissman, K. J. (2001). Polyketide biosynthesis: A millennium review. Natural Product Reports, 18, 380–416. Strieker, M., Nolan, E. M., Walsh, C. T., & Marahiel, M. A. (2009). Stereospecific synthesis of threo- and erythro-b-hydroxyglutamic acid during kutzneride biosynthesis. Journal of the American Chemical Society, 131, 13523–13530. Thomas, M. G., Burkart, M. D., & Walsh, C. T. (2002). Conversion of L-proline to pyrrolyl- 2-carboxyl-S-PCP during undecylprodigiosin and pyoluterin biosynthesis. Chemistry & Biology, 9, 171–184. Tailoring Enzymes Acting on Carry Protein-Tethered Substrates 343

Ueki, M., Galonic´, D. P., Vaillancourt, F. H., Garneau-Tsodikova, S., Yhe, E., Vosburg, D. A., et al. (2006). Enzymatic generation of the antimetabolite g, g-dichloroaminobutyrate by NRPS and mononuclear iron halogenase action in a strep- tomycete. Chemistry & Biology, 13, 1183–1191. Vaillancourt, F. H., Yeh, E., Vosburg, D. A., O’Connor, S. E., & Walsh, C. T. (2005). Cryp- tic chlorination by a non-haem iron enzyme during cyclopropyl amino acid biosynthesis. Nature, 436, 1191–1194. Vaillancourt, F. H., Yin, J., & Walsh, C. T. (2005). SyrB2 in syringomycin E biosynthesis is a II nonheme Fe a-ketoglutarate- and O2-dependent halogense. Proceedings of the National Academy of Sciences of the United States of America, 102, 10111–10116. Van Lanen, S. G., Dorrestein, P. C., Christenson, S. D., Liu, W., Ju, J., Kelleher, N. L., et al. (2005). Biosynthesis of the b-amino acid moiety of enediyne antitunor antibiotic C-1027 featuring b-amino acyl-S-carrier protein intermediates. Journal of the American Chemical Society, 127, 11594–11595. Van Lanen, S. G., Lin, S., Dorrestein, P. C., Kelleher, N. L., & Shen, B. (2006). Substrate specificity of the adenylation enzyme SgcC1 involved in the biosynthesis of the enediyne antitumor antibiotic C-1027. The Journal of Biological Chemistry, 281, 29633–29640. Van Lanen, S. G., & Shen, B. (2008). Biosynthesis of enediyne antitumor antibiotics. Current Topics in Medicinal Chemistry, 8, 448–459. Walsh, C. T., Chen, H., Keating, T. A., Hubbard, B. K., Losey, H. C., Luo, L., et al. (2001). Tailoring enzymes that modify nonribosomal peptides during and after chain elongation on NRPS assembly lines. Current Opinion in Chemical Biology, 5, 525–534. Weissman, K. J. (2009). Introduction to polyketide biosynthesis. Methods in Enzymology, 459, 3–16. Zhang, W., Ntai, I., Bolla, M., Malcolmson, S. J., Kahne, D., Kelleher, N. L., et al. (2011). Nine enzymes are required for assembly of the pacidamycin group of peptidyl nucleoside antibiotics. Journal of the American Chemical Society, 133, 5240–5243.