Natural Product Reports

Interrupted adenylation domains: unique bifunctional enzymes involved in nonribosomal peptide biosynthesis

Journal: Natural Product Reports

Manuscript ID: NP-REV-09-2014-000120.R1

Article Type: Highlight

Date Submitted by the Author: 12-Jan-2015

Complete List of Authors: Labby, Kristin; Beloit College, Chemistry Watsula, Stoyan; University of Michigan, Medicinal Chemistry Garneau-Tsodikova, Sylvie; University of Kentucky, Pharmaceutical Sciences

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Interrupted adenylation domains: unique bifunctional enzymes involved in nonribosomal peptide biosynthesis

Kristin J. Labby, a Stoyan G. Watsula,b and Sylvie Garneau-Tsodikova* c

Received (in XXX, XXX) Xth XXXXXXXXX 20XX, Accepted Xth XXXXXXXXX 20XX

5 DOI: 10.1039/b000000x

Covering up to 2014

Nonribosomal peptides (NRPs) account for a large portion of drugs and drug leads currently available in the pharmaceutical industry. They are one of two main families of natural products biosynthesized on megaenzyme assembly-lines composed of multiple modules that are, in general, each comprised of three 10 core domains and on occasion of accompanying auxiliary domains. The core adenylation (A) domains are known to delineate the identity of the specific chemical components to be incorporated into the growing NRPs. Previously believed to be inactive, A domains interrupted by auxiliary enzymes have recently been proven to be active and capable of performing two distinct chemical reactions. This highlight summarizes current knowledge on A domains and presents the various interrupted A domains found in a number of 15 nonribosomal peptide synthetase (NRPS) assembly-lines, their predicted or proven dual functions, and their potential for manipulation and engineering for chemoenzymatic synthesis of new pharmaceutical agents with increased potency.

the NRP to be formed as it is responsible for the selection and 1 Introduction activation of the proteinogenic or non-proteinogenic 1 20 2 Adenylation domain core signature sequences and 45 building block to be incorporated into the growing peptide chain structures prior to covalent attachment onto the 4’-phosphopantetheinyl 3 Interrupted adenylation domains (Ppant) arm of the active (holo) downstream T domain partner. The C domains, normally located between consecutive pairs of A 3.1 Overview of interrupted adenylation domains and T domains, serve to link the upstream and downstream 3.2 Adenylation domains with methyltransferase activity 50 building blocks tethered to the T domains that surround them. A

25 3.3 Adenylation domains with ketoreductase activity thioesterase (TE) domain, located at the C-terminus of the module acting last during the natural product biosynthesis 3.4 Adenylation domains with oxidase or monooxygenase normally releases the peptidic chain from the NRPS assembly- activity line. 4 Perspectives 55 In addition to the three core domains, in order to add 5 Acknowledgements functional groups and structural diversity to the natural products to be biosynthesized, NRPS modules may contain strategically 30 6 Notes and references embedded auxiliary domains, such as cyclization (Cy), dehydratase (DH), epimerase (E), formylase (F), ketoreductase 1. Introduction 60 (KR), methyltransferase (M), monooxygenase (MOx), oxidase Nonribosomal peptides (NRPs) are natural products that exhibit a (Ox), and reductase (R) domains. wide range of pharmaceutically relevant biological activities, More recently, A domains interrupted by portions of auxiliary including anti-cancer, anti-fungal, anti-malarial, anti-microbial, domains including M, KR, Ox, and MOx have been observed in the NRPSs responsible for the production of thiocoraline, 2 35 and anti-viral functions. NRPs are biosynthesized on 3 4 5 6 nonribosomal peptide synthetase (NRPS) assembly-lines, which 65 kutznerides, pyochelin, yersiniabactin, micacocidin, 7 8 9 10 11,12 are divided into multiple modules that are each generally enniatins, microcystin, micropeptin, tubulysin, cereulide, 13 14 15 16 composed of at least three core domains: a condensation (C), an valinomycin, myxothiazol, melithiazole, and indigoidine. adenylation (A), and a thiolation (T) domain (in the literature also Originally thought to be inactive as the auxiliary domains are usually not found to interrupt A domains or other NRPS domains, 40 referred to as peptidyl carrier protein (abbreviated as PCP, PC, or P) or carrier protein (CP)). 70 some of these interrupted A domains have now been found to The A domain plays a critical role in dictating the structure of take an active role in the biosynthesis of a variety of natural products, including kutznerides,17 thiocoraline, and cereulide.18

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Table 1. Highly conserved core motifs of A, M, KR, Ox and MOx domains of NRPSs. Domain Core(s) Consensus sequence Role

Adenylation a1 L(T/S)YxEL Structural. At N-terminus of domain, caps an α-helix. 19

a2 LKAGxAYL(V/L)P(L/I)D Structural. Properly aligns Gly78. 19 a3 LAYxxYTSG(S/T)TGxPKG Substrate binding. Acts as a loop and positions the β,γ-phosphates correctly. 19

a4 ΦDxS Substrate binding. Aromatic residue terminates an α -helix that forms side of acyl- binding pocket. Aromatic residue also switches conformation between two enzyme states. 19 a5 NxYGPTE Structural and substrate binding. Invariant glutamic acid coordinates Mg 2+ ion. Adenine ring of ATP is stacked against aromatic residue. 19 a6 GELxIGx(V/L)ARGYL Structural. Stabilizes distorted β -sheets in the N-terminal domain. 19

a7 Y(R/K)TGDL Substrate binding and catalytic. Aspartic acid residue is 100% conserved and hydrogen bonds with ATP through the ribose hydroxyls. 19

a8 GRxDxxxKxxGxxxELxxxE Structural and substrate binding. Arginine stabilizes the ribose through its hydroxyls. A hinge is formed at aspartic acid residue (or occasionally lysine). In the thioester- forming conformation, the glycine forms part of the pantetheine tunnel. 19

a9 (L/V)Px ΦM(L/V/I)P Catalytic. Stabilizes thioester-forming conformation by properly positioning residues to interact with T domain. 20 a10 NGK(V/L)DR Catalytic. In the adenylate-forming conformation, lysine is within the active site. 19

Methylation M1 VL(D/E)GxGxG S-adenosylmethionine (SAM) binding. Methyltransferase: catalyze N- or C-methylation of amino acid residues. M2 NELSxYRYxAV M3 VExSxARQxGxLD Oxidase Ox1 KYxYxSxGxxY(P/G)VQ Ox2 GxxxG(L/V)xxGxYYY(H/D) P Ox3 IxxxYG Ketoreductase KR GGxGxxGxxxA 21 YxxxN 21 Monooxygenase MOx1 GFxxxxxxExH 14 MOx2 VxPxxxPxxxxEExxxxDxxxx 14 GRxxxxxxxG Φ designates an aromatic amino acid residue and x is any residue.

22 In this highlight, we present an overview of these interrupted A 10 sequence motifs, a1-a10 (Table 1). These highly conserved core domains as well as what is known about the structures and core motifs serve a variety of functions, including structural, substrate recognition sequences of A domains in general. binding, and catalytic roles. While the ten signature sequence motifs are highly conserved

5 2. Adenylation domain core signature sequences across A domains, their structures are diverse (Fig. 1). A domains and structures 15 contain approximately 550 amino acid residues. The crystal structures reveal that A domains typically consist of a large N- Adenylation domains determine the selection of the next amino terminal domain containing conserved signature sequences a1-a7 acid residue to be incorporated along the NRPS assembly-line. A and a smaller C-terminal domain consisting of a8-a10. The active domains can be identified by ten conserved core signature site, where ATP binds, is located between these two subunits.

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1AMU: gramacidin synthetase 1 1PG3: acetyl-CoA synthetase 2VSQ: surfactin A synthetase

a5 a6

a6 a6 a7 a3 a10 a3 a7 a1 a5 a8 a2 a7 a8 a4 a9 a8 a5 a9 a4 a1 a3 a4 a2 a2 a10 a1 a10

3DHV: DltA ligase 3ITE: SidN synthetase

a6 a3 a6 a7 a7 a9 a5 a2 a5 a2 a8 a4 a8 a4 a1 a1 a3 a10

3RG2: enterobactin synthase component E (EntE) 4DG8: Pseudomonas aeruginosa PA1221 a4 a5 a6 a6 a7 a5 a1 a3 a7 a8 a10 a8 a2 a10 a9 a9 a2 a1 a4 a3

Fig. 1 A. Structures of adenylation domains from gramicidin synthetase 1 (1AMU) 23 and SidN siderophore synthetase (3ITE). 24 Crystal structures are also shown of other adenylate-forming enzymes: acetyl-CoA synthetase (1PG3), 25 surfactin A synthetase C (2VSQ), 26 D-alanine-poly(phosphoribitol) (DltA) ligase (3DHV), 27 enterobactin synthase component E (EntE) (3RG2), 28 and Pseudomonas aeruginosa PA1221 (4DG8). 29 Notes : (i) The 10 conserved 5 signature sequences of A domains with the a1-a10 core signature sequences highlighted in black (a1), red (a2), orange (a3), yellow (a4), green (a5), aqua (a6), dark blue (a7), purple (a8), magenta (a9), and brown (a10), respectively. (ii) The sequence of 1PG3 does not contain an a9 motif; the crystal structure of 3ITE does not include a9 or a10.

In recent years, a class of small (approximately 70 amino acid domains, in which an additional domain, or part of a domain, is residues) proteins called MbtH-like proteins found to be encoded inserted between two core sequences, most commonly a8 and a9. 30,31 10 within many NRPS gene clusters have been studied in detail. 30 In this section, we cover fourteen natural product scaffolds made These small proteins are reported to have strong interactions with by NRPSs containing interrupted A domains: thiocoraline, their corresponding A domains and are suspected to play a role in kutznerides, pyochelin, yersiniabactin, micacocidin A, enniatins, increasing binding affinity between the A domain and its amino micropeptins, microcystins, tubulysins, cereulide, valinomycin, acid substrate. MbtH-like proteins are not necessary for all A myxothiazols, melithiazoles, and indigoidine. These interrupted 32 15 domains expression and/or activity. They seem to have high 35 A domains are interrupted by methyltransferase (M), specificity for their particular A domain partners, but some ketoreductase (KR), oxidase (Ox), and monooxygenase (MOx) examples of weak complementarity are reported. 33 The roles of domains. MbtH-like proteins have been explored in a variety of 3.2 Adenylation domains with methyltransferase activity biochemical and structural studies, 17,33-46 but their exact functions 20 and mechanisms of action still remain elusive. The NRPs thiocoraline, kutznerides, pyochelin, yersiniabactin, 40 micacocidin A, enniatins, micropeptins, microcystins, and 3. Interrupted adenylation domains tubulysins are biosynthesized by NRPSs that contain A domains interrupted with M domains (Fig. 2 and Fig. 3). These M domains 3.1 Overview of interrupted adenylation domains are capable of S-, N-, or O-methylation (Fig. 4). Typically O- NRPs obtain their unique structures in part from the diversity of methyltransferases exist as stand-alone domains, as exemplified 47 the hundreds of viable substrates incorporated by A domains, 45 in type I polyketide biosynthesis. However, in NRPSs, M 25 and also from the incorporation of auxiliary domains into specific domains are normally embedded in modules of the assembly- modules of NRPS assembly-lines. As additional means to lines as intact full-length domains.48 We note that other A incorporate structural diversity, Nature has evolved interrupted A domains interrupted by methyltransferases exist; some are

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functional, some are not functional. 48 In the unique cases involved in the biosynthesis of yersiniabactin contains an M

presented in this section, portions of M domains are found 60 domain between a8 and a9 of its A 1 domain which also bears the embedded between different consensus sequences of A domains, Gly to Arg mutation within the SAM-binding motif. As expected, either between a2 and a3 or between a8 and a9 (Table 1 and Figs. this mutation prevents methylation of the yersiniabactin 5 2 and 3). nitrogen (indicated by an orange arrow in Fig. 2), The NRP thiocoraline is a bisintercalator with a twofold which corresponds to the methylated nitrogen in pyochelin. symmetric bicyclic scaffold that contains two rare S-methylated 65 RSc1806, the HMWP2 homologue found in Ralstonia 49,50 L-Cys moieties (Fig. 2). Originally, TioN, which consists of solanacearum GMI1000 and involved in micacocidin A an A domain interrupted between motifs a2 and a3 by part of an biosynthesis, also contains a non-functional M domain inserted 6 10 M domain containing an S-adenosylmethionine (SAM)-binding between a8 and a9 of its A 2 domain. Interestingly, another M sequence, was hypothesized to be non-functional due to this domain is inserted into a different protein in the micacocidin A 2 disruption. Upon further examination, however, and because of 70 NRPS gene cluster, RSc1811 (homologous to the latter half of its A site specificity for L-Cys (DLYDLSLV), TioN was HMWP1, Fig. 3). This M domain, located between a8 and a9 of

proposed to be responsible for side-chain S-methylation of this A4, does not bear the Gly to Arg mutation, so is most likely 51 15 residue just after its incorporation during biosynthesis (Fig. 4A). responsible for the methylation of the L-Cys residue added by A 4, Recently, it was reported that TioN, in the presence of its MbtH- though this NRPS has yet to be thoroughly characterized. like partner TioT, is capable of both adenylation of L-Cys 75 The most common interrupted A domains identified are those followed by its S-methylation. 32 This means of S-methylation is involved in N-methylation of peptide backbones. 48 While this is unique in NRPS biosynthesis and moreover, the position of not an exhaustive list, examples other than those discussed herein 55 56 57 20 interruption, between a2 and a3 is unprecedented. Furthermore, include the cyclosporines, actinomycin, PF1022A, the TioS thiocoraline biosynthesis module includes two A anabaenopeptilide, 58 complestatin, 59 pristinamycin, 60 thaxtomin, 61 62 domains interrupted by N-methyltransferase domains that 80 and barbamide. These N-methylated NRPs all use SAM as a methylate backbone amine nitrogens. 52 This more common type cosubstrate and are interrupted between a8 and a9. Examining the of interrupted A domain will briefly be discussed later. crystal structures of A domains, this position would be 25 Kutznerides are cyclic depsipeptides isolated from soil accommodating to an additional moiety (Fig. 1). The structure of actinomycetes and have antifungal and antimicrobial activity. 53 enniatin B is shown (Fig. 2) as a representative NRP structure Kutznerides feature an O-methylated L-Ser residue 85 with N-methylation of its backbone. Enniatins are depsipeptides

unprecedentedly incorporated by the interrupted A 4 domain of (where one or more amide bonds are replaced by ester bonds) KtzH (Figs. 3 and 4A).17 KtzH contains four A domains, the produced by NRPS gene clusters from the fungal genus Fusarium 30 second of which is interrupted between a8 and a9 by a SAM- (Fig. 3). Enniatin structures consist of six alternating residues of binding containing portion of an M domain. The importance of D-hydroxyisovaleric acid ( D-HIV) and N-methylated amino acids 7 the MbtH-like protein partner KtzJ for expression of 90 linked by amide and ester bonds.

KtzH(A 4MA 4T4) was recently reported and the order of Another family of NRP compounds featuring backbone N- adenylation of L-Ser by the A domain portion of KtzH(A 4MA 4T4) methylation are the cyclic heptapeptides microcystins from the 9 35 followed by loading onto the T 4 domain and further O- Microcystis cyanobacterial genus (Fig. 4B). Over 80 17 methylation by the M portion of KtzH(A 4MA 4T4) established. microcystins have been identified, including microcystin-LR Similarly to thiocoraline and kutznerides, the biosynthesis of 95 (Fig. 2). Microcystins feature a mix of natural and unnatural the NRP pyochelin involves an A domain interrupted by a SAM- amino acids; the most common scaffold is cyclic Adda-D-Glu- binding portion of an M domain, but this M domain has N- Mdha-D-Ala-L-X-D-MeAsp-L-Y (in which Adda is 3-amino-9- 4 40 methyltransferase activity. Pyochelin is a siderophore produced methoxy-2,6,8-trimethyl-10-phenyl-4,6-decadienoic acid, D- by Pseudomonas aeruginosa during iron starvation and MeAsp is 3-methylaspartic acid, Mdha is N-methyl- 54 contributes to the virulence of P. aeruginosa in animals. The 100 dehydroalanine, and X and Y are varying amino acids). PchF subunit of the four-protein, 11-domain pyochelin NRPS Installation of these N-methyl groups also occurs by M domains assembly-line contains a SAM-binding M domain located within embedded between a8 and a9 of A domains. Similar in structure

45 A2 between a8 and a9 (Fig. 3). L-Cys is incorporated by PchF, to microcystins are the cyanobacterial depsipeptides cyclized by the C 2 domain, and then, after AMP activation by the micropeptins. The micropeptin NRPS from M. aeruginosa K-139, A2 domain and reduction from to thiazolidine by PchG, 105 which produces micropeptin K139, contains an M domain within the M domain is responsible for its N-methylation. Mutation of a the A 6 domain (Fig. 3). A 6 incorporates Tyr and the embedded M key residue within the M domain signature sequence, Gly1167, to domain N-methylates it (Fig. 2). Micropeptin biosynthesis gene 50 arginine eliminated N-methyltransferase activity and prevented clusters from many other M. aeruginosa strains ( e.g. , NIES-843, subsequent release by TE from PchF. 4 Interestingly, PchE was NIVA-CYA 172/5, and PCC 7806) also contain M domains also found to contain a similar M domain insertion between a8 110 embedded between a8 and a9 of an A domain that are responsible 9 and a9 of A 1, but the conserved signature motif for SAM-binding for peptide backbone N-methylation. is another naturally contains an arginine in place of a key glycine residue cyanobacterial peptide that is structurally similar to microcystins 55 (Gly1130), much like the aforementioned PchG G1167R mutant. and micropeptins, but nodularin is composed of only five amino Furthermore these genes are similar to those involved in the acids in the peptide ring. It too contains N-methyl moieties added 63 biosynthesis of structurally similar yersiniabactin 115 by an M domain spliced into an A domain between a8 and a9. and micacocidin A (Fig. 2); Yersenia pestis HMWP2, which is Microcystins, micropeptins, and nodularin are potent inhibitors of

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MeS HO Cl 3- OH -Glu R R R R O O 1 2 3 4 H Kutzneride 1 (S) H H H Me O N N OH N N N Cl Kutzneride 2 (S) Cl H H Me H Kutzneride 3 (R) H H H Me S S O O HN H N Kutzneride 4 (R) H π-bond Me O O S S O OH N H Kutzneride 5 (R) H H H H N N N O N N O HO O OO Kutzneride 6 (R) OH π-bond Me H Kutzneride 7 (S) H H H H O O HN O O OH SMe O Kutzneride 8 (R) Cl H H Me R4 Kutzneride 9 N (S) H π-bond Me Thiocoraline N N R3 H OMe TioN = A M A TioS = A3 M A3 A4 M A4 R2 KtzH = A4 M A4 a2 a3 a8 a9 a8 a9 R1 a8 a9

OH S H S OH S H S OH S H S N N H N H N OH HN S N S O O O HO N HO N OH OH Pyochelin Yersiniabactin Micacocidin A

PchF = A2 M A2 HMWP2 = A1 M A1 RSc1811 = A4 M A4 a8 a9 a8 a9 a8 a9

O OH O NH N HN NH H2N O O HN O O HN OH O HN N O N MeO NH H H N O O O NH O O N O H O O O O N O O N O OH O N N N O O H O O NH OH NH CO 2H O HN NH 2 Enniatin B Micropeptin K139 Microcystin-LR

EnS = A M A 2 2 McnC = A6 M A6 McyA = A3 M A3 a8 a9 a8 a9 a8 a9

R O 2

O O O H N N N N N O S H O CO 2H

OR1

Tubulysin A R1 = CH 2CH(CH3)2; R2 = OH Tubulysin B R1 = CH 2CH 2CH 3; R2 = OH Tubulysin D R1 = CH 2CH(CH3)2; R2 = H Tubulysin E R1 = CH 2CH 2CH 3; R2 = H

TubB = A1 M A1 TubC = A3 M A3 a8 a9 a8 a9 Fig. 2 Structures of natural products with adenylation (A) domains interrupted by methyltransferase (M) domains in their biosynthetic NRPS machinery. Regions of the structure synthesized by the interrupted A domains are highlighted in color. See Fig. 3 for a definition of domain abbreviations.

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THIOCORALINE: TioO TioR TioS

T C1 A1 T1 E1 C2 A2 T2 C3 A3 M A3 T3 C4 A4 M A4 T4 TE a8 a9 a8 a9 TioN

A M A a2 a3 KUTZNERIDE: KtzE KtzG KtzH

A1 T1 E1 C2 T2 A2 KR T2’ C3 A3 T3 E3 C4 A4 M A4 T4 C5 A5 T5 E5 C6 A6 T6 TE a8 a9 PYOCHELIN: PchD PchE PchF PchG

A CP C1 A1 T1 C2 A26 M A2 T2 TE R a8 a9 YERSINIABACTIN: YbtE HMWP2 HMWP1 YbtU

A T CyC12 A16 M A1 T1 C2 T2 KS AT M KR CP C3 M T3 TE R a8 a9 MICACOCIDIN A: RSc1806 RSc1810 RSc1811 RSc1805

A1 T1 KS AT KR CP C2 A26 M A2 T2 C3 T3 KS M KR CP C4 A46 M A4 T4 TE R a8 a9 a8 a9 ENNIATIN B: EnS

C1 A1 T1 C2 A2 M A2 T2 T3 C3 a8 a9 MICROPEPTIN K139: McnA McnB McnC McnE

C1 A1 T1 C2 A2 T2 C3 A3 T3 C4 A4 T4 C5 A5 T5 C6 A6 M A6 T6 C7 A7 T7 TE a8 a9 MICROCYSTIN-LR: McyG McyD McyE

A1 T1 KS AT M KR CP KS AT M DH KR CP KS AT DH KR CP KS AT M CP AmT C2 A2 T2 C3

McyA McyB McyC

A3 M A3 T3 C4 A4 T4 E C5 A5 T5 C6 A6 T6 C7 A7 T7 TE a8 a9 TUBULYSIN: TubB TubC TubD

C1 A1 M A1 T1 C2 A2 T2 C3 A3 M A3 T3 KS AT S KR DH ER CP HC A4 T4 Ox a8 a9 a8 a9 or

KS AT KR DH ER CP HC A4 T4 Ox

TubE TubF

C5 A5 T5 KS AT KR M DH ER CP TE or KS AT M KR DH ER CP TE

Fig. 3 Structural organization of thiocoraline, kutzneride, pyochelin, yersiniabactin, micacocidin, enniatin B, micropeptin K139, microcystin-LR, and tubulysin NRPSs. Each domain of the NRPSs is depicted in a different color. Abbreviations used for each domain: A, adenylation; C, condensation; T, thiolation (T domains have also been termed peptidyl carrier protein [PCP, PC, or P] and carrier protein [CP] in the literature); KR, ketoreductase; M, 5 methyltransferase; MOx, monooxygenase; Ox, oxidase; TE, thioesterase; KS, β-ketoacyl synthase; AT, acyltransferase; AmT, aminotransferase; E, epimerization; DH, β-hydroxy-dehydratase; ER, enoyl reductase; HC, heterocyclization; CP, acyl carrier protein; Cy, cyclization; R, reductase; S, spacer.

eukaryotic serine/threonine protein phosphatases. 8 These methylated. It remains unclear how this methyl group is further cyanobacterial cyclic peptides are hepatotoxic and are a oxidized and acylated as observed in the final product. 64 significant threat to human and animal health because they 20 3.3 Adenylation domains with ketoreductase activity 10 contaminate the water when they are produced during (blue-green algae) blooms. Nature incorporates functionalities into NRPs using various Tubulysins (Fig. 2) are produced by an NRPS that contains mechanisms; for example aforementioned enniatin NRPS two interrupted A domains, both interrupted between a8 and a9 incorporates D-HIV directly via an A domain coding specifically with M domains (Fig. 3). 10 The first, located within TubB, is for D-HIV, while cereulide incorporates D-HIV via an A domain 15 responsible for the incorporation and subsequent N-methylation 25 that codes for α-ketoisovaleric acid, which is then modified by of pipecolic acid. The second interrupted A domain, located the interrupting KR domain (Fig. 5A). Cereulide is an emetic within TubC, incorporates valine, which is also subsequently N- toxin from Bacillius cereus , and is structurally similar to

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valinomycin from Streptomyces spp. Both cereulide and α-position by the MOx (Fig. 8B). The A 3 and C 3 domains of valinomycin are macrocyclic potassium ionophores. They both MtaG/MelG act before the MOx of the interrupted A domain (Fig are cyclododecadepsipeptides consisting of alternating α-keto 8B). 15 Biosynthesis of melithiazole A differs from myxothiazol A acids (six) and amino acids (six). The NRPSs of cereulide and in the identity of the starter unit (isobutyrate for Mel) added to 18 5 valinomycin are also very similar to one another (Fig. 6). Both 35 MtaB/MelB (Fig. 5) as well as in the formation of the terminal contain two A domains interrupted by KR domains between a8 amide (myxothiazol A) versus the terminal methyl ester and a9. These KR domains are responsible for the reduction of (melithiazole A) (Fig. 8B). 15 Additional genes MelK and MelJ the α-ketone to a hydroxyl group, which then forms an ester bond are responsible for melithiazole terminal methyl ester formation to the next residue incorporated (Fig. 7). (Fig. 8B). 66

A. Si de-c hain meth ylati on: A O O OH OAMP T O O H H TioN(A4) TioS( T4) H H O O O N N O KtzH(A4) KtzH(T4) O N N O O O NH NH S O O 2 2 O NH HR HR O O NH O ATP PP O R = S, L-Cys (Thiocoraline ) i NH 2 O O O R = O, L-Ser (Kutzneride) HR O O O HN O O HN O O O O TioN(M) TioN(M) TioN(M) O O H H N NH KtzH(M) KtzH(M) KtzH(M) N NH O O O O O O T OH TioN(A4) OAMP TioS( T4) Cereulide Valinomycin KtzH(A ) KtzH(T ) O 4 O 4 S NH NH 2 2 O CesA =A KR A CesB =A KR A Vlm1 =A KR A Vlm2 = A KR A Me R Me R 1 1 3 3 1 1 3 3 ATP PP i NH 2 a8 a9 a8 a9 a8 a9 a8 a9 Me R

B OMe B. Backbone methylation: T O McnC(A ) McnC(T ) S N O OH 6 OAMP 6 H2N McyA(A ) McyA(T ) NH O 3 O 3 S N S TubC(A3) TubC(T3) MeO NH 2 NH 2 NH 2 O O R R NH 2 Myxothiazol A O ATP PP i R HN McnC(M) McnC(M) McnC(M) MtaD = A2 Ox A2 MtaG = A3 MOx A3 NH 2 McyA(M) McyA(M) McyA(M) a8 a9 a4 a5 O TubC(M) TubC(M) TubC(M) Indigoidine OMe H N IndC/IgiD = A Ox A McnC(A6) McnC(T6) T S O OH OAMP a8 a9 McyA(A3) McyA(T3) O O N S MeO OMe TubC(A3) TubC(T3) S NH Me NH Me R R O Melithiazole A ATP PP i NH Me R MelD = A2 Ox A2 MelG = A3 MOx A3 R = CH 2OH, L-Ser (Microcystin) 40 a8 a9 a4 a5 R = H 2 C OH , L-Tyr (Micropeptin) Fig. 5 Structures of natural products with A domains interrupted by A. R = CH(CH ) , L-Val (Tubulysin) KR or B. Ox and MOx domains in their biosynthetic NRPS machinery. 3 2 OH OH Me When applicable, regions of the structure synthesized by the interrupted A Note : In Tubulysin biosynthesis, TubB(M) similarly converts O into O NH N domains are highlighted in color. See Fig. 3 for a definition of domain 45 10 abbreviations. Fig. 4 Representative examples of potential proposed pathways for interrupted A domain with methyltransferase activity during natural Indigoidine (Fig. 5B) is a blue pigment produced by the plant product biosynthesis. A. Side-chain methylation produces thiocoraline pathogenic Erwinia chrysanthemi 67 as well as by other and kutzneride. B. Backbone N-methylation produces micropeptin, bacteria including certain Photorhabdus ,68 Phaeobacter ,69 and 15 microcystin, and tubulysin. Streptomyces 70 species. Indigoidine is composed of two 3.4 Adenylation domains with oxidase or monooxygenase 50 molecules of 5-amino-3H-pyridine-2,6-one, which are produced activity by the small NRPS IndC (Fig. 6, Fig. 8C). ( Note: What is termed IndC in E. chrysanthemi is homologous to “IgiD” in The structurally similar myxobacterial secondary metabolites Phaeobacter ). The IndC A domain codes for L-glutamine as a myxothiazols and melithiazoles are both biosynthesized from substrate. The IndC A domain is interrupted between a8 and a9 20 combined polyketide synthases (PKSs)/NRPSs containing two 14,15 55 by a flavin-dependent Ox domain that installs a carbon-carbon interrupted A domains (Fig. 5B). The first interrupted A double bond either immediately after loading the substrate onto domains, the A 2 domains of MtaD or MelD are interrupted the T domain (Fig. 8C), or perhaps after intramolecular between a8 and a9 by an Ox domain (Fig. 6). They incorporate an cyclization and release. 71 The final dimerization step is proposed L-Cys residue, then cyclize and oxidize it, forming the resultant 65 to be non-enzymatic and yields the conjugated product 25 thiazole ring (Fig. 8A). The second interrupted A domains, A 3 60 indigoidine, which is thought to be produced by these pathogenic located within MtaG and MelG, are interrupted between a4 and bacterial species as a defence mechanism against reactive oxygen a5 by an MOx domain. The location of the MOx domain, inserted species. between a4 and a5, is very unusual and these are the only interrupted A domains spliced at this position known to date. 30 These A domains add glycine, which is then hydroxylated at the

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CER EU LIDE: CesA CesB

A1 KR A1 T1 C2 A2 T2 E C3 A3 KR A3 T3 C4 A4 T4 TE a8 a9 a8 a9 VALINOMYCIN: Vlm1 Vlm2

A1 KR A1 T1 C2 A2 T2 E C3 A3 KR A3 T3 C4 A4 T4 TE a8 a9 a8 a9 MYXOTHIAZOL A and MELITHIAZOLE A: MtaB MtaC/MelC MtaD/MelD

CP KS AT AT DH KR CP KS AT DH KR CP HC A1 T1 Ox HC A2 Ox A2 T2 KS AT DH KR CP a8 a9 MtaE MtaF MtaG/MelG

KS AT M KR CP KS AT M CP C3 A3 MOx A3 T3 TE a4 a5 INDIGOIDINE: IndC/IgiD

C A Ox A T TE a8 a9 Fig. 6 Structural organization of cereulide, valinomycin, myxothiazol A, melithiazole A, and indigoidine NRPSs. Each module of the NRPSs is depicted in a different color. See Fig. 3 for a definition of domain abbreviations.

5

Ketoreduction: provides yet another viable means of controlling biosynthesis of Vlm2 (A3) Vlm2 (T3) T OH Vlm1(A1) OAMP Vlm1(T1) novel NRPs, which has yet to be explored. O CesB( A3) O CesB( T3) S CesA( A ) CesA( T ) O 1 O 1 O For many of these systems containing interrupted A domains, R R O 25 mechanistic and structural details remain to be resolved. Within ATP PP i R Vlm2 (KR ) Vlm2 (KR ) Vlm2 (KR ) the family of siderophores, for example, if the inactive M Vlm1(KR ) Vlm1(KR ) Vlm1(KR ) domains within PchE or HMWP2 were mutated active (R back to CesB( KR ) CesB( KR ) CesB( KR ) CesA( KR ) CesA( KR ) CesA( KR ) G), would methyl transfer occur? In the case of PchE, an

Vlm2 (A3) Vlm2 (T3) additional reductase domain would possibly be required, but a Vlm1(A ) Vlm1(T ) OH 1 OAMP 1 T CesB( A3) CesB( T3) 30 second N-methylation could occur on that subsequent O O CesA( A1) CesA( T1) S OH OH thiazolidine. For yersiniabactin, it is reasonable to expect that an R R O ATP PP i OH M domain mutated to active would produce an N-methylated R thiazolidine analogue. R = CH 3, α-ketoproionic acid (Valinomycin, Vlm2) R = CH(CH3)2, α-ketoisovaleric acid (Valinomycin, Vlm1 and Cereulide, CesB ) To better exploit A domains and other NRPS motifs, we need R = CH 2CH(CH3)2, α-ketoisocapoic acid (Cereulide, CesA ) 35 to understand where we can cut/insert portions of other auxiliary Fig. 7 Proposed potential pathways for interrupted adenylation domain enzymes into A domains. The known interrupted A domains are with ketoreductase activity during valinomycin and cereulide biosynthesis. commonly disrupted between motifs a8 and a9, but in some instances, between a2 and a3 or a4 and a5. Are there other positions in A domains where one could insert additional 10 4. Perspectives 40 functionalities? What are the boundaries? These rules need to be Because of their modular architectures, NRPSs provide a understood and leave much to be explored, especially with platform for engineering the biosynthesis of NRPs beyond those regards to less common interruptions such as those between a2 72,73 found to exist in Nature. The most common and most and a3 as well as a4 and a5. The unique biosynthetic mechanisms successful method of engineering NRPSs is domain swapping, featured in the interrupted A domains discussed here ( e.g. , S-Me- 74,75 15 often termed combinatorial biosynthesis. Variants of the 45 L-Cys within thiocoraline and O-Me-L-Ser within kutznerides) antibiotic daptomycin have been produced by NRPSs containing are rare, but could be powerful for biosynthetic engineering. 76 exchanged domains. Additionally, modification of A domain Furthermore, it remains to be answered whether or not domains active sites can be used to incorporate alternative amino acid other than M, KR, Ox, or MOx may be inserted to produce 77-80 substrates, including unnatural amino acids. Alteration of A functional A domains capable of achieving two separate chemical 20 domain specificity can lead to “tailor made enzyme systems”. 50 reactions. We, and others, seek answers to these questions; our The existence of A domains interrupted with auxiliary enzymes laboratory is currently working towards a better understanding

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A. Oxidation:

OH OAMP T2 T1 O O + MtaD(A2)/MelD(A2) MtaD(T2)/MelD(T2) MtaD(HC )/MelD(HC ) NH 2 NH 2 S S HS HS O O ATP PP i NH R L-Cys 2 HS

T2 T2 T2 T2 + H B S O MtaD(HC )/MelD(HC ) S MtaD(HC )/MelD(HC ) S MtaD(Ox )/MelD(Ox ) S O O H O O N R N H N N H OH B HS + H H R R B S R S S FMN

N N R = (Myxothiaxol A) R = (Melithiazole A) S H S

T2 B. Monooxygenase action: S O NH T T T 2 2 2 2 O O Glycine incorporation H S MtaG(A3)/MelG(A3) S MtaG(MOx)/MelG(MOx) S O O O MeO OMe MtaG(C3)/MelG(C3) HO NH NH MeO OMe O O R Myxothiazol A R MeO OMe MeO OMe

R R

NH 2 OH OMe S N O MelJ O MelK O R = (Myxothiaxol A) N S

S MeO OMe MeO OMe MeO OMe N R = (Melithiazole A) N H S R R R

Melithiazole A C. Synthesis of indigoidine:

T T T O O IndC( A)/IgiD(A) + S IndC( Ox )/IgiD(Ox ) S S HO NH 2 H H2N O H2N O NH 2 NH 2 FAD FADH2 NH 2 L-glutamine O O

O

H2N T NH O O O S non-enzymatic H H N H N dimerization O 2 NH 2 NH HN O O 2 O2 H2O2 NH 2 H H O Indigoidine Fig. 8 Proposed pathway for interrupted adenylation domains with A. oxidase (Ox) and B. monooxygenase (MOx) activity during myxothiazol and melithiazole biosynthesis. C. Proposed biosynthesis of indigoidine Note: oxidation may happen before (as depicted here) or after the cyclization step.

and engineering of a variety of interrupted A domains. manuscript. Support for this work is provided by an NSF CAREER award MCB-1149427 (S.G.-T.) and by startup funds

5 5. Acknowledgements 10 from the University of Kentucky. S.G.W. was supported by a University of Michigan NSF funded interdisciplinary REU The authors would like to acknowledge the work on adenylation program DBI-1156741. domains of those not cited in this highlight due to the scope of the

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