Skip to main content
NCBI home page
NIHPA Author Manuscripts logoLink to NIHPA Author Manuscripts
. Author manuscript; available in PMC: 2013 Aug 1.
Published in final edited form as: Curr Opin Chem Biol. 2012 May 6;16(3-4):362–369. doi: 10.1016/j.cbpa.2012.04.008

Complexity Generation during Natural Product Biosynthesis using Redox Enzymes

Peng Wang 1,†, Xue Gao 1,†, Yi Tang 1,2,*
PMCID: PMC3415589  NIHMSID: NIHMS372036  PMID: 22564679

Abstract

Redox enzymes such as FAD-dependent and cytochrome P450 oxygenases play indispensible roles in generating structural complexity during natural product biosynthesis. In the pre-assembly steps, redox enzymes can convert garden variety primary metabolites into unique starter and extender building blocks. In the post-assembly tailoring steps, redox cascades can transform nascent scaffolds into structurally complex final products. In this review, we will discuss several recently characterized redox enzymes in the biosynthesis of polyketides and nonribosomal peptides.

Introduction

The structural complexity observed among natural products is a testament to nature's amazing ability to perform chemistry with biological catalysts. Combinatorial biosynthesis is the approach to reengineer natural product pathways towards generation of new compounds in either the native or heterologous hosts through the manipulation of biosynthetic enzymes [1]. While increasing microbial genome sequences and synthetic biological tools in recent years have pushed combinatorial biosynthesis to a new frontier, it remains crucial to understand the enzymatic basis of complexity generation in order to (come close to) match the synthetic prowess of natural pathways. For major natural product families, such as polyketides and nonribosomal peptides, nature injects three levels of structural complexity when converting precursors (input) into the final product (output) (Figure 1). In the pre-modification step, simple primary metabolites such as acyl-CoAs and amino acids are modified into more elaborate precursors by dedicated enzymes [2, 3]. These precursors are incorporated into the nascent natural product scaffold by highly programmed machineries (second level), such as the assembly-line like polyketide synthase (PKS) and nonribosomal peptide synthetase (NRPS) [4]. The third level of complexity generation is performed by post-modification enzymes to complete the bioactive products [5]. While many different types of enzymes are recruited in the pre- and post-modification steps, redox enzymes such as FAD-dependent oxygenases, oxidoreductases, cytochrome P450 oxygenases, and NADPH-dependent reductases are ubiquitous and arguably catalyze the most diverse and sophisticated transformations. Especially in the post-modification steps, efficient redox cascades can regioselectively and stereoselectively morph the relatively simple structures into highly complex final products. In this review, we will discuss selected examples of recently discovered redox enzymes in the pre- and post-modification steps. Complexity generation in the assembly-line step is not discussed and can be found in numerous recent reviews [4, 6].

Figure 1.

Figure 1

Open in a new tab

Complexity generation during natural product biosynthesis can take place at three different stages.

Pre-assembly modification in polyketide biosynthesis

Polyketides are a group of natural products synthesized by PKSs using successive Claisen-like condensation reactions [4]. Prior to entering the polyketide assembly line, recruitment of starter and extender acyl thioesters is required. A variety of starter units are incorporated for polyketides produced by different families of PKSs [7]. Extender units for polyketide biosynthesis are selected by acyltransferases in PKSs in the form of CoA thioesters of dicarboxylic acids. The most common malonyl-CoA and methylmalonyl-CoA are siphoned from primary metabolism pool of the cell. In recent years, a growing numbers of polyketide natural products were discovered to contain acyl-malonyl building blocks synthesized by crotonyl-CoA carboxylase/reductase (CCR) homologs (Scheme 1a) [8]. Through reductive carboxylation of the β carbons of a variety of α-β unsaturated fatty acyl-CoAs, CCRs can synthesize a number of dicarboxylic acids, including ethyl-[9], chloroethyl-[10••], butyryl-, pentyl-, isopentyl-, hexylmalonyl-CoA [11••].

Scheme 1.

Scheme 1

Open in a new tab

Representative pathways to generate natural diversity in polyketide biosynthesis.

The first dedicated CCR homolog in natural product biosynthetic pathways was SalG found in the salinosporamide A gene cluster from the marine bacterium Salinispora tropica [10••]. SalG synthesizes chloroethylmalonyl-CoA, which is selected as a building block by the PKS SalA. The function of SalG was confirmed by gene inactivation and in vitro assay using 4-chlorocrotonyl-CoA as the substrate [10••]. SalG was subsequently shown to also generate 4-bromoethylmalonyl-CoA and 4-fluoroethylmalonyl-CoA from the corresponding 4-halocrotonyl-CoAs, which was utilized by downstream enzymes to produce bromosalinosporamide [12] and fluorosalinosporamide [13], respectively. Since then a growing number of CCR homologs have been discovered in PKS pathways, including those of FK506 [14], sanglifehrins [15], divergolides [16], and ansalactams [17] etc.

The detailed mechanism of CCR-catalyzed carboxylative reduction of (E)-crotonyl-CoA to (2S)-ethylmalonyl-CoA was revealed by Alber and coworkers [18]. The reaction is initiated by a hydride transfer from NADPH onto the β carbon followed by the anti carboxylation with CO2. The structure of hexylmalonyl-CoA synthase CinF involved in cinnabaramides biosynthesis was recently reported [19•]. The structure of CinF in complex with 2-octenoyl-CoA provided insights into the mechanism and substrate specificity of the CCR homolog. Two smaller amino acid residues Gly362 and Ala163 in the CinF binding pocket are important for accommodating the larger 2-octenoyl chain. The corresponding residues in CCR from S. coelicolor are Phe370 and Ile171, which sterically prevent the binding of substrates larger than crotonyl-CoA. CinF small to large point-mutants Gly362Phe and Ala163IIe each lost activities toward 2-octenoyl-CoA, but remained active towards crotonyl-CoA [19•]. The accumulation of CCR homologs and the newly gained understanding of CCR substrate specificity will enable dramatic expansion of polyketide extender unit pools. With concerted engineering of acyltransferase specificity and downstream PKS flexibility, these new extender units may lead to significantly more structural complexity in the final products.

Post-assembly modification in polyketide biosynthesis

Following the assembly-line like synthesis of the polyketide backbone, post-assembly modifications by different tailoring enzymes are required to convert the compound into its bioactive form. Among them, redox enzymes play crucial roles in rearranging the scaffolds into highly complex, sometimes unrecognizable final products. The biochemical mechanisms of two elusive oxidative cascades were recently established and are highlighted below.

Polyketides containing spiroacetal cores are widely found in nature, including reveromycin A [11••], griseorhodin A [20] etc. Compounds in this family exhibit a broad spectrum of biological activities, such as the potent induction of apoptosis in osteoclasts by reveromycin A. While the stereospecific spiroacetal cores are crucial for the observed activities, the enzymatic basis of the transformation from linear precursors has been unresolved until recently for the cyclization step in reveromycin A [11••]. Two enzymes, a dihydroxy ketone synthase (RevG) and a spiroacetal synthase (RevJ), are involved in stereoselective spiroacetal formation (Scheme 1b). Knockout of RevG led to accumulation of the acyclic precursor RM-A1a 1 that contains the 11, 15, 19-triol. In vitro assay with RevG and 1 led to formation of an unstable intermediate that contains the dihydroxy ketone moiety, which can spontaneous form the spiroacetal without stereocontrol. Addition of the previously unknown enzyme RevJ resulted in dehydrative cyclization of the dihydroxy ketone into exclusively the 15S product 2 that is found in the final reveromycin A structure [11••]. Interestingly, homologs of RevG and RevJ are not found in the known gene clusters of other spiroacetal-containing polyketides, suggesting different mechanisms of stereocontrol may be required to generate different cyclic scaffolds.

The gilvocarcins are a group of antitumor natural products bearing a unique benzo[d]naphtho[1,2-b]pyran-6-one chromophore linked with a variety of C-linked deoxysugar moieties [21]. The unique chromophore is rearranged from an angucycline polyketide via multiple oxidative modifications. Rohr and coworkers performed one-pot total enzymatic synthesis of the aglycon defucogilvocarcin M 7 using purified enzymes and various redox partners/cofactors (Scheme 1c) [22••]. In the “reaction pot”, the ensemble of PKS enzymes assembled acetyl-CoA and malonyl-CoA into the angucycline scaffold 3, which was tailored into 7 by a collection of four redox enzymes (GilOI, GilOII, JadF, GilR) and two methyltransferases (GilM, GilMT) in the presence of cofactors FAD, NADPH, S-adenosyl methionine (SAM), and cofactor regeneration enzymes. The precise control of reaction components through removal of individual tailoring enzymes in the pot, as well as the elimination of nonspecific reactions from endogenous cellular enzymes under in vivo conditions, enabled recovery of true intermediates in the reaction cascade. Interestingly, the inability to process the angucycline UWM6 under any combinations of the tailoring enzymes indicated that UWM6 is in fact a shunt product, instead of the long-standing assignment as an intermediate. This finding also led to the assignment of the oxygenase JadF (the soluble replacement of GilOIV) in the release, decarboxylation and dehydration of the ACP-tethered polyketide product 3 to yield 4. In addition, GilOI was identified as a bifunctional enzyme catalyzing both C12 oxidation and 4a,12b-dehydration of 4 to yield 5, which undergoes oxidative cleavage catalyzed by GilOII to afford the dibenzochromen-6-one core. The last step in the synthesis of 7 from the hemiacetal 6 is catalyzed by the oxidoreductase GilR, which can also catalyze the conversion of pregilvocarcin V to the gilvocarcins V [23]. Crystal structure of GilR in complex with pregilvocarcin V was recently solved, which showed the cofactor FAD is covalently attached to the enzyme through His65 and Cys125 [24]. Two amino acids Tyr445 and Tyr448 located in the catalytic pocket were proposed and confirmed by mutagenesis to play crucial roles in catalysis. With the roles of the redox enzymes assigned in this cascade, one gains more appreciation of the nature's remarkable ability to control reactive intermediates en route to highly complex structures.

Pre-assembly modification in nonribosomal peptide biosynthesis

Nonribosomal peptides (NRPs) are a group of structurally and functionally diverse natural products which are synthesized by NRPSs [4]. A typical module of NRPS consists of an adenylation (A) domain, thiolation (T) domain and condensation (C) domain. Each A domain activates an amino acid as an aminoacyl adenylate, which is transferred to the phosphopantetheinyl arm of the T domain and incorporated into the NRPs [25]. Much of the structural complexity seen in NRPs is generated through the incorporation of nonproteinogenic amino acids, many of which are hydroxylated versions of the natural amino acids. Hydroxylation of amino acids can either place on the free amino acid, or on the activated aminoacyl moiety that is attached to the T domain (Scheme 2). For example, N5-hydroxyornithine (8) is found in siderophores coelichelin and erythrocelin, and is the product of l-ornithine N-hydroxylation by FAD-dependent monooxygeneases CchB and EtcB, respectively [26, 27]. In contrast, epoxidation of the succinamoyl building block by the α-ketoglutarate dependent DdaC to afford epoxysuccinamoyl (9) takes place after Nβ-fumaramoyl-l-2,3-diaminopropionate is transferred to the NRPS DdaD [28]. T-domain dependent oxidation is also observed in the formation of 3, 5-dihydroxy-4-methylanthranilate (10) found in sibiromycin. Following NRPS-independent modification of 3-hydroxykynurenine to yield 3-hydroxy-4-methylanthranilic acid (3H4MAA), 3H4MAA is transferred to the NRPS SibE and is hydroxylated by the FAD-dependent hydroxylase SibG to give 10 [29].

Scheme 2.

Scheme 2

Open in a new tab

Representative chemical structures generated by pre-assembly modifications in nonribosomal peptide biosynthesis.

Recently, oxygenases using different mechanisms were identified to catalyze the aromatic hydroxylation of tyrosine (Tyr) and phenylalanine (Phe). SfmD was characterized as a heme containing peroxidase which regioselectivly hydroxylates 3-methyltyrosine to 3-hydroxy-5-methyltyrosine (11) in the biosynthesis of saframycin A, using H2O2 as oxidant [30]. SgcC is a monooxygenase involved in the biosynthesis of the antitumor enediyne C-1207 and was shown to catalyze the hydroxylation of β-Tyr in a T-domain dependent fashion [31]. SgcC is able to hydroxylate (S)-3-chloro-β-tyrosine tethered to SgcC2 at the C-5 position to afford (S)-3-chloro-5-hydroxy-β-tyrosyl-Sgc2 (12) in presence of O2 and FADH2. In additional to 12, SgcC can also hydroxylate meta-substituted bromo-, iodo-, fluoro-, and methyl-β-tyrosyl-Sgc2. 2-hydroxylphenylalanine was found as one of the building blocks in the pentapeptide antibiotics pacidamycins [32]. A phenylalanine hydroxylase (PecX) was demonstrated to be an Fe(II)-dependent enzyme that regioselectively synthesize 13 from l-Phe using tetrahydrobiopterin as a cofactor [32, 33]. Comparing with another group of Phe 4-hydroxylases (Phe4Hs) which catalyzes the formation of l-Tyr, Cys187 and Thr202 in the binding pocket of PacX are replaced by Phe and Gly, respectively. A PacX C187F/T202G mutant was shown to have dramatically altered hydroxylation regiospecificity to afford >90% L-Tyr instead.

SyrP is an Fe(II) and α-ketoglutarate dependent hydroxylase that acts on an aspartic residue tethered to the eighth module of SyrE to generate L-threo-3-OH-Asp (14) in the biosynthesis of syringomycin E [34]. In the biosynthesis of the antifungal hexadepsipeptide kutznerides, both L-threo-hydroxyl and L-erythro-hydroxyl glutamate derivatives can be incorporated [35•]. After L-glutamic acid is transferred to the T domain of KtzH, the oxygenases KtzO and KtzP can catalyze β-hydroxylation to form 15 and 16, respectively. Unlike the above examples, AsnO was demonstrated to catalyze the hydroxylation of free L-asparagine to yield L-3-hydroxyasparagine 17, which is activated and incorporated by the NRPS to form the calcium dependent antibiotic (CDA) peptidyl chain [36]. Based on the AsnO crystal structure, Marahiel and coworkers engineered a D241N AsnO mutant that can hydroxylate Asp instead of Asn [37].

Post-assembly modification in nonribosomal peptide biosynthesis

Fungal indole alkaloids derived from tryptophans are NRPs consisting of relatively few amino acids. While the NRPSs construct the starting structural frameworks, which can be pyrazinoquinazolines and diketopiperizines, the seemingly exponential increase in structural complexity of the final products can be attributed to the oxidative modifications catalyzed by a few redox enzymes. In particular, epoxidation of the 2,3-double bond in the indole ring is a highly effective strategy to activate the C2 carbon and build multicyclic ring systems. For example, fumiquinazoline F 18 is the nascent NRP product of the tryptoquialanine (TQA) and fumiquinazoline A 20 biosynthetic pathways (Scheme 3a) [38•, 39]. Epoxidation of 18 by a FAD-dependent monooxygenase (Af12060 in pathway of 20) initiates annulation of the indole ring by an amino acid residue (e.g. alanine) tethered to a monomodule NPRS 21. Biochemical analysis suggests the stereoselective attack of the iminium 19 by the nucleophilic amine of 21 is catalyzed by a C-like domain in the monomodule NRPS [40]. Subsequent attack of the indole nitrogen on the alanyl-thioester forms the 6-5-5 imidazolindolone present in 20. A monocovalent flavoprotein (Af12070) can then dehydrogenate the C-N bond in the pyrazinone ring in 20, which can trigger different events including hydrolytic cleavage of the ring towards the formation of TQA, or spontaneous intramolecular cyclization to yield the spirohemiaminal fumiquinazoline C, which can be converted to the more stable aminal fumiquinazoline D [41•]. Similarly, epoxidation of the indole ring in the synthesis of notoamides, a family of prenylated fungal indole alkaloids containing the bicyclo [2.2.2] diazaoctane core, is catalyzed by a FAD-dependent monooxygenease NotB (Scheme 3b) [42]. Formation of the 2,3-epoxide initiates either a Pinacol-like rearrangement to afford notoamide C or annulation of C-2 by the tryptophan amide to yield the hexacyclic notoamide D.

Scheme 3.

Scheme 3

Open in a new tab

Representative pathways to generate natural complexity in nonribosomal peptide biosynthesis and other systems.

Whereas the above examples illustrate how post-NRPS oxidation can lead to dramatic change in alkaloid structure, reductive modifications have also been noted in generation of unusually complex alkaloid-like natural products, as exemplified in the formation of the pentacyclic tetrahydroisoquinoline saframycin A [43•]. A single R domain in the NRPS SfmC reductively released three different peptidyl thioesters in a NADPH-dependent fashion to generate the corresponding aldehyde intermediates. Through Pictet-Spengler reactions catalyzed by the C domain, the aldehydes are cyclized with peptidyl thioesters reloaded on SfmC T-domain to afford the unique saframycin ring systems.

Newly discovered Redox enzymes in other systems

In addition to their importance in polyketide and nonribosomal peptide biosynthesis, redox enzymes with novel functions are indispensable in the modification of other classes of natural products. For example, the mononuclear iron enzyme hydroxypropylphosphonic acid epoxidase (HppE) catalyzes the final step of fosfomyin biosynthesis, in which HppE generates a C1 radical intermediate of (S)-2-hydroxypropylphosphonic acid (S-HPP) that can cyclize into the epoxide (Scheme 3c). Interestingly, HppE instead generates a C2 radical intermediate in the presence of the R-enantiomer, which can form the ketone 2-oxo-propylphosphonic acid [44]. The structures of HppE in complex with either enantiomer revealed variations in the positioning of the two compounds with respect to the reactive Fe (III)-superoxo species [45••]. The different orientations of the C1 and C2 carbons in the active site therefore dictate the regioselectivity of hydrogen abstraction step.

Tirandamycin A and B are dienoyl tetramic acids that contain heavily modified bicyclic ketal cores [46••]. Following the biosynthesis of tirandamycin C by a hybrid PKS-NRPS pathway, the bicyclic core is oxidized by a P450 monooxygenase (TamI) and a FAD-dependent oxidase TamL to yield tirandamycin A and B (Scheme 3d). In vitro reconstitution by Sherman and coworkers elegantly confirmed TamI and TamL are sufficient to catalyze the dazzling multi-step oxidative cascade. In particular, TamI alone can catalyze the hydroxylation at C10 and C18, as well as epoxidation of C11-C12. TamI was also shown to be able to complete the entire oxidative cascade single-handedly by performing double hydroxylation of C10, which can spontaneously form the ketone intermediate tirandamycin D, albeit the second hydroxylation at C10 is much less efficient. TamL is hence recruited to oxidize the monohydroxylated tirandamycin E into tirandamycin D. The remarkable versatility of TamI, along with recently confirmed activities a P450 monooxygenase that converts amorpha-4,11-diene to artemisinic acid [47]; and LovA from the lovastatin biosynthetic [48], fully demonstrates how nature can use a single P450 enzyme to catalyze multiple oxidative modifications in a biosynthetic pathway and generate structural complexity in natural products [47].

Conclusions

Redox enzymes play indispensable roles in generating structural complexity during natural product biosynthesis. Continued characterization of these enzymes will undoubtedly expand our molecular toolbox for combinatorial biosynthesis. However, it remains challenging to recombine redox enzymes from different pathways towards the rational enzymatic synthesis of new compounds, largely attributed to the limited substrate specificities of the enzymes. Another difficulty in using these enzymes as synthetic biology parts is our inability to predict substrates and products of uncharacterized redox enzymes uncovered from microbial genome sequencing. Protein engineering and biochemical characterization are required to overcome these limitations and allow us to truly realize the impressive catalytic power of these enzymes.

Highlights (for review).

Redox enzymes play important roles in pre- and post-assembly modifications.

Both PKS and NRPS pathways use redox enzymes to introduce structural complexity

Recent discoveries of novel redox enzymes are discussed

Acknowledgements

Due to space constraint, we apologize for omission of other relevant works. Research on combinatorial biosynthesis in our lab is supported by funds from NIH, the Alfred Sloan Foundation and the David and Lucile Packard Foundations.

Footnotes

Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimersthat apply to the journal pertain.

References and recommended reading

Papers of particular interest, published within the period of review, have been highlighted as:

• of special interest

•• of outstanding interest

  • 1.Pickens LB, Tang Y, Chooi YH. Metabolic engineering for the production of natural products. Annu Rev Chem Biomol Eng. 2011;2:211–236. doi: 10.1146/annurev-chembioeng-061010-114209. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 2.Chan YA, Podevels AM, Kevany BM, Thomas MG. Biosynthesis of polyketide synthase extender units. Nat Prod Rep. 2009;26:90–114. doi: 10.1039/b801658p. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 3.Wilkinson B, Micklefield J. Chapter 14. Biosynthesis of Nonribosomal Peptide Precursors. In: Hopwood DA, editor. Methods Enzymol; Complex Enzymes in Microbial Natural Product Biosynthesis, Part A: Overview Articles and Peptides. Academic Press; 2009. pp. 353–378. [DOI] [PubMed] [Google Scholar]
  • 4.Fischbach MA, Walsh CT. Assembly-line enzymology for polyketide and nonribosomal peptide antibiotics: Logic, machinery, and mechanisms. Chem Rev. 2006;106:3468–3496. doi: 10.1021/cr0503097. 5. [DOI] [PubMed] [Google Scholar]
  • 5.Olano C, Mendez C, Salas JA. Post-PKS tailoring steps in natural product-producing actinomycetes from the perspective of combinatorial biosynthesis. Nat Prod Rep. 2010;27:571–616. doi: 10.1039/b911956f. [DOI] [PubMed] [Google Scholar]
  • 6.Walsh CT. The chemical versatility of natural-product assembly lines. Acc Chem Res. 2008;41:4–10. doi: 10.1021/ar7000414. [DOI] [PubMed] [Google Scholar]
  • 7.Hertweck C. The biosynthetic logic of polyketide diversity. Angew Chem Int Ed Engl. 2009;48:4688–4716. doi: 10.1002/anie.200806121. [DOI] [PubMed] [Google Scholar]
  • 8.Wilson MC, Moore BS. Beyond ethylmalonyl-CoA: the functional role of crotonyl-CoA carboxylase/reductase homologs in expanding polyketide diversity. Nat Prod Rep. 2012;29:72–86. doi: 10.1039/c1np00082a. [DOI] [PubMed] [Google Scholar]
  • 9.Erb TJ, Berg IA, Brecht V, Muller M, Fuchs G, Alber BE. Synthesis of C5-dicarboxylic acids from C2-units involving crotonyl-CoA carboxylase/reductase: the ethylmalonyl-CoA pathway. Proc Natl Acad Sci USA. 2007;104:10631–10636. doi: 10.1073/pnas.0702791104. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 10••.Eustaquio AS, McGlinchey RP, Liu Y, Hazzard C, Beer LL, Florova G, Alhamadsheh MM, Lechner A, Kale AJ, Kobayashi Y, et al. Biosynthesis of the salinosporamide A polyketide synthase substrate chloroethylmalonyl-coenzyme A from S-adenosyl-L-methionine. Proc Natl Acad Sci USA. 2009;106:12295–12300. doi: 10.1073/pnas.0901237106. [Eustaquio et al. characterized the first dedicated CCR homolog SalG involved in natural product biosynthesis. This work showed 4-chlorocrotonyl-CoA is the substrate of SalG.] [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 11••.Takahashi S, Toyoda A, Sekiyama Y, Takagi H, Nogawa T, Uramoto M, Suzuki R, Koshino H, Kumano T, Panthee S, et al. Reveromycin A biosynthesis uses RevG and RevJ for stereospecific spiroacetal formation. Nat Chem Biol. 2011;7:461–468. doi: 10.1038/nchembio.583. [Using reveromycin A as an example, Takahashi et al. provided the first enzymatic basis of spiroaacetal formation in polyketides. This work showed formation of spiroacetal from the triol proceeds through a dihydroxy ketone intermeidate and is catalyzed by RevG and RevJ.] [DOI] [PubMed] [Google Scholar]
  • 12.Liu Y, Hazzard C, Eustaquio AS, Reynolds KA, Moore BS. Biosynthesis of salinosporamides from alpha,beta-unsaturated fatty acids: implications for extending polyketide synthase diversity. J Am Chem Soc. 2009;131:10376–10377. doi: 10.1021/ja9042824. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 13.Eustaquio AS, Moore BS. Mutasynthesis of fluorosalinosporamide, a potent and reversible inhibitor of the proteasome. Angew Chem Int Ed Engl. 2008;47:3936–3938. doi: 10.1002/anie.200800177. [DOI] [PubMed] [Google Scholar]
  • 14.Mo S, Kim DH, Lee JH, Park JW, Basnet DB, Ban YH, Yoo YJ, Chen SW, Park SR, Choi EA, et al. Biosynthesis of the allylmalonyl-CoA extender unit for the FK506 polyketide synthase proceeds through a dedicated polyketide synthase and facilitates the mutasynthesis of analogues. J Am Chem Soc. 2011;133:976–985. doi: 10.1021/ja108399b. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 15.Qu XD, Jiang N, Xu F, Shao L, Tang GL, Wilkinson B, Liu W. Cloning, sequencing and characterization of the biosynthetic gene cluster of sanglifehrin A, a potent cyclophilin inhibitor. Molecular Biosystems. 2011;7:852–861. doi: 10.1039/c0mb00234h. [DOI] [PubMed] [Google Scholar]
  • 16.Xu ZL, Ding L, Hertweck C. A branched extender unit shared between two orthogonal polyketide pathways in an endophyte. Angew Chem Int Ed Engl. 2011;50:4667–4670. doi: 10.1002/anie.201008265. [DOI] [PubMed] [Google Scholar]
  • 17.Wilson MC, Nam SJ, Gulder TAM, Kauffman CA, Jensen PR, Fenical W, Moore BS. Structure and biosynthesis of the marine streptomycete ansamycin ansalactam a and its distinctive branched chain polyketide extender unit. J Am Chem Soc. 2011;133:1971–1977. doi: 10.1021/ja109226s. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 18.Erb TJ, Brecht V, Fuchs G, Muller M, Alber BE. Carboxylation mechanism and stereochemistry of crotonyl-CoA carboxylase/reductase, a carboxylating enoyl-thioester reductase. Proc Natl Acad Sci USA. 2009;106:8871–8876. doi: 10.1073/pnas.0903939106. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 19•.Quade N, Huo LJ, Rachid S, Heinz DW, Muller R. Unusual carbon fixation gives rise to diverse polyketide extender units. Nat Chem Biol. 2012;8:117–124. doi: 10.1038/nchembio.734. [The first X-ray crystal structure of CCR homolog in complex with 2-octenylcrotonyl-CoA. The structural basis of the reaction mechanism and substrate specificity are discussed in detail.] [DOI] [PubMed] [Google Scholar]
  • 20.Yunt Z, Reinhardt K, Li A, Engeser M, Dahse HM, Gutschow M, Bruhn T, Bringmann G, Piel J. Cleavage of four carbon-carbon bonds during biosynthesis of the griseorhodin a spiroketal pharmacophore. J Am Chem Soc. 2009;131:2297–2305. doi: 10.1021/ja807827k. [DOI] [PubMed] [Google Scholar]
  • 21.Kharel MK, Pahari P, Shepherd MD, Tibrewal N, Nybo SE, Shaaban KA, Rohr J. Angucyclines: Biosynthesis, mode-of-action, new natural products, and synthesis. Nat Prod Rep. 2012;29:264–325. doi: 10.1039/c1np00068c. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 22••.Pahari P, Kharel MK, Shepherd MD, van Lanen SG, Rohr J. Enzymatic total synthesis of defucogilvocarcin M and its implications for gilvocarcin biosynthesis. Angew Chem Int Ed Engl. 2012;51:1216–1220. doi: 10.1002/anie.201105882. [Pahari et al. has reported the enzymatic total synthesis of a natural product using 15 purified enzymes in a one pot reaction. By examining products from different combinations of reactions, a new pathway for gilvocarcin aglycon formation is established.] [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 23.Kharel MK, Pahari P, Lian H, Rohr J. GilR, an unusual lactone-forming enzyme involved in gilvocarcin biosynthesis. Chembiochem. 2009;10:1305–1308. doi: 10.1002/cbic.200900130. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 24.Noinaj N, Bosserman MA, Schickli MA, Piszczek G, Kharel MK, Pahari P, Buchanan SK, Rohr J. The crystal structure and mechanism of an unusual oxidoreductase, GilR, involved in gilvocarcin V biosynthesis. J Biol Chem. 2011;286:23533–23543. doi: 10.1074/jbc.M111.247833. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 25.Schwarzer D, Finking R, Marahiel MA. Nonribosomal peptides: From genes to products. Nat Prod Rep. 2003;20:275–287. doi: 10.1039/b111145k. [DOI] [PubMed] [Google Scholar]
  • 26.Pohlmann V, Marahiel MA. Delta-amino group hydroxylation of L-ornithine during coelichelin biosynthesis. Org Biomol Chem. 2008;6:1843–1848. doi: 10.1039/b801016a. [DOI] [PubMed] [Google Scholar]
  • 27.Robbel L, Helmetag V, Knappe TA, Marahiel MA. Consecutive enzymatic modification of ornithine generates the hydroxamate moieties of the siderophore erythrochelin. Biochemistry. 2011;50:6073–6080. doi: 10.1021/bi200699x. [DOI] [PubMed] [Google Scholar]
  • 28.Hollenhorst MA, Bumpus SB, Matthews ML, Bollinger JM, Kelleher NL, Walsh CT. The nonribosomal peptide synthetase enzyme DdaD tethers N(β)-fumaramoyl-l-2,3-diaminopropionate for Fe(II)/α-ketoglutarate-dependent epoxidation by DdaC during dapdiamide antibiotic biosynthesis. J Am Chem Soc. 2011;133:1609–1609. doi: 10.1021/ja1072367. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 29.Giessen TW, Kraas FI, Marahiel MA. A four-enzyme pathway for 3,5-dihydroxy-4-methylanthranilic acid formation and incorporation into the antitumor antibiotic sibiromycin. Biochemistry. 2011;50:5680–5692. doi: 10.1021/bi2006114. [DOI] [PubMed] [Google Scholar]
  • 30.Tang MC, Fu CY, Tang GL. Characterization of SfmD as a heme peroxidase that catalyzes the regioselective hydroxylation of 3-methyltyrosine to 3-hydroxy-5-methyltyrosine in saframycin a biosynthesis. J Biol Chem. 2012;287:5112–5121. doi: 10.1074/jbc.M111.306316. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 31.Lin S, Van Lanen SG, Shen B. 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. J Am Chem Soc. 2008;130:6616–6623. doi: 10.1021/ja710601d. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 32.Zhang WJ, Ames BD, Walsh CT. Identification of phenylalanine 3-hydroxylase for meta-tyrosine biosynthesis. Biochemistry. 2011;50:5401–5403. doi: 10.1021/bi200733c. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 33.Gruschow S, Rackham EJ, Goss RJM. Diversity in natural product families is governed by more than enzyme promiscuity alone: Establishing control of the pacidamycin portfolio. Chem Sci. 2011;2:2182–2186. [Google Scholar]
  • 34.Singh GM, Fortin PD, Koglin A, Walsh CT. beta-Hydroxylation of the aspartyl residue in the phytotoxin syringomycin E: Characterization of two candidate hydroxylases AspH and SyrP in Pseudomonas syringae. Biochemistry. 2008;47:11310–11320. doi: 10.1021/bi801322z. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 35•.Strieker M, Nolan EM, Walsh CT, Marahiel MA. Stereospecific synthesis of threo- and erythro-beta-hydroxyglutamic acid during kutzneride biosynthesis. J Am Chem Soc. 2009;131:13523–13530. doi: 10.1021/ja9054417. [Sterieker et al. reported that two oxygenases are present in the kutzneride pathway to specifically hydroxylate glutamic acid to threo- and beta-hydroxyglutamic acids.] [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 36.Strieker M, Kopp F, Mahlert C, Essen LO, Marahiel MA. Mechanistic and structural basis of stereospecific C beta-hydroxylation in calcium-dependent antibiotic, a daptomycin-type lipopeptide. ACS Chem Biol. 2007;2:187–196. doi: 10.1021/cb700012y. [DOI] [PubMed] [Google Scholar]
  • 37.Strieker M, Essen LO, Walsh CT, Marahiel MA. Non-heme hydroxylase engineering for simple enzymatic synthesis of L-threo-hydroxyaspartic acid. Chembiochem. 2008;9:374–376. doi: 10.1002/cbic.200700557. [DOI] [PubMed] [Google Scholar]
  • 38•.Gao X, Chooi Y-H, Ames BD, Wang P, Walsh CT, Tang Y. Fungal indole alkaloid biosynthesis: Genetic and biochemical investigation of the tryptoquialanine pathway in Penicillium aethiopicum. J Am Chem Soc. 2011;133:2729–2741. doi: 10.1021/ja1101085. [Gao et al. has investigated the biosynthesis of fungal indole alkalod tryptoquialanine using both genetic and biochemical methods. Characterizations of intermediates accumulated from gene inactivation studies delineated the complex biosynthetic pathway.] [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 39.Ames BD, Liu X, Walsh CT. Enzymatic processing of fumiquinazoline F: A tandem oxidativeacylation strategy for the generation of multicyclic scaffolds in fungal indole alkaloid biosynthesis. Biochemistry. 2010;49:8564–8576. doi: 10.1021/bi1012029. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 40.Haynes SW, Ames BD, Gao X, Tang Y, Walsh CT. Unraveling terminal C-domain-mediated condensation in fungal biosynthesis of imidazoindolone metabolites. Biochemistry. 2011;50:5668–5679. doi: 10.1021/bi2004922. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 41•.Ames BD, Haynes SW, Gao X, Evans BS, Kelleher NL, Tang Y, Walsh CT. Complexity generation in fungal peptidyl alkaloid biosynthesis: Oxidation of fumiquinazoline A to the heptacyclic hemiaminal fumiquinazoline C by the flavoenzyme Af12070 from Aspergillus fumigatus. Biochemistry. 2011;50:8756–8769. doi: 10.1021/bi201302w. [An outstanding example of complexity generation from a single FAD-dependent oxygenase during the biosynthesis of multicyclic fungal indole alkaloids.] [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 42.Li S, Finefield JM, Sunderhaus JD, McAfoos TJ, Williams RM, Sherman DH. Biochemical characterization of NotB as an FAD-dependent oxidase in the biosynthesis of notoamide indole alkaloids. J Am Chem Soc. 2012;134:788–791. doi: 10.1021/ja2093212. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 43•.Koketsu K, Watanabe K, Suda H, Oguri H, Oikawa H. Reconstruction of the saframycin core scaffold defines dual Pictet-Spengler mechanisms. Nat Chem Biol. 2010;6:408–410. doi: 10.1038/nchembio.365. [This work showed assembly of the saframycin core is facilitated by the iterative activities of the reductive and condensation domains of the a NRPS module.] [DOI] [PubMed] [Google Scholar]
  • 44.Zhao Z, Liu P, Murakami K, Kuzuyama T, Seto H, Liu HW. Mechanistic studies of HPP epoxidase: Configuration of the substrate governs its enzymatic fate. Angew Chem Int Ed Engl. 2002;41:4529–4532. doi: 10.1002/1521-3773(20021202)41:23<4529::AID-ANIE4529>3.0.CO;2-2. [DOI] [PubMed] [Google Scholar]
  • 45••.Yun D, Dey M, Higgins LJ, Yan F, Liu HW, Drennan CL. Structural basis of regiospecificity of a mononuclear iron enzyme in antibiotic fosfomycin biosynthesis. J Am Chem Soc. 2011;133:11262–11269. doi: 10.1021/ja2025728. [Yun et al. presented detailed structural analyses of HHP epoxidase in complex with S-HHP and R-HHP. The conformational differences of the two substrates explains how a single enzyme can catalyze two different reactions when presented with the two enantiomers.] [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 46••.Carlson JC, Li S, Gunatilleke SS, Anzai Y, Burr DA, Podust LM, Sherman DH. Tirandamycin biosynthesis is mediated by co-dependent oxidative enzymes. Nat Chem. 2011;3:628–633. doi: 10.1038/nchem.1087. [This work showcases the remarkable versatility of a P450 oxygenase that can catalyze four different oxidations reactions during tirandamycin biosynthesis.] [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 47.Ro DK, Paradise EM, Ouellet M, Fisher KJ, Newman KL, Ndungu JM, Ho KA, Eachus RA, Ham TS, Kirby J, et al. Production of the antimalarial drug precursor artemisinic acid in engineered yeast. Nature. 2006;440:940–943. doi: 10.1038/nature04640. [DOI] [PubMed] [Google Scholar]
  • 48.Barriuso J, Nguyen DT, Li JW, Roberts JN, MacNevin G, Chaytor JL, Marcus SL, Vederas JC, Ro DK. Double oxidation of the cyclic nonaketide dihydromonacolin L to monacolin J by a single cytochrome P450 monooxygenase, LovA. J Am Chem Soc. 2011;133:8078–8081. doi: 10.1021/ja201138v. [DOI] [PubMed] [Google Scholar]

RESOURCES