The hidden enzymology of bacterial natural product biosynthesis

Abstract

Bacterial natural products display astounding structural diversity, which, in turn, endows them with a remarkable range of biological activities that are of significant value to modern society. Such structural features are generated by biosynthetic enzymes that construct core scaffolds or perform peripheral modifications, and can thus define natural product families, introduce pharmacophores and permit metabolic diversification. Modern genomics approaches have greatly enhanced our ability to access and characterize natural product pathways via sequence-similarity-based bioinformatics discovery strategies. However, many biosynthetic enzymes catalyse exceptional, unprecedented transformations that continue to defy functional prediction and remain hidden from us in bacterial (meta)genomic sequence data. In this Review, we highlight exciting examples of unusual enzymology that have been uncovered recently in the context of natural product biosynthesis. These suggest that much of the natural product diversity, including entire substance classes, awaits discovery. New approaches to lift the veil on the cryptic chemistries of the natural product universe are also discussed.

Fig. 1. Bacterial natural product chemical diversity.

Natural product (NP) examples described in this Review are illustrated. Compounds are coloured according to the section within this Review in which they are discussed. Orange, polyketide synthase/nonribosomal peptide synthetase-derived NPs; purple, terpenes; cyan, ribosomally synthesized and post-translationally modified peptides (RiPPs); magenta, NPs with non-signature biosynthetic origins. Note that closthioamide (28) and 6-thioguanine (29) are coloured magenta but are not RiPPs. 1, Obafluorin; 2, kutzneride 1; 3, curacin A; 4, nocardicin A; 5, pyrroindomycin A; 6, spinosyn A; 7, TMC-86A; 8, ikarugamycin; 9, saframycin A; 10, rhizoxin; 11, pederin; 12, leinamycin; 13, metatricycloene; 14, oocydin B; 15, albicidin; 16, saxitoxin; 17, dynemicin A; 18, clostrubin; 19, 12-epi-hapalindole U; 20, sodorifen; 21, longestin; 22, teleocidin B; 23, pentalenolactone; 24, bottromycin; 25, klebsazolicin; 26, saalfelduracin; 27, thioviridamide; 28, closthioamide; 29, 6-thioguanine; 30, nosiheptide; 31, streptide; 32, polytheonamide B; 33, microvionin; 34, ammosamide; 35, crocagin A; 36, andrimid; 37, belactosin C; 38, bicyclomycin; 39, indolmycin; 40, roseoflavin.

Fig. 2. Remarkable transformations during nonribosomal peptide synthetase substrate biosynthesis.

a | Proposed mechanism for the l-Thr transaldolase (l-TTA), ObaG, during obafluorin biosynthesis. Grey panel: pyridoxal-phosphate binds l-Thr to form an external aldimine that undergoes retro-aldol cleavage to yield a glycine enolate. An aldol-type reaction with 4-nitrophenyacetaldehyde (4-NPA) yields (2S,3R)-2-amino-3-hydroxy-4-(4-nitrophenyl)butanoate (AHNB). The new bond formed in this transformation is indicated in red. Blue panel: other natural products that incorporate l-TTA-derived, β-OH-α-amino acid substrates (proposed for alanylclavam)^–. In each case, aldehyde and l-Thr-substrate-derived moieties are highlighted in orange and blue, respectively. b | The KtzT-catalysed haem-dependent cyclization of l-N⁵-OH-Orn to form l-piperazate (grey panel). KtzI is an l-Orn N-hydroxylase. Blue panel: other natural products that are purported to employ a KtzT homologue or a mechanism involving N-hydroxylation to generate an activated intermediate susceptible to intramolecular attack by an amino group in the formation of N–N bonds during their biosynthesis^–. The relevant corresponding moieties are highlighted in blue.

Fig. 3. Noncanonical polyketide synthase/nonribosomal peptide synthetase reactions involving tethered thioester intermediates.

a | Enoyl reductase (ER)-catalysed cyclopropanation during curacin biosynthesis. The curacin and jamaicamide biosynthetic pathways diverge at the β-branching step, which is initiated by decarboxylase (ECH₂)-catalysed decarboxylation, resulting in α–β double-bond and β–γ double-bond formation in respective curacin and jamaicamide enoyl-γ-chloro-acyl carrier protein (ACP) intermediates^,. Subsequent activity of an unusual cis-acting ER domain in CurF catalyses cyclopropane ring formation (highlighted in orange). The homologous ER domain in JamJ is unreactive towards its respective ECH₂ product (unmodified ECH₂ thioester product, highlighted in blue). The new bond formed to close the cyclopropane ring is highlighted in red. b | On-line tailoring reactions catalysed by divergent condensation (C) and thioesterase (TE) domains during nocardicin biosynthesis. The terminal C domain in NocB catalyses initial elimination of water, followed by cyclization to yield the β-lactam pharmacophore. The resulting thioester intermediate is subsequently transferred to the NocB TE domain, which performs an unprecedented epimerization of the l-(p-hydroxyphenyl)glycine (l-Hpg) moiety, before canonical hydrolysis to release nocardicin G. Nonribosomal peptide synthetase domains catalysing the specific transformations illustrated are highlighted and new bonds formed to generate the β-lactam ring by the C domain are shown in red. Polyketide synthase domains and nonribosomal peptide synthetase domains are highlighted in red and blue, respectively. Trans-acting enzymes are highlighted in purple. A, adenylation; AT, acyltransferase; ECH₁, dehydratase; Hal, halogenase; HMGS, 3-hydroxy-3-methylglutaryl CoA synthase; KS, ketosynthase; PCP, peptidyl carrier protein.

Fig. 4. Unusual post-polyketide synthase/nonribosomal peptide synthetase enzymology.

a | Enzymatic [4+2] cycloadditions during pyrroindomycin biosynthesis. Grey panel: Diels–Alder-type cyclizations catalysed by PyrE3 (orange) and PyrI4 (blue). New bonds formed are highlighted in red. Blue panel: six-membered rings (highlighted in blue) installed by PyrI4 homologues during abyssomycin and versipelostatin biosynthesis. b | Grey panel: TmcF catalyses a remarkable decarboxylation–dehydrogenation–oxygenation transformation to generate the epoxyketone moiety present in TMC-86A. The new bond formed to close the epoxide ring is highlighted in red. Blue panel: select examples of natural product proteasome inhibitors that are proposed to employ TmcF homologues during their biosynthesis to install α/β-epoxyketone warhead moieties^–. Epoxyketone moieties introduced by TmcF and its homologues are highlighted in orange.

Fig. 5. Polyketide synthase-catalysed and nonribosomal peptide synthetase-catalysed transformations that define novel natural product families.

a | Members of the polycyclic tetramate macrolactam family of polyketides are produced by the activities of only three enzymes, as illustrated for ikarugamycin^–. New bonds formed during successive cyclizations are highlighted in red. Steps 1–3 illustrate amide bond formation between thioester intermediates X and Y by the IkaA C domain (step 1), transfer of the resulting thioester intermediate to the IkaA thioesterase domain active site Ser residue (step 2) and thioesterase-catalysed intramolecular attack to yield the pyrroline moiety with concomitant release of intermediate Z (step 3). It is not clear whether formation of the six-membered ring is spontaneous or enzyme-catalysed. The timing and nature of the isomerization event that results in the introduction of a cis double-bond in the final ikarugamycin pathway product is also not clear; however, the IkaC homologue OX4 is proposed to be responsible for cis double-bond introduction during the biosynthesis of heat-stable antifungal factor, a polycyclic tetramate macrolactam produced by Lysobacter enzymogenes^–. b | A single nonribosomal peptide synthetase module catalyses iterative Pictet–Spengler-type cyclizations and reductions of structurally different intermediates to assemble the characteristic scaffold of the tetrahydroisoquinoline antibiotics. Structurally different SfmC reductase (R) domain peptidyl thioester intermediates are indicated by X–Z. Adenylations of 3-OH-O,5-dimethyl-l-Tyr residues before condensation (C)-domain-catalysed cyclization by the SfmC adenylation (A) domain are indicated in grey. New bonds formed by the C domain are highlighted in purple. Individual substrates are highlighted in different colours so that their fate can be tracked through the mechanism illustrated. ACP, acyl carrier protein; AT, acyltransferase; DH, dehydratase; KR, ketoreductase; KS, ketosynthase; PCP, peptidyl carrier protein; TE, thioesterase.

Fig. 6. Newly characterized on-line trans-acyltransferase polyketide synthase transformations.

a | Ketoreductase (KS)-catalysed vinolygous β-branching and lactonization during rhizoxin biosynthesis^,. New bonds formed during this transformation are highlighted in red. b | Pyran synthase (PS)-domain-catalysed cyclic ether formation during pederin biosynthesis. The new bond formed to close the pyran ring is highlighted in red. c | Sulfur insertion catalysed by the sequential activities of cis-acting domain of unknown function (DUF) and rare cysteine lyase domains during leinamycin biosynthesis. d | Oxygen insertion into the growing oocydin polyketide backbone catalysed by the trans-acting Baeyer–Villiger monooxygenase, OocK. OocK and its flavin cofactor are coloured purple. Further processing of the OocK-modified moiety is proposed to be due to additional enzymatic activities in the oocydin producer. Trans-acyltransferase polyketide synthase domains catalysing the specific transformations illustrated and the resulting chemical moieties generated are highlighted in orange. ACP, acyl carrier protein; B, branching domain; DH, dehydratase; KR, ketoreductase; SH, cysteine lyase; TE, thioesterase.

Fig. 7. Unusual transformations from terpene biosynthesis.

a | Cyclization of the common 3-geranyl-3-isocyanylvinyl indolenine intermediate (X) catalysed by Stig cyclases. The remarkable transformation catalysed involves a rare Cope-like rearrangement, a 6-exo-trig cyclization and an electrophilic aromatic substitution^–. Different Stig cyclases possess specific stereoselectivity and regioselectivity, as illustrated by the different products generated by HpiC1, FamC2 and FimC5. b | Proposed mechanism of oxidative rearrangement in the conversion of pentalenolactone F into pentalenolactone by the cytochrome P450, PntF^,. The mechanism of transient neopentyl cation intermediate formation remains to be experimentally verified.

Fig. 8. Post-translational modifications of ribosomally synthesized and post-translationally modified peptide natural products.

A | The three characterized ATP-dependent, amide backbone modifications currently known to be catalysed by members of the YcaO superfamily. Grey panel: azole and azoline formation (part Aa, orange), amidine formation (part Ab, blue) and thioamide formation (part Ac, magenta). Blue panel: examples of ribosomally synthesized and post-translationally modified peptides (RiPPs) comprising one or more of the above YcaO-catalysed modifications are also illustrated and each modification has been highlighted with its respective colour. B | Summary of post-translational modifications catalysed by members of the radical S-adenosyl methionine (rSAM) superfamily in the context of RiPP biosynthesis. Colours are used to highlight new bonds formed and/or the fate of particular atoms following (rSAM) enzyme activity.

Fig. 9. Formation of the side-ring system during the biosynthesis of nosiheptide.

3-Methyl-2-indolic acid (MIA) is formed by the rearrangement of l-Trp catalysed by NosL and is subsequently introduced into the nosiheptide side-ring system by NosIJK^–. These enzymes transfer the MIA moiety to an unmodified Cys residue in a linear pentathiazolyl nosiheptide intermediate. NosN is proposed to methylate MIA at the C4 position to generate a key methylene radical intermediate that is subsequently linked via an ester bond to Glu6 in the pentathiazolyl intermediate (X). The S-adenosyl methionine (SAM)-derived methyl group introduced by NosN is highlighted in blue and the bond formed with Glu6 to close the nosiheptide side-ring system is highlighted in red.

Fig. 10. Unusual enzymology from natural product pathways that lack signature biosynthetic genes.

a | Biosynthesis of indolmycin from l-Arg and indolmycenic acid. Following Ind4 oxidation of l-Arg, the resulting unstable imine product is selectively reduced by the d-specific, NADH-dependent reductase Ind5, preventing off-pathway reactions, such as deamination, from occurring. Ind3 catalyses an ATP-dependent condensation of d-4,5-dehydroarginine with indolmycenic acid, resulting in the formation of the indolmycin oxazoline ring (new bond highlighted in red). Ind6 (embedded in a complex with Ind5) performs an unusual gatekeeping role to ensure release of the correct leaving group, again to prevent the generation of off-pathway products. Ind5 and Ind6 are highlighted for their respective transformations. The N-methyltransferase Ind7 completes biosynthesis. b | Roseoflavin biosynthesis from riboflavin. The ATP-dependent flavokinase RibC first catalyses riboflavin-5′-phosphate (RP) formation. RosB (orange) performs the subsequent three transformations to generate 8-demethyl-8-amino-riboflavin-5′-phosphate (AFP) in the presence of thiamine and l-Glu, with 2-oxoglutarate (2-OG) produced as a by-product^,. The RibC-installed phosphate group is subsequently removed by a cryptic phosphatase, before sequential N-methylations catalysed by the S-adenosyl methionine (SAM)-dependent dimethyltransferase RosA (blue). X represents a cryptic hydrogen acceptor. Modifications catalysed by RosA and RosB have been highlighted in their respective colours.