A ketoreductase domain in the PksJ protein of the bacillaene assembly line carries out both α- and β-ketone reduction during chain growth

Calderone, Christopher T.; Bumpus, Stephanie B; Kelleher, Neil L.; Walsh, Christopher T.; Magarvey, Nathan A.

doi:10.1073/pnas.0806305105

Cited by 43 publications

(38 citation statements)

References 27 publications

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“…AulA is suggested to incorporate L-isoleucic acid (= 2-hydroxy-3-methylvaleric acid). The domain architecture indicates the substrate undergoes no other chemical modification besides a reductive step after being tethered to the PCP domain by the PKS-type ketoreductase domain (KR), as reported for other natural products, such as pyridomycin [11], cereulide, valinomycin [12], and bacillaene [13]. Hence, the molecule to be recognized and activated by AulA would be 3-methyl-2-oxovaleric acid ( 3 ).…”

Section: Resultsmentioning

confidence: 99%

A non-canonical peptide synthetase adenylates 3-methyl-2-oxovaleric acid for auriculamide biosynthesis

Braga¹,

Hoffmeister²,

Nett³

2016

Beilstein J. Org. Chem.

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Auriculamide is the first natural product known from the predatory bacterium Herpetosiphon aurantiacus. It is composed of three unusual building blocks, including the non-proteinogenic amino acid 3-chloro-L-tyrosine, the α-hydroxy acid L-isoleucic acid, and a methylmalonyl-CoA-derived ethane unit. A candidate genetic locus for auriculamide biosynthesis was identified and encodes four enzymes. Among them, the non-canonical 199 kDa four-domain nonribosomal peptide synthetase, AulA, is extraordinary in that it features two consecutive adenylation domains. Here, we describe the functional characterization of the recombinantly produced AulA. The observed activation of 3-methyl-2-oxovaleric acid by the enzyme supports the hypothesis that it participates in the biosynthesis of auriculamide. An artificially truncated version of AulA that lacks the first adenylation domain activated this substrate like the full-length enzyme which shows that the first adenylation domain is dispensable. Additionally, we provide evidence that the enzyme tolerates structural variation of the substrate. α-Carbon substituents significantly affected the substrate turnover. While all tested aliphatic α-keto acids were accepted by the enzyme and minor differences in chain size and branches did not interfere with the enzymatic activity, molecules with methylene α-carbons led to low turnover. Such enzymatic plasticity is an important attribute to help in the perpetual search for novel molecules and to access a greater structural diversity by mutasynthesis.

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Section: Resultsmentioning

confidence: 99%

A non-canonical peptide synthetase adenylates 3-methyl-2-oxovaleric acid for auriculamide biosynthesis

Braga¹,

Hoffmeister²,

Nett³

2016

Beilstein J. Org. Chem.

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“…To date, more and more building blocks, such as fatty acids, α-ketoacids, and α-hydroxyacids originating from amino acid metabolism as well as polyketide-derived units can also be used by NRPS assembly lines in the hybrid systems (4,5), which has greatly enriched the diversity of the peptide natural products. It has been shown previously that there are two strategies for the biosynthesis of α-hydroxyacyl units in nonribosomal peptides: either α-hydroxyacids were directly adenylated by the A domain (29) or α-ketoacids were activated and reduced by ketoreductase to the corresponding α-hydroxyacids while tethered to a PCP domain on the NRPS assembly line (30,31). We have now characterized a unique NRPS extender unit derived from ketose phosphates through (α,β-dihydroxyethyl)-ThDP and a lipoyl group-tethered Fig.…”

Section: Discussionmentioning

confidence: 99%

Hijacking a hydroxyethyl unit from a central metabolic ketose into a nonribosomal peptide assembly line

Peng

Song

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

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Nonribosomal peptide synthetases (NRPSs) usually catalyze the biosynthesis of peptide natural products by sequential selection, activation, and condensation of amino acid precursors. It was reported that some fatty acids, α-ketoacids, and α-hydroxyacids originating from amino acid metabolism as well as polyketide-derived units can also be used by NRPS assembly lines as an alternative to amino acids. Ecteinascidin 743 (ET-743), naphthyridinomycin (NDM), and quinocarcin (QNC) are three important antitumor natural products belonging to the tetrahydroisoquinoline family. Although ET-743 has been approved as an anticancer drug, the origin of an identical two-carbon (C 2 ) fragment among these three antibiotics has not been elucidated despite much effort in the biosynthetic research in the past 30 y. Here we report that two unexpected two-component transketolases (TKases), NapB/NapD in the NDM biosynthetic pathway and QncN/QncL in QNC biosynthesis, catalyze the transfer of a glycolaldehyde unit from ketose to the lipoyl group to yield the glycolicacyl lipoic acid intermediate and then transfer the C 2 unit to an acyl carrier protein (ACP) to form glycolicacyl-S-ACP as an extender unit for NRPS. Our results demonstrate a unique NRPS extender unit directly derived from ketose phosphates through (α,β-dihydroxyethyl)-thiamin diphosphate and a lipoyl group-tethered ester intermediate catalyzed by the TKase-ACP platform in the context of NDM and QNC biosynthesis, all of which also highlights the biosynthesis of ET-743. This hybrid system and precursor are distinct from the previously described universal modes involving the NRPS machinery. They exemplify an alternate strategy in hybrid NRPS biochemistry and enrich the diversity of precursors for NRPS combinatorial biosynthesis.primary metabolism | secondary metabolism | xylulose-5-phosphate | fructose-6-phosphate

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“…These include biosynthetic enzymes for the myxobacterial metabolites soraphen (24) [99][100][101], myxothiazol (25) [102], melithiazol (26) [103], chondramide (27) [104], thuggacin (28) [105], aurafurone (29) [106], stigmatellin (30) [107], and salinosporamide A (31) [108,109] (Fig. 8).…”

Section: Tandem Acyltransferase Domainsmentioning

confidence: 99%

“…Ketoreduction at the a-position would be an unusual transformation for a KR. However, KRs acting at nonstandard (other than b) positions are also known from other PKS pathways, for example in bacillaene (2) biosynthesis [25].…”

Section: Tandem Acyltransferase Domainsmentioning

confidence: 99%

The Catalytic Diversity of Multimodular Polyketide Synthases: Natural Product Biosynthesis Beyond Textbook Assembly Rules

Gulder

Freeman

Piel

2011

Topics in Current Chemistry

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Bacterial multimodular polyketide synthases (PKSs) are responsible for the biosynthesis of a wide range of pharmacologically active natural products. These megaenzymes contain numerous catalytic and structural domains and act as biochemical templates to generate complex polyketides in an assembly line-like fashion. While the prototypical PKS is composed of only a few different domain types that are fused together in a combinatorial fashion, an increasing number of enzymes is being found that contain additional components. These domains can introduce remarkably diverse modifications into polyketides. This review discusses our current understanding of such noncanonical domains and their role in expanding the biosynthetic versatility of bacterial PKSs.

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A ketoreductase domain in the PksJ protein of the bacillaene assembly line carries out both α- and β-ketone reduction during chain growth

Cited by 43 publications

References 27 publications

A non-canonical peptide synthetase adenylates 3-methyl-2-oxovaleric acid for auriculamide biosynthesis

A non-canonical peptide synthetase adenylates 3-methyl-2-oxovaleric acid for auriculamide biosynthesis

Hijacking a hydroxyethyl unit from a central metabolic ketose into a nonribosomal peptide assembly line

The Catalytic Diversity of Multimodular Polyketide Synthases: Natural Product Biosynthesis Beyond Textbook Assembly Rules

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