Enzyme-catalysed oxidations are some of the most common transformations in primary and secondary metabolism. The vancomycin biosynthetic enzyme DpgC belongs to a small class of oxygenation enzymes that are not dependent on an accessory cofactor or metal ion. The detailed mechanism of cofactor-independent oxygenases has not been established. Here we report the first structure of an enzyme of this oxygenase class in complex with a bound substrate mimic. The use of a designed, synthetic substrate analogue allows unique insights into the chemistry of oxygen activation. The structure confirms the absence of cofactors, and electron density consistent with molecular oxygen is present adjacent to the site of oxidation on the substrate. Molecular oxygen is bound in a small hydrophobic pocket and the substrate provides the reducing power to activate oxygen for downstream chemical steps. Our results resolve the unique and complex chemistry of DpgC, a key enzyme in the biosynthetic pathway of an important class of antibiotics. Furthermore, mechanistic parallels exist between DpgC and cofactor-dependent flavoenzymes, providing information regarding the general mechanism of enzymatic oxygen activation.
The pyrroloquinoline alkaloid family of natural products that includes the immunosuppressant lymphostin has long been postulated to arise from tryptophan. We now report the molecular basis of lymphostin biosynthesis in three marine Salinispora species that maintain conserved biosynthetic gene clusters harboring a hybrid nonribosomal peptide synthetase-polyketide synthase central to lymphostin assembly. Through a series of experiments involving gene mutations, stable isotope profiling, and natural product discovery, we report the assembly line biosynthesis of lymphostin and nine new analogues that exhibit potent mTOR inhibitory activity.
Prephenate is the direct precursor of phenylpyruvate and 4-hydroxyphenylpyruvate in the biogenesis of phenylalanine and tyrosine by action of the decarboxylative, aromatizing enzymes prephenate dehydratase and dehydrogenase, respectively. The recent characterization of BacA in bacilysin biosynthesis as a non-aromatizing decarboxylase reveals a new route from prephenate in the biosynthesis of nonproteinogenic amino acids. This study describes two additional enzymes, AerD from Planktothrix agardhii and SalX from Salinispora tropica, that utilize the central building block prephenate for flux down distinct pathways to amino acid products, representing a new metabolic fate for prephenate and establishing a new family of non-aromatizing prephenate decarboxylases.Prephenate has been known for decades as the product of the celebrated 3,3-sigmatropic rearrangement of chorismate, a reaction that is classically followed by formation of the aromatic amino acids phenylalanine and tyrosine (1). Prephenate dehydratase and prephenate dehydrogenase are the enzymes that direct prephenate to Phe and Tyr by decarboxylation and aromatization of the cyclohexenyl ring with concomitant loss of the C-7 hydroxide or hydride, respectively. Recently we characterized the Bacillus subtilis prephenate dehydratase homolog BacA, which is involved in the biosynthesis of bacilysin (1, Figure 1), and catalyzes the first step of a 4-enzyme pathway that transforms prephenate to a distinct non-aromatic amino acid product (2). Like prephenate dehydratase, BacA decarboxylates prephenate at C-4 yet uniquely protonates C-6 or C-6' to yield the endocyclic diene dihydro-4-hydroxyphenylpyruvate (H 2 HPP, 5, Figure 2A) (2). This diene is then subject to nonenzymatic isomerization to the exocyclic diene in a reaction that can be dramatically accelerated by BacB to yield the indicated H 2 HPP regioisomer 6. Here we identify two homologs to BacA from distinct microbial secondary metabolite pathways that catalyze the same non-aromatizing decarboxylation of prephenate, thus establishing a new family of enzymes. The first of these is AerD from the cyanobacterium Planktothrix agardhii, which is involved in the production of the nonribosomal glycopeptides
The enzyme DpgC belongs to a small class of oxygenases not dependent on accessory cofactors for activity. DpgC is in the biosynthetic pathway for the nonproteinogenic amino acid 3,5-dihydroxyphenylglycine in actinomycetes bacteria responsible for the production of the vancomycin/teicoplanin family of antibiotic natural products. The X-ray structure of DpgC [Widboom, P. W., Fielding, E. N., Liu, Y., and Bruner, S. D. (2007) Nature 447, 342-345] confirmed the absence of cofactors and defined a novel hydrophobic dioxygen binding pocket adjacent to a bound substrate analogue. In this paper, the role specific amino acids play in substrate recognition and catalysis is examined through biochemical and structural characterization of site-specific enzyme mutations and alternate substrates. The results establish the importance of three amino acids, Arg254, Glu299, and Glu189, in the chemistry of DpgC. Arg254 and Glu189 join to form a specific contact with one of the phenolic hydroxyls of the substrate, and this interaction plays a key role in both substrate recognition and catalysis. The X-ray crystal structure of Arg254Lys was determined to address the role this residue plays in the chemistry. In addition, characterization of alternate substrate analogues demonstrates the presence and position of phenol groups are necessary for both enzyme recognition and downstream oxidation chemistry. Overall, this work defines the mechanism of substrate recognition and specificity by the cofactor-independent dioxygenase DpgC.
The enzyme DpgC is included in the small family of cofactor-independent dioxygenases. The chemistry of DpgC is uncommon as the protein binds and utilizes dioxygen without the aid of a metal or organic cofactor. Previous structural and biochemical studies identified the substrate-binding mode and the components of the active site that are important in the catalytic mechanism. In addition, the results delineated a putative binding pocket and migration pathway for the co-substrate dioxygen. Here, structural biology is utilized, along with site-directed mutagenesis, to probe the assigned dioxygen-binding pocket. The key residues implicated in dioxygen trafficking were studied to probe the process of binding, activation and chemistry. The results support the proposed chemistry and provide insight into the general mechanism of dioxygen binding and activation.
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