Insights into heterocyclization from two highly similar enzymes - PubMed (original) (raw)

Insights into heterocyclization from two highly similar enzymes

John A McIntosh et al. J Am Chem Soc. 2010.

Abstract

The cyanobactin biosynthetic pathways pat and tru, isolated from metagenomes of marine animals, lead to diverse natural products containing heterocycles derived from Cys, Ser, and Thr. Previous work has shown that PatD and TruD are extremely broad-substrate heterocyclase enzymes. These enzymes are virtually identical in their N-terminal putative catalytic domains, but only approximately 77% identical in their C-terminal putative substrate-binding domains. Here, we show that these differences allow the enzymes to control regioselectivity of posttranslational modifications, helping to control product chemistry in this hypervariable family of marine natural products.

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Figures

Figure 1

Figure 1

Biosynthesis of heterocyclic cyanobactins. A. Enzymes modify ribosomal precursor peptides into mature products. Trunkamide group: top; patellamide group: bottom. B. An alignment of PatD and TruD proteins, showing putative catalytic and peptide-binding domains. Catalytic domains are >99% identical; peptide-binding domains are >77% identical.

Figure 2

Figure 2

Enzyme selectivity defined by metagenome sequence and chemical analysis. Natural products from the pat (top) and tru (bottom) pathways contain modified Cys, Ser, and Thr residues in the defined positions. y-axis: % of natural products containing these modifications. x-axis: amino acid position in cassette. Empty space above the bars denotes hypervariability: diverse amino acids occupy these positions in isolated natural products.

Figure 3

Figure 3

Reaction characterization and substrate specificity of TruD and PatD. A. MALDI MS of reaction products. B. Position of modifications identified by ESI-MS/MS. C. Representative SDS-PAGE gel showing band shifts of precursor peptides 1–4. The left of each triplet is a standard of the peptide, middle is the TruD-modified peptide, and right is the PatD-modified peptide.

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