Glycopeptide antibiotic biosynthesis: enzymatic assembly of the dedicated amino acid monomer (S)-3,5-dihydroxyphenylglycine

Abstract

Four proteins, DpgA-D, required for the biosynthesis by actinomycetes of the nonproteinogenic amino acid monomer (S)-3,5-dihydroxyphenylglycine (Dpg), that is a crosslinking site in the maturation of vancomycin and teicoplanin antibiotic scaffolds, were expressed in Escherichia coli, purified in soluble form, and assayed for enzymatic activity. DpgA is a type III polyketide synthase, converting four molecules of malonyl-CoA to 3,5-dihydroxyphenylacetyl-CoA (DPA-CoA) and three free coenzyme A (CoASH) products. Almost no turnover was observed for DpgA until DpgB was added, producing a net k(cat) of 1-2 min(-1) at a 3:1 ratio of DpgB:DpgA. Addition of DpgD gave a further 2-fold rate increase. DpgC had the unusual catalytic capacity to convert DPA-CoA to 3,5-dihydroxyphenylglyoxylate, which is a transamination away from Dpg. DpgC performed a net CH(2) to C=O four-electron oxidation on the Calpha of DPA-CoA and hydrolyzed the thioester linkage with a k(cat) of 10 min(-1). Phenylacetyl-CoA was also processed, to phenylglyoxylate, but with about 500-fold lower k(cat)/K(M). DpgC showed no activity in anaerobic incubations, suggesting an oxygenase function, but had no detectable bound organic cofactors or metals. A weak enoyl-CoA hydratase activity was detected for both DpgB and DpgD.

Figure 1

Vancomycin and teicoplanin family members containing the nonproteinogenic amino acids Hpg and Dpg. The Hpg and Dpg residues in each structure are indicated by their residue numbers.

Figure 2

Coomassie-stained 4–15% SDS-polyacrylamide gradient gel of purified DpgA–D. Lane 1, DpgA; lane 2, DpgB; lane 3, DpgC; lane 4, DpgD.

Figure 3

Acceleration in rate of formation of DPA-CoA by DpgA upon addition of DpgB. (a) HPLC traces of reactants and products upon incubation of malonyl-CoA with various combinations of DpgA and DpgB for 1 h. Enzymes in each incubation: 1, DpgA; 2, DpgB; 3, DpgA/DpgB; 4, DPA-CoA standard. (b) Rate of CoASH production by DpgA (5 μM) at various concentrations of DpgB. (Inset) Rates of CoASH production by DpgA (5 μM) in the presence (dashed line) or absence (solid line) of DpgB (15 μM). k_obs = mM/min.

Figure 4

Conversion of DPA-CoA to 3,5-dihydroxyphenylglyoxylate by DpgC. (a) HPLC traces of reactants and products upon incubation of various substrates with DpgC for 1 h. Substrates in each incubation: 1, malonyl-CoA and DpgA/DpgB; 2, DPA-CoA; 3, HPA-CoA; 4, PA-CoA; 5, phenylglyoxylate standard; 6, DPA-CoA/HPA-CoA/PA-CoA standards. (b) HPLC traces of reactants and products upon incubation of (R)- and (S)-mandelyl-CoA in the presence or absence of DpgC for 1 h. 1, (R)-mandelyl-CoA with DpgC; 2, (R)-mandelyl-CoA; 3, (S)-mandelyl-CoA with DpgC; 4, (S)-mandelyl-CoA. (c) HPLC traces of time points of incubation of DPA-CoA with DpgC in the absence (traces 1–3) and then presence (traces 4–5) of O₂. 1, 5 min; 2, 30 min; 3, 1 h; 4, 5 min after exposure to O₂; 5, 1 h after exposure.

Figure 5

Schemes of proposed in vivo activities of DpgA–D. (a) DpgA. In this scheme, the consumption of four malonyl-CoA molecules by DpgA leads to the production of three CoASH molecules and one hydrated DPA-CoA precursor. (b) DpgB/DpgD. This scheme depicts DpgB and DpgD as dehydratases that act on the product of DpgA to produce DPA-CoA. (c) DpgC. This scheme depicts a possible mechanism through a peroxide intermediate for the novel oxidase activity of DpgC. The lack of DPA-CoA consumption/CoASH production by DpgC in the absence of O₂ suggests that the thioesterase activity occurs after the oxidase activity, as shown in the scheme.