Mechanistic insights into the biosynthesis of polyketide antibiotics

Abstract

Anthracyclines are a group of aromatic polyketide compounds with significant medical importance due to their antineoplastic properties. Doxorubicin and daunorubicin members of this family are among the two most commonly used anticancer drugs. However, these compounds exhibit severe side effects like cardiotoxicity and multi-drug resistance. A promising approach towards the production of modified anthracyclines with improved toxicity profiles appears to be combinatorial biosynthesis, including the redesign of biosynthetic enzymes however, structural and mechanistic information of the biosynthetic enzymes is necessary for the redesigning approach.

The main focus of this thesis is the structural and functional studies of several anthracycline biosynthetic enzymes. The crystal structures of two polyketide cyclases, SnoaL and AknH were determined to 1.35Å and 1.9Å respectively. These enzymes share very similar α+β structural folds and catalyze a novel type of intramolecular aldol condensation reaction using acid/base chemistry. Moreover, comparison of the cyclase structures, followed by site-directed mutagenesis provided insights into the structural basis of stereoselectivity of products in AknH.

Several enzymes from aromatic polyketide biosynthetic pathways, including SnoaL/AknH provide illustrative examples of divergent evolution. The related enzymes usually share the same fold but contain different catalytic machineries to catalyze diverse reactions.

AknOx is a FAD-dependent oxidoreductase which is involved in the sugar modification in aclacinomycins and catalyzes two consecutive oxidation reactions. The structure of AknOx to 1.65Å resolution was obtained from a pseudomerohedrally twinned crystal by MAD. AknOx crystals show an unusual multi-domain twinning with six twin domains Investigation of the active site and mutagenesis of the proposed residues revealed a unique feature in AknOx compared to other flavoenzymes. The enzyme contains a dual active site with two different sets of catalytic residues for catalyzing two consecutive reactions.