Crystal Structure of the Acyltransferase Domain of the Iterative Polyketide Synthase in Enediyne Biosynthesis

• Published in: The Journal of biological chemistry Vol. 287; no. 27; pp. 23203 - 23215
• Main Authors: Liew, Chong Wai, Nilsson, Martina, Chen, Ming Wei, Sun, Huihua, Cornvik, Tobias, Liang, Zhao-Xun, Lescar, Julien
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 29-06-2012; American Society for Biochemistry and Molecular Biology
• Subjects: Acetyl Coenzyme A; Acetyl Coenzyme A - metabolism; Acyl Carrier Protein; Acyltransferases - chemistry; Acyltransferases - genetics; Acyltransferases - metabolism; Amino Acid Motifs; Anti-Bacterial Agents - chemistry; Antibiotics; Catalytic Domain; Cloning, Molecular; Crystallography; Enediynes - metabolism; Enzyme Structure; Glycerol - chemistry; Micromonospora - enzymology; Micromonospora - genetics; Polyketide Synthases - chemistry; Polyketide Synthases - genetics; Polyketide Synthases - metabolism; Polyketides; Protein Structure and Folding; Protein Structure, Secondary; Protein Structure, Tertiary; Structure-Activity Relationship; Substrate Specificity; Enzyme Structure; Acyl Carrier Protein; Polyketides; Antibiotics; Acetyl Coenzyme A; Crystallography
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M112.362210

Biosynthesis of the enediyne natural product dynemicin in Micromonospora chersina is initiated by DynE8, a highly reducing iterative type I polyketide synthase that assembles polyketide intermediates from the acetate units derived solely from malonyl-CoA. To understand the substrate specificity and the evolutionary relationship between the acyltransferase (AT) domains of DynE8, fatty acid synthase, and modular polyketide synthases, we overexpressed a 44-kDa fragment of DynE8 (hereafter named ATDYN10) encompassing its entire AT domain and the adjacent linker domain. The crystal structure at 1.4 Å resolution unveils a α/β hydrolase and a ferredoxin-like subdomain with the Ser-His catalytic dyad located in the cleft between the two subdomains. The linker domain also adopts a α/β fold abutting the AT catalytic domain. Co-crystallization with malonyl-CoA yielded a malonyl-enzyme covalent complex that most likely represents the acyl-enzyme intermediate. The structure explains the preference for malonyl-CoA with a conserved arginine orienting the carboxylate group of malonate and several nonpolar residues that preclude α-alkyl malonyl-CoA binding. Co-crystallization with acetyl-CoA revealed two noncovalently bound acetates generated by the enzymatic hydrolysis of acetyl-CoA that acts as an inhibitor for DynE8. This suggests that the AT domain can upload the acyl groups from either malonyl-CoA or acetyl-CoA onto the catalytic Ser651 residue. However, although the malonyl group can be transferred to the acyl carrier protein domain, transfer of the acetyl group to the acyl carrier protein domain is suppressed. Local structural differences may account for the different stability of the acyl-enzyme intermediates.
Background: DynE8 is an iterative polyketide synthase (PKS) that assembles polyketide intermediates from acetate units derived from malonyl-CoA.
Results: We report the first acyltransferase (ATDYN10) crystal structure for an iterative PKS.
Conclusion: ATDYN10 protects the malonyl-enzyme, but not the acetyl-enzyme intermediate, from hydrolysis and facilitates the transfer of malonyl to the acyl carrier protein.
Significance: This differs from the dual specificity exhibited by acyltransferases of mammalian FAS and other iterative PKSs.