Structure and Functional Analysis of RifR, the Type II Thioesterase from the Rifamycin Biosynthetic Pathway

• Published in: The Journal of biological chemistry Vol. 284; no. 8; pp. 5021 - 5029
• Main Authors: Claxton, Heather B., Akey, David L., Silver, Monica K., Admiraal, Suzanne J., Smith, Janet L.
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 20.02.2009; American Society for Biochemistry and Molecular Biology
• Subjects: Actinomycetales - enzymology; Acyl Coenzyme A - chemistry; Acyl Coenzyme A - metabolism; Amycolatopsis mediterranei; Bacterial Proteins - chemistry; Bacterial Proteins - metabolism; Catalytic Domain - physiology; CRYSTAL STRUCTURE; Crystallography, X-Ray; Fatty Acid Synthases - chemistry; Fatty Acid Synthases - metabolism; FUNCTIONAL ANALYSIS; HYDROLYSIS; KINETICS; MATERIALS SCIENCE; PEPTIDES; Polyketide Synthases - chemistry; Polyketide Synthases - metabolism; Protein Structure, Secondary - physiology; Protein Structure, Tertiary - physiology; Rifamycins - biosynthesis; SUBSTRATES; Thiolester Hydrolases - chemistry; Thiolester Hydrolases - metabolism
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M808604200

Two thioesterases are commonly found in natural product biosynthetic clusters, a type I thioesterase that is responsible for removing the final product from the biosynthetic complex and a type II thioesterase that is believed to perform housekeeping functions such as removing aberrant units from carrier domains. We present the crystal structure and the kinetic analysis of RifR, a type II thioesterase from the hybrid nonribosomal peptide synthetases/polyketide synthase rifamycin biosynthetic cluster of Amycolatopsis mediterranei. Steady-state kinetics show that RifR has a preference for the hydrolysis of acyl units from the phosphopantetheinyl arm of the acyl carrier domain over the hydrolysis of acyl units from the phosphopantetheinyl arm of acyl-CoAs as well as a modest preference for the decarboxylated substrate mimics acetyl-CoA and propionyl-CoA over malonyl-CoA and methylmalonyl-CoA. Multiple RifR conformations and structural similarities to other thioesterases suggest that movement of a helical lid controls access of substrates to the active site of RifR.