Structure and functional analysis of RifR, the type II thioesterase from the rifamycin biosynthetic pathway.
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.
Claxton
Heather B
HB
Life Science Institute, University of Michigan, Ann Arbor, Michigan 48109, USA.
Akey
David L
DL
Silver
Monica K
MK
Admiraal
Suzanne J
SJ
Smith
Janet L
JL
D000192:Actinomycetales / Q000201:enzymology*; D000214:Acyl Coenzyme A / Q000737:chemistry* / Q000378:metabolism; D001426:Bacterial Proteins / Q000737:chemistry* / Q000378:metabolism; D020134:Catalytic Domain / Q000502:physiology; D018360:Crystallography, X-Ray; D064429:Fatty Acid Synthases / Q000737:chemistry* / Q000378:metabolism; D007700:Kinetics; D048630:Polyketide Synthases / Q000737:chemistry / Q000378:metabolism; D017433:Protein Structure, Secondary / Q000502:physiology; D017434:Protein Structure, Tertiary / Q000502:physiology*; D012294:Rifamycins / Q000096:biosynthesis; D013869:Thiolester Hydrolases / Q000737:chemistry* / Q000378:metabolism
D000214:Acyl Coenzyme A; D001426:Bacterial Proteins; D012294:Rifamycins; D048630:Polyketide Synthases; D064429:Fatty Acid Synthases; D013869:Thiolester Hydrolases; C022738:thioesterase II