title: Structural basis of functional group activation by sulfotransferases in complex metabolic pathways.
abstract: Sulfated molecules with diverse functions are common in biology, but sulfonation as a method to activate a metabolite for chemical catalysis is rare. Catalytic activity was characterized and crystal structures were determined for two such "activating" sulfotransferases (STs) that sulfonate β-hydroxyacyl thioester substrates. The CurM polyketide synthase (PKS) ST domain from the curacin A biosynthetic pathway of Moorea producens and the olefin synthase (OLS) ST from a hydrocarbon-producing system of Synechococcus PCC 7002 both occur as a unique acyl carrier protein (ACP), ST, and thioesterase (TE) tridomain within a larger polypeptide. During pathway termination, these cyanobacterial systems introduce a terminal double bond into the β-hydroxyacyl-ACP-linked substrate by the combined action of the ST and TE. Under in vitro conditions, CurM PKS ST and OLS ST acted on β-hydroxy fatty acyl-ACP substrates; however, OLS ST was not reactive toward analogues of the natural PKS ST substrate bearing a C5-methoxy substituent. The crystal structures of CurM ST and OLS ST revealed that they are members of a distinct protein family relative to other prokaryotic and eukaryotic sulfotransferases. A common binding site for the sulfonate donor 3'-phosphoadenosine-5'-phosphosulfate was visualized in complexes with the product 3'-phosphoadenosine-5'-phosphate. Critical functions for several conserved amino acids in the active site were confirmed by site-directed mutagenesis, including a proposed glutamate catalytic base. A dynamic active-site flap unique to the "activating" ST family affects substrate selectivity and product formation, based on the activities of chimeras of the PKS and OLS STs with exchanged active-site flaps.
authors.affiliation: Life Sciences Institute and Department of Biological Chemistry, University of Michigan, Ann Arbor, MI 48109, USA.
authors.forename: Jennifer Gehret
authors.initials: JG
authors.lastname: McCarthy
authors.forename: Eli B
authors.initials: EB
authors.lastname: Eisman
authors.forename: Sarang
authors.initials: S
authors.lastname: Kulkarni
authors.forename: Lena
authors.initials: L
authors.lastname: Gerwick
authors.forename: William H
authors.initials: WH
authors.lastname: Gerwick
authors.forename: Peter
authors.initials: P
authors.lastname: Wipf
authors.forename: David H
authors.initials: DH
authors.lastname: Sherman
authors.forename: Janet L
authors.initials: JL
authors.lastname: Smith
mesh_terms: D055162:Biocatalysis; D008958:Models, Molecular; D015394:Molecular Structure; D013379:Substrate Specificity; D015238:Sulfotransferases / Q000737:chemistry / Q000378:metabolism*; D046940:Synechococcus / Q000378:metabolism
chemical_list: D015238:Sulfotransferases