Structural Basis of Functional Group Activation by Sulfotransferases in Complex Metabolic Pathways

• Published in: ACS chemical biology Vol. 7; no. 12; pp. 1994 - 2003
• Main Authors: McCarthy, Jennifer Gehret, Eisman, Eli B, Kulkarni, Sarang, Gerwick, Lena, Gerwick, William H, Wipf, Peter, Sherman, David H, Smith, Janet L
• Format: Journal Article
• Language: English
• Published: United States American Chemical Society 21.12.2012
• Subjects: Biocatalysis; Models, Molecular; Molecular Structure; Substrate Specificity; Sulfotransferases - chemistry; Sulfotransferases - metabolism; Synechococcus - metabolism
• ISSN: 1554-8929; 1554-8937
• DOI: 10.1021/cb300385m

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.