Structure and function of human PPTase: implications from ternary complexes

Abstract

Themodification of invariant Ser residues in acyl carrier proteins (ACPs) by transfer of a 4-phosphopantetheinemoiety is essential to all living organisms. The reactions is carried out by a diverse group of enzymes collectively characterized as 4-phosphopantetheine transferases (PPTases), also known as holo-ACP synthases, yielding holo-carrier proteins by group transfer of the 4-phosphopantetheine moiety from coenzyme A (CoA) as donor substrate. Recent molecular cloning and biochemical characterization of human PPTase revealed that it is a cytosolic, monomeric enzyme with broad acceptor substrate specificity, and requires Mg for catalysis. In order to better understand its mode of action, human PPTase was crystallised and structures of the enzyme, its complex with CoA, and in complex with CoA and cytosolic fatty acid synthase (FAS) ACP were determined. The enzyme, which adopts the same fold observed in bacterial group II PPTases, consists of two nearly identical domains with the active site located in a deep cleft between the domains. ACP binds with its essential Ser pointing towards the bound CoA, interacts with the enzyme on a large surface and is positioned primarily by hydrophobic forces. CoA-binding is mediated by a Mg ion complexed by Glu residues. Mutagenesis studies indicate that in addition to the residues involved in Mg-binding, a base catalyst Lys and a His are essential for catalysis. Although the shape of FASACP is complementary to the large basin on the enzyme near the active site, there are still features on the binding surface not explained by the ternary complex. It is tempting to speculate that these regions play a role in binding mitochondrial ACP, since it has been demonstrated that human PPTase can activate ACP from the mitochondrial fatty acid synthesis machinery as well. Progress on establishing the structure of this complex will also be reported. m09.p04