Structural and Mechanistic Insight into the Listeria monocytogenes Two-enzyme Lipoteichoic Acid Synthesis System

Ivan Campeotto,Matthew G Percy,James T Macdonald,Andreas Förster,Paul S Freemont,Angelika Gründling

doi:10.1074/jbc.m114.590570

Abstract

Lipoteichoic acid (LTA) is an important cell wall component required for proper cell growth in many Gram-positive bacteria. In Listeria monocytogenes, two enzymes are required for the synthesis of this polyglycerolphosphate polymer. The LTA primase LtaP(Lm) initiates LTA synthesis by transferring the first glycerolphosphate (GroP) subunit onto the glycolipid anchor and the LTA synthase LtaS(Lm) extends the polymer by the repeated addition of GroP subunits to the tip of the growing chain. Here, we present the crystal structures of the enzymatic domains of LtaP(Lm) and LtaS(Lm). Although the enzymes share the same fold, substantial differences in the cavity of the catalytic site and surface charge distribution contribute to enzyme specialization. The eLtaS(Lm) structure was also determined in complex with GroP revealing a second GroP binding site. Mutational analysis confirmed an essential function for this binding site and allowed us to propose a model for the binding of the growing chain.

Highlights

Listeria monocytogenes lipoteichoic acid is synthesized by the LtaP/LtaS two-enzyme system
Apo-structures of eLtaPLm and LtaSLm—To identify differences between Lipoteichoic acid (LTA) synthase and primase enzymes, the soluble extracellular enzymatic domains eLtaPLm and eLtaSLm were overexpressed and purified from E. coli and their crystal structures were determined at 1.75- and 3.0-Å resolution, respectively
Both enzymes were monomers in solution, as assessed by size exclusion chromatography, eLtaPLm crystallized with two molecules in the asymmetric unit and eLtaSLm with five molecules in the asymmetric unit (Table 3)

Summary

Background

Listeria monocytogenes lipoteichoic acid is synthesized by the LtaP/LtaS two-enzyme system. Results: Structural analysis reveals a second glycerolphosphate binding site in LtaS important for in vitro and in vivo enzyme function. The LTA primase LtaPLm initiates LTA synthesis by transferring the first glycerolphosphate (GroP) subunit onto the glycolipid anchor and the LTA synthase LtaSLm extends the polymer by the repeated addition of GroP subunits to the tip of the growing chain. Lipoteichoic acid (LTA) is an important cell wall component found in many Gram-positive bacteria, including human pathogens such as Staphylococcus aureus and Listeria monocytogenes. The PGP backbone chain is polymerized by lipoteichoic acid synthase or LtaS-type enzymes (1). This class of enzyme uses the membrane lipid phosphatidylglycerolphosphate (PG) as a substrate, hydrolyzes the glycerolphosphate (GroP) head group of this lipid and subsequently adds it to the tip of the growing chain (9, 10). Taken together the structural and functional data allowed us to propose a revised mechanism for LTA biosynthesis in Gram-positive bacteria

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION