Structure-guided enzymology of the lipid A acyltransferase LpxM reveals a dual activity mechanism

Abstract

Gram-negative bacteria possess a characteristic outer membrane, of which the lipid A constituent elicits a strong host immune response through the Toll-like receptor 4 complex, and acts as a component of the permeability barrier to prevent uptake of bactericidal compounds. Lipid A species comprise the bulk of the outer leaflet of the outer membrane and are produced through a multistep biosynthetic pathway conserved in most Gram-negative bacteria. The final steps in this pathway involve the secondary acylation of lipid A precursors. These are catalyzed by members of a superfamily of enzymes known as lysophospholipid acyltransferases (LPLATs), which are present in all domains of life and play important roles in diverse biological processes. To date, characterization of this clinically important class of enzymes has been limited by a lack of structural information and the availability of only low-throughput biochemical assays. In this work, we present the structure of the bacterial LPLAT protein LpxM, and we describe a high-throughput, label-free mass spectrometric assay to characterize acyltransferase enzymatic activity. Using our structure and assay, we identify an LPLAT thioesterase activity, and we provide experimental evidence to support an ordered-binding and "reset" mechanistic model for LpxM function. This work enables the interrogation of other bacterial acyltransferases' structure-mechanism relationships, and the assay described herein provides a foundation for quantitatively characterizing the enzymology of any number of clinically relevant LPLAT proteins.LpxM | acyltransferase | lipid A | RapidFire mass spectrometry | phosphopantetheine ejection assay

Diacylglycerol acyltransferase 1 is activated by phosphatidate and inhibited by SnRK1‐catalyzed phosphorylation

“…Once oleoyl‐CoA concentrations greater than 5 μM are reached, the plot prefers a combined model for the Hill equation and substrate inhibition (Table S1), which was recently used to account for the effects of increasing oleoyl‐CoA concentration on the activity of recombinant BnaDGAT1 in yeast microsomes (Xu et al ., ). The combined model was previously proposed for another acyltransferase that exhibited the same kinetic behavior (Dovala et al ., ). The effect of bovine serum albumin (BSA) on the substrate saturation plot was also investigated (Figure S1(b)) as BSA is sometimes used as an additive in in vitro DGAT assays (Little et al ., ; Weselake, ), although there is no general consensus on its usage.…”

How lipid droplets “TAG” along: Glycerolipid synthetic enzymes and lipid storage

“…Since these initial studies, additional structures of LPLAT family members have been determined that have provided new insight into the binding site of acyl-CoA [188,189]. These include the structure of Mycobacterium smegmatis PatA, which uses palmitoyl-CoA in the biosynthesis of mycobacterial phosphatidyl-myo-inositol mannosides [188].…”

Multiple mechanisms contribute to increased neutral lipid accumulation in yeast producing recombinant variants of plant diacylglycerol acyltransferase 1

“…In addition to the proposed acyl-CoA-binding motif FYXDWWN in the hydrophobic segment (46), the hydrophilic N-terminal domain has been shown to associate with acyl-CoA via an allosteric interaction (14,20,21,44). Substrate inhibition could potentially arise when two substrate molecules bind to the enzyme to form a catalytically inactive ES2 complex by blocking second substrate binding (24) or product release (47) or when the substrate binding leads to the formation of less thermodynamically favored ES complex that turns over slowly (48 -50). In this study, it is hypothesized that the substrate inhibition of plant DGAT1 is caused by the formation of ES2 complex.…”