Abstract
LCAT (lecithin:cholesterol acyltransferase) catalyzes the transacylation of a fatty acid of lecithin to cholesterol, generating a cholesteryl ester and lysolecithin. The knowledge of LCAT atomic structure and the identification of the amino acids relevant in controlling its structure and function are expected to be very helpful to understand the enzyme catalytic mechanism, as involved in HDL cholesterol metabolism. However - after an early report in the late ‘90 s - no recent advance has been made about LCAT three-dimensional structure. In this paper, we propose an LCAT atomistic model, built following the most up-to-date molecular modeling approaches, and exploiting newly solved crystallographic structures. LCAT shows the typical folding of the α/β hydrolase superfamily, and its topology is characterized by a combination of α-helices covering a central 7-strand β-sheet. LCAT presents a Ser/Asp/His catalytic triad with a peculiar geometry, which is shared with such other enzyme classes as lipases, proteases and esterases. Our proposed model was validated through different approaches. We evaluated the impact on LCAT structure of some point mutations close to the enzyme active site (Lys218Asn, Thr274Ala, Thr274Ile) and explained, at a molecular level, their phenotypic effects. Furthermore, we devised some LCAT modulators either designed through a de novo strategy or identified through a virtual high-throughput screening pipeline. The tested compounds were proven to be potent inhibitors of the enzyme activity.
Highlights
Protein members of the a/b hydrolase superfamily, present in all living organisms, share the same structural architecture but do not have common functions
The LCAT reaction consists in a trans-esterification, in which a fatty acid at the sn-2 position of phosphatidylcholine, or lecithin, is transferred to the free hydroxyl group of cholesterol, and in the meantime phosphatidylcholine is converted into lysophosphatidylcholine
Submitting the entire LCAT sequence to the Fold Recognition of PSIPRED server [23] did not identify any useful entry, and the best matching protein with a solved structure was a hydrolase from Lactobacillus plantarum (PDB ID 3LP5, UniProt ID: F9UMW5)
Summary
Protein members of the a/b hydrolase superfamily, present in all living organisms, share the same structural architecture but do not have common functions. This implies that the same fold has been used through evolution for a number of different functions including the catalytic activity as, for instance, hydrolase and esterase [1]. The canonical fold of this superfamily consists of an 8stranded, mainly parallel, b-sheet surrounded by a-helices, in which the second strand is oriented in the antiparallel direction. LCAT (phosphatidylcholine-sterol acyltransferase, EC 2.3.1.43) belongs to the a/b hydrolase folding superfamily and shares the Ser/Asp-Glu/His triad with lipases, esterases and proteases, as already thoroughly discussed by Peelman et al in 1998 [3]. At an atomic level, the mechanism is not yet accurately described [3]
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