A Calcium-gated Lid and a Large β-Roll Sandwich Are Revealed by the Crystal Structure of Extracellular Lipase from Serratia marcescens

Abstract

Lipase LipA from Serratia marcescens is a 613-amino acid enzyme belonging to family I.3 of lipolytic enzymes that has an important biotechnological application in the production of a chiral precursor for the coronary vasodilator diltiazem. Like other family I.3 lipases, LipA is secreted by Gram-negative bacteria via a type I secretion system and possesses 13 copies of a calcium binding tandem repeat motif, GGXGXDXUX (U, hydrophobic amino acids), in the C-terminal part of the polypeptide chain. The 1.8-Å crystal structure of LipA reveals a close relation to eukaryotic lipases, whereas family I.1 and I.2 enzymes appear to be more distantly related. Interestingly, the structure shows for the N-terminal lipase domain a variation on the canonical ␣/␤ hydrolase fold in an open conformation, where the putative lid helix is anchored by a Ca 2؉ ion essential for activity. Another novel feature observed in this lipase structure is the presence of a helical hairpin additional to the putative lid helix that exposes a hydrophobic surface to the aqueous medium and might function as an additional lid. The tandem repeats form two separated parallel ␤-roll domains that pack tightly against each other. Variations of the consensus sequence of the tandem repeats within the second ␤-roll result in an asymmetric Ca 2؉ binding on only one side of the roll. The analysis of the properties of the ␤-roll domains suggests an intramolecular chaperone function.

Crystal structure of a family I.3 lipase from Pseudomonas sp. MIS38 in a closed conformation

“…1A). This structure highly resembles the SML structure [12], except for the lid structures (residues 46-74 and 146-167) (Fig. 1B).…”

“…Further deletion of the repeats or mutation abolishes the repeats' ability to form b-roll motif, and greatly decreases secretion efficiency, proteolysis stability, and enzymatic activity [9,10]. These results allowed us to propose that the b-roll motif has a chaperone-like function [2,12]. Because the parallel b-sheet of one side of the first b-roll motif is extended to form a long parallel b-sheet with three b-strands (b6-b8) of the lipase domain (Fig.…”

“…The C-domain of PML contains two b-roll motifs, laterally stacked together forming the so-called b-roll sandwich [12], similar to that of SML. The first b-roll motif consists of residues 373-417, containing five RTX repeats and binds three Ca 2+ ions that are ''dry'', i.e.…”

“…The crystal structure of Serratia marcescens LipA (SML), which is a family I.3 lipase and shows the amino acid sequence identity of 61% to PML, has recently been determined [12]. The SML structure consists of the N-terminal lipase domain and C-terminal RTX domain.…”

The C Terminus of Tubulin, a Versatile Partner for Cationic Molecules

“…Interestingly, the zone of the greatest chemical shift perturbations observed on the ␣Tub410C peptide upon calcium binding are within the RTX consensus (Fig. 7C) (63). This may indicate that the C terminus of tubulin behaves truly as a calcium consensus region.…”