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-A 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 alpha/beta 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 beta-roll domains that pack tightly against each other. Variations of the consensus sequence of the tandem repeats within the second beta-roll result in an asymmetric Ca(2+) binding on only one side of the roll. The analysis of the properties of the beta-roll domains suggests an intramolecular chaperone function.
Highlights
Lipases (EC 3.1.1.3) hydrolyze the ester bonds of long-chain acylglycerides [1]
Lipases of families I.1 and I.2 are clearly homologous with amino acid sequence identities above 30%, the family I.3 enzymes exhibit only a very low sequence similarity to the former two families
Whereas family I.1 and I.2 lipases are secreted by a type II secretion system (T2SS,3 named general secretory pathway) (14 –16), the family I.3 lipases are transported by a type I secretion system (T1SS) (16 – 18)
Summary
T2SS, type 2 secretion system; T1SS, type 1 secretion system; RTX, repeats in toxins. The number n of the repeats correlates positively with the molecular weight of the protein. This so-called RTX signature (repeats in toxins) [20] is responsible for Ca2ϩ binding. The nonpolar amino acids (abbreviated U) at position 8 build the hydrophobic core of the -roll. This peculiar structure is unstable in the absence of Ca2ϩ but does fold spontaneously in the presence of calcium concentrations in the mM range [24, 25]. From inspection of the sequence, S. marcescens LipA possesses about 12–14 repeats of the glycine-rich sequence motif that come in two blocks spanning residues 369 – 418 and 489 –564. We have determined the high resolution crystal structure of LipA from S. marcescens
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