Human secretory group IIa phospholipase A2 (hIIa-PLA2) contains a large number of prominent cationic patches on its molecular surface and has exceptionally high affinity for anionic surfaces, including anionic membranes. To identify the cationic amino acid residues that support binding of hIIa-PLA2 to anionic membranes, we have performed extensive site-directed mutagenesis of this protein and measured vesicle binding and interfacial kinetic properties of the mutants using polymerized liposomes and nonpolymerized anionic vesicles. Unlike other secretory PLA2s, which have a few cationic residues that support binding of enzyme to anionic membranes, interfacial binding of hIIa-PLA2 is driven in part by electrostatic interactions involving a number of cationic residues forming patches on the putative interfacial binding surface. Among these residues, the amino-terminal patch composed of Arg-7, Lys-10, and Lys-16 makes the most significant contribution to interfacial adsorption, and this is supplemented by contributions from other patches, most notably Lys-74/Lys-87/Arg-92 and Lys-124/Arg-127. For these mutants, complete vesicle binding occurs in the presence of high vesicle concentrations, and under these conditions the mutants display specific activities comparable to that of wild-type enzyme. These studies indicate that electrostatic interactions between surface lysine and arginine residues and the interface contribute to interfacial binding of hIIa-PLA2 to anionic vesicles and that cationic residues closest to the opening of the active-site slot make the most important interactions with the membrane. However, because the wild type binds extremely tightly to anionic vesicles, it was not possible to exactly determine what fraction of the total interfacial binding energy is due to electrostatics.

Human nonpancreatic (group IIa) secreted phospholipase A2 (human sPLA2) is associated with a number of inflammatory disorders in which the extracellular concentrations of this enzyme can become highly elevated. It is probable that the enzyme normally acts as an acute-phase protein whose function is to facilitate the removal of infectious organisms or damaged host cells as part of the normal inflammatory response. The enzyme shows negligible activity with phosphatidylcholine (PC) vesicles and cell membranes, presumably reflecting the enzyme's lack of ability to bind productively to such condensed neutral interfaces. Mammalian pancreatic enzymes show modest activity with such interfaces and contain a unique tryptophan at position 3, which is part of the presumptive interfacial binding surface of these enzymes. Human sPLA2 does not contain tryptophan. The amphiphilic indole side chain of tryptophan is noted for its ability to penetrate the lipid interface of membranes, and tryptophan residues appear to be associated with the ability of lipases and phospholipases A2 to bind to and hydrolyze such interfaces. We have investigated in detail the properties of a V3W mutant of human sPLA2, which has a unique tryptophan on the interfacial binding surface of this enzyme. Although this enzyme shows a modest ( approximately 50%) reduction in activity when anionic substrates are used under standard assay conditions, the activity of the enzyme on phosphatidylcholine vesicles and cell membranes is dramatically increased compared with human sPLA2. This is particularly the case with small unilamellar vesicles of PC, where activity is enhanced over 250-fold compared to the almost zero activity expressed by human sPLA2. This enhanced activity is best explained by increased interfacial binding and activation of the V3W mutant and is not due to enhanced active-site binding and hydrolysis. The results highlight the important role that tryptophan residues can play in interfacial binding, particularly to condensed zwitterionic interfaces. The interfacial characteristics of the mutant human enzyme now resemble more closely the mammalian pancreatic enzymes that already have a tryptophan at position 3.

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Isoprenoids are a large and diverse group of metabolites with interesting properties such as flavour, fragrance and therapeutic properties. They are produced via two pathways, the mevalonate pathway or the 2-C-methyl-D-erythritol-4-phosphate (MEP) pathway. While plants are the richest source of isoprenoids, they are not the most efficient producers. Escherichia coli and yeasts have been extensively studied as heterologous hosts for plant isoprenoids production. In the current study, we describe the usage of the food grade Lactococcus lactis as a potential heterologous host for the production of sesquiterpenes from a local herbaceous Malaysian plant, Persicaria minor (synonym Polygonum minus). A sesquiterpene synthase gene from P. minor was successfully cloned and expressed in L. lactis. The expressed protein was identified to be a β-sesquiphellandrene synthase as it was demonstrated to be functional in producing β-sesquiphellandrene at 85.4% of the total sesquiterpenes produced based on in vitro enzymatic assays. The recombinant L. lactis strain developed in this study was also capable of producing β-sesquiphellandrene in vivo without exogenous substrates supplementation. In addition, overexpression of the strain’s endogenous 3-hydroxy-3-methylglutaryl coenzyme-A reductase (HMGR), an established rate-limiting enzyme in the eukaryotic mevalonate pathway, increased the production level of β-sesquiphellandrene by 1.25–1.60 fold. The highest amount achieved was 33 nM at 2 h post-induction.

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