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  • br Acknowledgements This study was

    2019-07-15


    Acknowledgements This study was financially supported by the National Special Research Programs for Non-Profit Trades (Agriculture) (Grant No. 201303045) and the Basic Foundation for Scientific Research of the State-level Public Welfare Institutes of China (Grant No. 2013JB10).
    Introduction Lactobacillus plantarum is a lactic CFDA-SE bacteria that is broadly used as a starter in fermented foods and also as a gastrointestinal probiotic being able to adhere and colonize human intestinal mucosa, playing a critical role in competitive exclusion of pathogenic bacteria [1], [2], [3]. This adherence is related to the cell surface characteristics and more specifically, to the expression of surface proteins that directly mediate binding mechanisms to human intestinal cells [4], [5]. Alfa-enolase (α-Eno) from Lactobacillus plantarum is a classical glycolytic enzyme that is usually found in the cytoplasm, however, it has unexpectedly been found on the outer surface of the cell wall [3], [6] where it displays different, seemingly unrelated functions such as adhesion to epithelial cells and extracellular matrix proteins through binding to human plasminogen and fibronectin [5], [7]. Owing to these moonlighting functions, α-Eno has now been reclassified as a multifunctional-anchorless-surface CFDA-SE protein [4], [8] that is also thought to have a role in cellular stress in response to different hostile environments [9], [10]. Linoleic acid (LA; 18:2 ο9Z,12Z), along with other polyunsaturated fatty acids, are stress and toxic factors to many bacterial cells causing the inhibition of cell growth and deterioration of cellular membranes by blocking native fatty acid biosynthesis via the inhibition of enoyl-ACP reductase [11], [12], [13]. In order to prevent this toxicity, bacteria belonging to Lactobacillus genus, carry on a biohydrogenation process which is the complete reduction of double bonds on the carbon chain, producing non-toxic saturated fatty acids as the final product [14]. During biohydrogenation, LA is converted to saturated non-toxic stearic acid (SA; 18:0) [15] with the accumulation of various intermediate molecules such as conjugated linoleic acids (CLA; 18:2 οxX,yY) which are LA isomers with conjugated double bonds in either cis or trans configuration [16]. The predominant isomer produced by Lactobacillus plantarum is the bioactive molecule 9-cis-11-trans-CLA (rumenic acid; 18:2 ο9Z,11E) which has been associated with a variety of health promoting effects for humans such as antiobesity and antiadipogenic activities [17], anticarcinogenic activity [18] and modulation of immune functions [19]. It has been demonstrated that 9-cis-11-trans-CLA synthesis in Lactobacillus plantarum occurs through a newly discovered and intricate metabolism consisting of multiple reactions catalyzed by multiple proteins [20]. The first step is hydration of the ο9Z double bond of LA which is mediated by a 64.7kDa peripheral-membrane bound hydratase that produces 10-hydroxy-12-cis-octadecenoic acid (10-HOE; 18:1 ο12Z) as the only product [21], [22]. Once 10-HOE is produced, it can suffer various reactions (oxidation of hydroxyl group, reduction of oxo group, dehydration and isomerization) catalyzed by the partially characterized linoleate isomerase complex (CLA-DH, CLA-DC, CLA-DH and CLA-ER), to finally produce monoenoic acids and conjugated linoleic acid isomers (CLA; 18:2 οxX,yY) [20]. These proteins, implicated in dehydration of the hydroxy-enoic acid and formation of bioactive isomers of CLA in Lactobacillus plantarum, have just been studied recently and hence, the participation of other proteins catalyzing similar or equivalent reactions cannot be disregarded. In this work, we identified a protein isolated from Lactobacillus plantarum ATCC 8014 cells, which possess the ability to produce 9-cis-11-trans-CLA from LA when combined with the membrane-associated protein fraction. Mass spectrometric analysis showed that the isolated native enzyme is the multifunctional α-enolase. We determined that α-Eno is not capable of using directly LA as a substrate but instead uses 10-HOE, previously produced by the membrane fraction, to finally form bioactive CLA. We also performed biochemical optimization studies to elucidate the conditions for maximum formation of 9-cis-11-trans-CLA and maximum stability of α-Eno when catalyzing this reaction. Furthermore, we propose a three-dimensional arrangement of the protein and carried out molecular docking analysis with the substrate and the product in order to identify the binding sites of these molecules which were characterized as two hydrophobic superficial pockets located at opposite ends of the protein connected through a superficial channel where the catalysis of dehydration and isomerization of 10-HOE occurs. These results prove that multifunctional α-Eno is a side participant in the biohydrogenation process and plays a role in cell detoxification from polyunsaturated fatty acids such as LA, along with the linoleate isomerase complex.