Poster Presentation The 43rd Lorne Conference on Protein Structure and Function 2018

Structural determination of Streptomyces antibioticus phospholipase D engineered to produce 1-phosphatidylinositol (#226)

Ariela Samantha 1 , Jasmina Damnjanovic 2 , Yugo Iwasaki 2 , Alice Vrielink 1
  1. School of Molecular Sciences, The University of Western Australia, Perth, Western Australia, Australia
  2. School of Bioagricultural Sciences, Nagoya University, Nagoya, Japan

Phospholipases are class of enzymes that hydrolyse acyl esters and phosphate esters of phospholipids. Transphosphatidylase activity of phospholipase is a unique reaction which can only be catalysed by phospholipase D (PLD). This phosphatidylalcohol-generating reaction has long been utilized for industrial phospholipid synthesis [1].

PLDs are produced by many organisms, however Streptomyces antibioticus phospholipase D (SaPLD) is prefered for phospholipid synthesis due to its excellent transphosphatidylation activity and storage stability [2]. Moreover, SaPLD can be engineered to produce novel artificial or naturally available but limited phospholipids, such as phosphatidylinositol (PI) [3]. PI production becomes important especially since some reports show that PI has various therapeutical values, and therefore is gaining interest to be used as dietary supplements to improve health or treat specific medical conditions [4].

Wild-type, SaPLD is unable to synthesize PI. This is possibly due to steric hindrance in the active site towards bulky molecules such as myo-inositol [5]. Although engineered SaPLD enzymes that can accept myo-inositol and synthesize PI have been obtained by site-directed saturation mutagenesis [6,7], the positional specificity of the enzymes was insufficient. Through several rounds of mutagenesis, an engineered variant of the enzyme was finally obtained. The mutant: 186T, 187N, 191Y, 385R can specifically produce 1-PI, the only natural PI isomer, with isomeric purity of  >97% [8].

Crystallographic structure determination of the mutant PLD with and without myo­­-inositol will explain the mechanism that underlies the accommodation of this bulky acceptor molecule and the resulting positional specificity of the phospholipid product. This work will support further strategies to optimize PLD for the production of structured and pure 1-PI suitable for various industrial purposes.

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