Jasmina Damnjanović

Phospholipase D as a catalyst: Application in phospholipid synthesis, molecular structure and protein engineering

Phospholipase D (PLD) is a useful enzyme for its transphosphatidylation activity, which enables the enzymatic synthesis of various phospholipids (PLs). Many reports exist on PLD-mediated synthesis of natural and tailor-made PLs with functional head groups, from easily available lecithin or phosphatidylcholine. Early studies on PLD-mediated synthesis mainly employed enzymes of plant origin, which were later supplanted by ones from microorganisms, especially actinomycetes. Many PLDs are members of the PLD superfamily, having one or two copies of a signature sequence, HxKxxxxD or HKD motif, in the primary structures. PLD superfamily members share a common core structure, and thereby, a common catalytic mechanism. The catalysis proceeds via two-step reaction with the formation of phosphatidyl-enzyme intermediate. Both of the two catalytic His residues are critical in the reaction course, where one acts as a nucleophile, while the other functions as a general acid/base. PLD is being engineered to improve its activity and stability, alter head group specificity and further identify catalytically important residues. Since the knowledge on PLD enzymology is constantly expanding, this review focuses on recent advances in the field, regarding PLD-catalyzed synthesis of bioactive PLs, deeper understanding of substrate recognition and binding mechanism, altering substrate specificity, and improving thermostability. We introduced some of our recent results in combination with existing facts to further deepen the story on the nature of this useful enzyme.

Deletion of a dynamic surface loop improves stability and changes kinetic behavior of phosphatidylinositol‐synthesizing Streptomyces phospholipase D

Supplementary phosphatidylinositol (PI) was shown to improve lipid metabolism in animals, thus it is interesting for pharmaceutical and nutritional applications. Homogenous PI can be produced in transphosphatidylation of phosphatidylcholine (PC) with myo‐inositol catalyzed by phospholipase D (PLD). Only bacterial enzymes able to catalyze PI synthesis are Streptomyces antibioticus PLD (SaPLD) variants, among which DYR (W187D/Y191Y/Y385R) has the best kinetic profile. Increase in PI yield is possible by providing excess of solvated myo‐inositol, which is achievable at high temperatures due to its highly temperature‐dependent solubility. However, high‐temperature PI synthesis requires the thermostable PLD. Previous site‐directed combinatorial mutagenesis at the residues of DYR having high B‐factor yielded the most improved variant, D40H/T291Y DYR, obtained by the combination of two selected mutations. D40 and T291 are located within dynamic surface loops, D37‐G45 (termed D40 loop) and G273‐T313. Thus, in this work, thermostabilization of DYR SaPLD was attempted by rational design based on deletion of the D40 loop, generating two variants, Δ37‐45 DYR and Δ38‐46 DYR PLD. Δ38‐46 DYR showed highest thermostability as its activity half‐life at 70°C proved 11.7 and 8.0 times longer than that of the DYR and Δ37‐45 DYR, respectively. Studies on molecular dynamics predicted Δ38‐46 DYR to have the least average RMSD change as temperature dramatically increases. At 60 and 70°C, both mutants synthesized PI in a twofold higher yield compared to the DYR, while at the same time produced less of the hydrolytic side‐product, phosphatidic acid. Biotechnol. Bioeng. 2014;111: 674–682.

Improving thermostability of phosphatidylinositol-synthesizing Streptomyces phospholipase D.

Aimed to produce thermostable phosphatidylinositol (PI)-synthesizing phospholipase D (PLD), we initiated site-directed combinatorial mutagenesis followed by high-throughput screening. Previous site-directed combinatorial mutagenesis of wild-type Streptomyces PLD produced a mutant, DYR (W187D/Y191Y/Y385R) with PI-synthesizing ability. Deriving PI as a product of transphosphatidylation between phosphatidylcholine and myo-inositol, with myo-inositol in excess at high-temperature reaction conditions can increase yield due to enhanced solubility of this substrate. Thus, we improved DYRs thermostability by introduction of random mutations into selected amino acid positions having high B-factor. Screening of the libraries under restricted conditions yielded single-point mutants, specifically D40H, T291Y and R329G. Combinations of these point mutations yielded double (D40H/T291Y, D40H/R329G and T291Y/R329G) and triple (D40H/T291Y/R329G) mutants. PI synthesis at elevated temperatures pointed at D40H/T291Y as the most efficient enzyme. Circular dichroism analysis revealed D40H/T291Y to have increased melting temperature and postponed onset of thermal unfolding compared with DYR. Thermal tolerance study at 65°C confirmed D40H/T291Ys thermostability as its half-inactivation time was 8.7 min longer compared with DYR. This mutant had significantly less root-mean-square deviation change compared with DYR and showed no change in root-mean-square fluctuation when temperature shifts from 40 to 60°C, as determined by molecular dynamics analysis. Acquired different degrees of thermostability were also observed for several other DYR mutants.

Directing positional specificity in enzymatic synthesis of bioactive 1-phosphatidylinositol by protein engineering of a phospholipase D

Phosphatidylinositol (PI) holds a potential of becoming an important dietary supplement due to its effects on lipid metabolism in animals and humans manifested as a decrease of the blood cholesterol and lipids, and relief of the metabolic syndrome. To establish an efficient, enzymatic system for PI production from phosphatidylcholine and myo‐inositol as an alcohol acceptor, our previous study started with the wild‐type Streptomyces antibioticus phospholipase D (SaPLD) as a template for generation of PI‐synthesizing variants by saturation mutagenesis targeting positions involved in acceptor accommodation, W187, Y191, and Y385. The isolated variants generated PI as a mixture of positional isomers, among which only 1‐PI exists in nature. Thus, the current study has focused to improve positional specificity of W187N/Y191Y/Y385R SaPLD (NYR) which generates PI as a mixture of 1‐PI and 3‐PI in the ratio of 76/24, by subjecting four residues of its acceptor‐binding site to saturation mutagenesis. Subsequent screening pointed at NYR‐186T and NYR‐186L as the most improved variants producing PI with a ratio of 1‐/3‐PI = 93/7 and 87/13, respectively, at 37°C. Lowering the reaction temperature further improved the specificity of both variants to 1‐/3‐PI > 97/3 at 20°C with no change in total PI yield. Structure model analyses imply that G186T and G186L mutations increased rigidity of the acceptor‐binding site, thus limiting the possible orientations of myo‐inositol. The two newly isolated PLDs are promising for future application in large‐scale 1‐PI production. Biotechnol. Bioeng. 2016;113: 62–71.

Enzymatic Modification of Phospholipids by Phospholipase D

Abstract Phospholipase D (PLD) is a useful enzyme because of its transphosphatidylation activity, which enables the head group modification of phospholipids (PLs). For this purpose, various reaction systems have been developed, including solvent–buffer biphasic, anhydrous solvent, heterogeneous phase, mixed micelle, and “green” solvent systems, which are used according to the purpose of the synthesis. PLD-mediated reactions enable the syntheses of natural and unnatural types of PLs with various functional head groups, from easily available lecithin or phosphatidylcholine. The majority of PLDs are members of the PLD superfamily, containing one or two the so-called HKD motif in their primary sequence. The superfamily members share a common core structure and, thereby, a common catalytic mechanism. Protein engineering of PLD contributes to the improvement of their activity, stability, and substrate specificity. The alteration of head group specificity enables the preparation of PLs that cannot be synthesized by wild-type PLDs, thereby expanding the applicability of this enzyme class.