Head group specificity of phospholipase D isoenzymes from poppy seedlings (Papaver somniferum L.)

Oblozinsky, Marek; Ulbrich‐Hofmann, Renate; Bezáková, Lýdia

doi:10.1007/s10529-004-7853-x

Cited by 32 publications

(13 citation statements)

References 19 publications

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“…An uncommon head group specificity has been found for two PLD isoenzymes from poppy seedlings (Oblozinsky et al 2005). Both enzymes showed the same ratio in rates of hydrolysis toward PC, PG, PS and PI (1:0.5:0.3:0.1) and were inactive toward PE.…”

Section: Substrate Specificity and Transesterification Potential Of Pldmentioning

confidence: 89%

Phospholipase D and its application in biocatalysis

et al. 2005

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Phospholipase D (PLD) from plants or microorganisms is used as biocatalyst in the transformation of phospholipids and phospholipid analogs in both laboratory and industrial scale. In recent years the elucidation of the primary structure of many PLDs from several sources, as well as the resolution of the first crystal structure of a microbial PLD, have yielded new insights into the structural basis and the catalytic mechanism of this catalyst. This review summarizes some new results of PLD research in the light of application.

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Section: Substrate Specificity and Transesterification Potential Of Pldmentioning

confidence: 89%

Phospholipase D and its application in biocatalysis

et al. 2005

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“…However, conformation and size of myo ‐inositol make it difficult to fit properly into the acceptor binding site of most of the PLDs. As a result, only two wild‐type plant PLDs, PLD‐B from poppy seedlings and spinach leaves have been reported to catalyze PI synthesis (Mandal et al, ; Oblozinsky et al, ), but these enzymes are not easily available for commercial applications. Another reason for poor reactivity of PLDs towards myo ‐inositol is the fact that PLDs prefer primary over secondary alcohols (Damnjanović and Iwasaki, and references therein).…”

Section: Introductionmentioning

confidence: 99%

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

Damnjanović

Nakano

Iwasaki

2013

Biotech & Bioengineering

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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.

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“…To date, two plant PLDs have been reported to catalyze PI‐synthesis 6. The PI‐synthesizing rate of PLD from poppy seedlings is reported to be 66±7.2 nmol min −1 mg −1 ,6a that is, 16 times lower than that of DYR. Therefore, we may mention that, although their PC‐hydrolyzing activity was lower than that of WT‐PLD, our mutant PLDs have the highest PI‐synthesizing activity recognized so far.…”

Section: Resultsmentioning

confidence: 94%

Isolation of Phospholipase D Mutants Having Phosphatidylinositol‐Synthesizing Activity with Positional Specificity on myo‐Inositol

Masayama¹,

Tsukada²,

Ikeda³

et al. 2009

ChemBioChem

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ENZYME-MEDIATED SYNTHESIS OF PHOSPHATIDYLINOSITOL: Engineered phospholipase D enzymes enable the synthesis of phosphatidylinositol by transphosphatidylation. The 1- or 3-hydroxy group of myo-inositol is selectively reacted. Phospholipase D (PLD) mutants that have phosphatidylinositol (PI)-synthesizing activity with positional selectivity towards 1- or 3-OH groups of myo-inositol have been isolated. A mutant PLD library, in which site-directed saturation mutations were introduced in vitro at positions 187, 191, and 385 of the wild-type PLD of Streptomyces antibioticus, was screened for PI-synthesizing mutants. TLC and HPLC analyses of the PI synthesized by the isolated mutant PLDs revealed that three mutants, namely 187D/191Y/385R (DYR), 187A/191Y/385R (AYR), and 187M/191Y/385R (MYR), selectively generated 1- or 3-PI among the other possible PI positional isomers. Taking into account the consensus sequence of the three mutants, a series of mutants, 187X/191Y/385R (XYR), was constructed and analyzed. Almost all the XYR mutants generated 1(3)-PI selectively, thus suggesting that the Y385R mutation contributed to the selectivity for the 1(3)-PI synthesis. The XYR mutants showed similar phosphatidylcholine-hydrolyzing activity among the mutants, but the PI-synthesizing activities were different depending on the amino acid at position 187. In particular, aromatic amino acids at position 187 greatly reduced the PI-synthesizing activity. The ratios of 1-PI versus 3-PI in the PIs synthesized with the XYR mutants were analyzed by selective hydrolysis with PI-specific phospholipase C. It was found that 187H/191Y/385R (HYR) generated 1-PI more than 3-PI (ratio=7:3), whereas 187T/191Y/385R (TYR) generated 1-PI less than 3-PI (ratio=2:8). This confirmed that the amino acid at position 187 determined the selectivity between 1-PI and 3-PI formation.

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Head group specificity of phospholipase D isoenzymes from poppy seedlings (Papaver somniferum L.)

Cited by 32 publications

References 19 publications

Phospholipase D and its application in biocatalysis

Phospholipase D and its application in biocatalysis

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

Isolation of Phospholipase D Mutants Having Phosphatidylinositol‐Synthesizing Activity with Positional Specificity on myo‐Inositol

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