Optimization of the Kinetic Resolution of the dl‐Phosphomonoesters of Threonine and Serine by Random Mutagenesis of the Acid Phosphatase from Salmonella enterica

Abstract

Acid phosphatases are enzymes with a broad substrate specificity showing hydrolytic activity towards several different organic phosphate monoesters, such as nucleotides and sugar phosphates. The acid phosphatase from Salmonella enterica ser. typhimurium LT2 (PhoN-Se) is able to hydrolyze O-phospho-dl-threonine to yield l-threonine with a very high enantioselectivity (E > 200). When O-phospho-dl-serine was hydrolyzed by PhoN-Se, d-serine was formed, however, the ee values rapidly dropped to 50 %. Random mutagenesis by error-prone PCR was performed on the phosphatase in order to increase its enantioselectivity in the formation of d-serine. Two variants with increased selectivity from a library of 9600 mutants have been found, N151D and V78L showing E values of 18.1 and 4.1, respectively, compared to 3.4 for the wild-type (WT) enzyme.Keywords: acid phosphatase; directed evolution; enantioselectivity; enzyme catalysis; phosphomonoester hydrolysis; random mutagenesis Enzymes are attractive tools in catalysis and efficiently complement (traditional) chemical methods. Some enzymes are commercially available [1,2] but the substrate specificity and enantioselectivity are not always sufficient. Finding an enzyme that shows high stereoselectivity towards a broad range of synthetically useful molecules is desirable. Bacterial non-specific acid phosphatases (NSAPs) [3,4] are a group of secreted enzymes with a broad substrate specificity that shows hydrolytic activity towards several different organic phosphate monoesters. These enzymes are also capable of transferring a phosphate group from a donor (phosphomonoester and pyrophosphate) to a wide range of acceptors (alcohols). [5,6] The acid phosphatase from Shigella flexneri (PhoN-Sf) [7] shows enantioselectivity in the phosphorylation of d-and l-glucose by pyrophosphate having a higher affinity for d-glucose.[6] To broaden the application range of phosphatases, we investigated the possible use of PhoN-Sf and PhoN-Se (the acid phosphatase from Salmonella enterica ser. typhimurium LT2) [8] in the kinetic resolution of phosphomonoesters and optimized the hydrolysis reaction by applying random mutagenesis.Here we report on the formation of l-threonine and d-serine from O-phosphorylated dl-threonine and dl-serine by PhoN-Sf and PhoN-Se. PhoN-Se shows reasonable reaction rates in dephosphorylation of both O-phospho-dl-threonine and O-phospho-dlserine as monitored by HPLC (Figure 1). Therefore the pH dependency of the enantioselectivity of this enzyme was investigated in more detail. Hydrolysis rates are not affected to a large extent between pH 3.9 and 5.0, but they clearly diminish at pH 6.25 and no activity is found at pH 3.3 (not shown). To our surprise, only l-threonine was detected within 5 h of incubation for pH values between 3.9 and 5.0 (~32 % conversion) and O-phospho-d-threonine was not hydrolyzed, giving an ee value of 100 %. [The detection limit of d-threonine in this system is 0.75 mM. In this case, no d-threonine detected means that less than 1 mM of d-threo...

Exploiting Acid Phosphatases in the Synthesis of Phosphorylated Monoalcohols and Diols

“…Mevalonolactone ( rac ‐ 13 ) and charged substrates, like lactic ( rac ‐ 14 ) and tartaric acid ( 15 ) (both enantiomers tested separately) were not converted. Although O ‐phospho‐ D , L ‐serine could be stereoselectively hydrolyzed by a mutant of PhoN‐Se,29 the wild‐type enzyme was not active in the phosphorylation of rac ‐serine ( 16 ). Finally, oximes 17 – 19 did not undergo phosphorylation.…”

Improvement of an Acid Phosphatase/DHAP‐Dependent Aldolase Cascade Reaction by Using Directed Evolution

“…It is striking that the mutation V78L is found three times. The V78 residue is probably very important in tuning the phosphorylation and dephosphorylation reaction, because we had previously found the identical mutation in a directed evolution experiment in which it showed a higher enantioselectivity in the dephosphorylation of O ‐phospho‐ dl ‐serine 16…”

Key advances in biocatalytic phosphorylations in the last two decades: Biocatalytic syntheses in vitro and biotransformations in vivo (in humans)

“…Acid phosphatase from Salmonella enterica has been demonstrated to catalyze enantioselective hydrolysis of O-phospho-DL-threonine, whereby only the L-enantiomer was hydrolyzed and O-phospho-D-threonine was left unchanged. [61] The excellent enantioselectivities of a bacterial phosphotriesterase and mutants, which have been measured for a range of enantiomeric pairs of phosphorus-containing esters, show their application potential for the kinetic resolution of racemic phosphate esters, phosphonate esters, and phosphinate esters. [62] A simple stereoselective hydrolytic resolution catalyzed by phosphotriesterase from Pseudomonas diminuta has been used for the preparation of diastereochemically pure phosphorylated nucleosides having a chiral phosphorus center, which are valuable chiral phosphoramidate precursors to antiviral prodrugs.…”