Protein engineering of class-A non-specific acid phosphatase (PhoN) of Salmonella typhimurium: Modulation of the pH-activity profile

Makde, Ravindra D.; Dikshit, Krati; Kumar, Vinay

doi:10.1016/j.bioeng.2006.06.004

Cited by 14 publications

(14 citation statements)

References 26 publications

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“…The associated remarkable change in phosphorylation activity could be explained by a subtle conformational change, because the side chain of residue 78 is in close proximity to H158, which is involved in protonation of the leaving group. Exchange of isoleucine 78 for an aspartic acid or histidine in PhoN‐Se MD6001 causes a shift of the pH optimum towards alkaline pH in the hydrolysis of p NPP 17. As shown in Figure 1, we did not observe a shift in pH for the variant V78L.…”

Section: Resultsmentioning

confidence: 64%

“…Because mutation of residue 78 in a related phosphatase had resulted in an alkaline pH shift in the hydrolysis of p NPP,17 the pH profile of the variant V78L in DHA phosphorylation with PP i was studied (Figure 1) and compared to that of the WT PhoN‐Se to explore the possibility that the mutant enzyme might show a shift in its pH optimum. The variant shows higher DHA phosphorylation activity than the WT PhoN‐Se at all pH values tested, but the pH profile is not affected.…”

Section: Resultsmentioning

confidence: 99%

“…Figure 4 shows the positions of the activity‐enhancing amino acid substitutions in a 3D structure model generated from the known X‐ray structure of PhoN‐Se MD6001 17. The substitution of valine 78 to a leucine is a small change in secondary structure: only one CH 2 group is added.…”

Section: Resultsmentioning

confidence: 99%

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

Herk¹,

Hartog²,

Babich³

et al. 2009

ChemBioChem

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To enhance the phosphorylating activity of the bacterial nonspecific acid phosphatase from Salmonella enterica ser. typhimurium LT2 towards dihydroxyacetone (DHA), a mutant library was generated from the native enzyme. Three different variants that showed enhanced activity were identified after one round of epPCR. The single mutant V78L was the most active and showed an increase in the maximal DHAP concentration to 25 % higher than that of the wild-type enzyme at pH 6.0. This variant is 17 times more active than the wild-type acid phosphatase from Salmonella enterica ser. typhimurium LT2 in the acid phosphatase/aldolase cascade reaction at pH 6.0 and is also six times more active than the phosphatase from Shigella flexneri that we previously used.

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Section: Resultsmentioning

confidence: 64%

Section: Resultsmentioning

confidence: 99%

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

Herk¹,

Hartog²,

Babich³

et al. 2009

ChemBioChem

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“…typhymurium MD6001. [13] In this protein, exchanging the isoleucine 78 for an aspartic acid resulted in a pH shift towards more basic pH. The authors explain this by an increase in pK a of the H158 involved in the protonation of the leaving group due to electrostatic effects from the D78, which stabilized the protonated form of H158.…”

mentioning

confidence: 93%

“…Figure 2 fits well with simulations described by Chen et al [9] The acid phosphatases form Salmonella enterica ser. typhimurium MD6001 [13] and Escherichia blattae [14] have been crystallized and their 3D structures have been resolved. Our PhoN-Se shows 94 % and 38 % identity, respectively, and 3D models have been build.…”

mentioning

confidence: 99%

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

Herk¹,

Hartog²,

Ruijssenaars³

et al. 2007

Adv Synth Catal

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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...

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Hydrolysis and Formation of PO Bonds

Wever

Herk

2012

Enzyme Catalysis in Organic Synthesis

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Protein engineering of class-A non-specific acid phosphatase (PhoN) of Salmonella typhimurium: Modulation of the pH-activity profile

Cited by 14 publications

References 26 publications

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

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

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

Hydrolysis and Formation of PO Bonds

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