(Below N is a link to NCBI taxonomic web page and E link to ESTHER at designed phylum.) > cellular organisms: NE > Bacteria: NE > Proteobacteria: NE > Gammaproteobacteria: NE > Pseudomonadales: NE > Pseudomonadaceae: NE > Pseudomonas: NE > Pseudomonas sp. ECU1011: NE
Molecular evidence
Database
No mutation 5 structures(e.g. : 4OB6, 4OB7, 4OB8... more)(less) 4OB6: Complex structure of esterase rPPE S159A/W187H and substrate (S)-Ac-CPA, 4OB7: Crystal structure of esterase rPPE mutant W187H, 4OB8: Crystal structure of a novel thermostable esterase from Pseudomonas putida ECU1011, 4OU4: Crystal structure of esterase rPPE mutant S159A complexed with (S)-Ac-CPA, 4OU5: Crystal structure of esterase rPPE mutant S159A/W187H No kinetic
LegendThis sequence has been compared to family alignement (MSA) red => minority aminoacid blue => majority aminoacid color intensity => conservation rate title => sequence position(MSA position)aminoacid rate Catalytic site Catalytic site in the MSA GSPGVEQHTKAFLEALEQGGGKPLEQLSPKDARAVLTGAQASVKVDLSGI EVKERTIQANGQSIKLQVVRPANVKGELPVFMFFHGGGWVLGDFPTHQRL IRDLVVGSGAVAVYVDYTPSPESHYPTAINQAYAATQWVAEHGKEIGVDG KRLAVAGNSVGGNMAAVVALKAKEAGTPALRFQLLLWPVTDASFETASYK QFADGHFLTTGMMKWFWDNYTTDAKAREQIYASPLRASSEQLKGLPPALV QTAEFDVLRDEGEAYARKLNAAGVTVTSVRYNGMIHDYGLLNPLSQVPAV KAAMRQAGTELKVHLQ
References
Title: Crystal structures of Pseudomonas putida esterase reveal the functional role of residues 187 and 287 in substrate binding and chiral recognition Dou S, Kong XD, Ma BD, Chen Q, Zhang J, Zhou J, Xu JH Ref: Biochemical & Biophysical Research Communications, 446:1145, 2014 : PubMed
A recombinant carboxylesterase (rPPE) from Pseudomonas putida ECU1011 was previously cloned and engineered to give a potential application for resolving chiral alpha-hydroxy acids including mandelic acids and derivatives. Two variants rPPEW187H and rPPED287A showed a approximately 100-fold increase in activity towards rac-2-acetoxy-2-(2'-chlorophenyl) acetate (rac-AcO-CPA), but rPPED287A had a significant decrease in enantioselectivity (E=8.7) compared to rPPEW187H and the wild-type rPPE (rPPEWT) (E>200). Here we report the crystal structures of rPPEWT and rPPEW187H, both by themselves and in complex with the substrate, to elucidate the structural basis of this phenomenon. An inactive mutation of nucleophile residue S159A was introduced to obtain the structure of rPPES159A/W187H complexed with (S)-AcO-CPA. The structural analysis reveals that the side chain of residue Asp287 in rPPEWT would have a potential steric conflict with (S)-AcO-CPA when the substrate binds at the active site of the enzyme. However, the mutation W187H could facilitate the relocation of Asp287, while D287A directly eliminates the hindrance of Asp287, both of which offer sufficient space for the binding and hydrolysis of substrate. Moreover, Asp287 generates one site of the "three-point attachment model" as a hydrogen-bond donor that determines the excellent enantioselectivity of rPPE in chiral recognition, and D287A would obviously destroy the hydrogen bond and result in the low enantioselectivity of rPPED287A.
Title: Increased Catalyst Productivity in alpha-Hydroxy Acids Resolution by Esterase Mutation and Substrate Modification Ma BD, Kong XD, Yu HL, Zhang ZJ, Dou S, Xu YP, Ni Y, Xu JH Ref: ACS Catal, 4:1026, 2014 : PubMed
Optically pure alpha-hydroxy acids and their derivatives are versatile chiral building blocks in the pharmaceutical industry. In this study, the potential of a recombinant Pseudomonas putida esterase (rPPE01) for the enzymatic resolution of -acetoxy acids was significantly improved by combinatorial engineering of both the biocatalyst and substrate. Semirational design based on homologous modeling and molecular docking provided a single-point variant, W187H, whose kcat/KM for sodium 2-acetoxy-2-(2'-chlorophenyl)acetate (Ac-CPA-Na) was increased 100-fold, from 0.0611 to 6.20 mM-1 s-1, while retaining its excellent enantioselectivity and broad substrate spectrum. Biocatalyst deactivation under the operating conditions was decreased by using the potassium salt of Ac-CPA instead of Ac-CPA-Na. With 0.5 g L-1 of lyophilized cells containing rPPE01-W187H, 500 mM (R,S)-Ac-CPA-K was selectively deacylated with 49.9% conversion within 15 h, giving satisfactory enantiomeric excesses (ee) for both the S product (>99% ee) and the remaining R substrate (98.7% ee). Consequently, the amount of (S)-2-hydroxy-2-(2'-chlorophenyl)acetate prepared per unit weight of lyophilized cells was improved by a factor of 18.9 compared with the original productivity of the wild-type esterase. Further enzymatic resolution of other important hydroxy acids at the 100 mL scale demonstrated that the rPPE01-W187H-based bioprocess is versatile and practical for the large-scale preparation of chiral -hydroxy acids
Title: Bioproduction of chiral mandelate by enantioselective deacylation of alpha-acetoxyphenylacetic acid using whole cells of newly isolated Pseudomonas sp. ECU1011 Ju X, Yu HL, Pan J, Wei DZ, Xu JH Ref: Applied Microbiology & Biotechnology, 86:83, 2010 : PubMed
Substrate-directed screening was carried out to find bacteria that could deacylate O-acetylated mandelic acid from environmental samples. From more than 200 soil isolates, we identified for the first time that Pseudomonas sp. ECU1011 biocatalytically deacylated (S)-alpha-acetoxyphenylacetic acid with high enantioselectivity (E > 200), yielding (S)-mandelic acid with 98.1% enantiomeric excess (ee) at a 45.5% conversion rate. The catalytic deacylation of (S)-alpha-acetoxyphenylacetic acid by the resting cell was optimized using a single-factor method to yield temperature and pH optima of 30 degrees C and 6.5, respectively. These optima help to reduce the nonselective spontaneous hydrolysis of the racemic substrate. It was found that substrate concentrations up to 60 mM could be used. 2-Propanol was used as a moderate cosolvent to help the substrate disperse in the aqueous phase. Under optimized reaction conditions, the ee of the residual (R)-alpha-acetoxyphenylacetic acid could be improved further, to greater than 99%, at a 60% conversion rate. Furthermore, using this newly isolated strain of Pseudomonas sp. ECU1011, three kinds of optically pure analogs of (S)-mandelic acid and (R)-alpha-acetoxyphenylacetic acid were successfully prepared at high enantiomeric purity.
Pseudomonas sp. ECU1011 Alpha/beta hydrolase fold-3 domain protein
