High-resolution Structure of ybfF from Escherichia coli K12: A Unique Substrate-binding Crevice Generated by Domain Arrangement

Park, Suk-Youl; Lee, Sang-Hak; Lee, Jieun; Nishi, Kosuke; Kim, Yong‐Sung; Jung, Chul-Ho; Kim, Ji Soo

doi:10.1016/j.jmb.2007.12.062

Cited by 26 publications

(54 citation statements)

References 37 publications

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“…In summary, previous structural and functional studies have shown that YbfF is an ␣/␤ hydrolase fold superfamily member with esterase activity toward palmitoyl-CoA, malonyl-CoA, pnitrophenyl butyrate (pNP-C4), and several triacylglycerides (16,19,25). Herein, we report that YbfF also hydrolyzes 1-phenylethyl acetate, IPG butyrate, and IPG caprylate and exhibits pronounced enantioselectivity for these substrates.…”

Section: Resultsmentioning

confidence: 83%

“…S1A in the supplemental material). The recently solved X-ray crystal structure of YbfF shows that the enzyme is monomeric (25). To confirm that recombinant His-tagged YbfF is also a monomer in solution, we performed gel filtration chromatography.…”

Section: Resultsmentioning

confidence: 99%

“…To obtain insight into the binding mode of these IPG esters, the R enantiomers of substrates 1 and 3 were modeled into the active site of YbfF. The crystal structure of YbfF was recently solved by Park and coworkers (25). They have reported the presence of an activesite cavity that is capable of accommodating large molecules of at least six carbon atoms or the pNP ring structure.…”

Section: Resultsmentioning

confidence: 99%

“…They have reported the presence of an activesite cavity that is capable of accommodating large molecules of at least six carbon atoms or the pNP ring structure. They also describe the existence of a thin hydrophobic pocket deep enough to fit at least a three-hydrocarbon moiety (e.g., the butyrate moiety of pNP-C4) and a surface cleft along the domain interface (i.e., a crevice) large enough to accommodate molecules with longer hydrocarbon chains or the nucleotide part of the substrates palmitoyl-CoA and malonyl-CoA (25). Indeed, our molecular docking experiments performed with YbfF suggest that the small hydrocarbon chain of substrate (R)-1 can bind in the thin hydrophobic pocket and the longer hydrocarbon chain of substrate (R)-3 is accommodated along the crevice (Fig.…”

Section: Resultsmentioning

confidence: 99%

“…While it has previously been shown by structural and functional studies that YbfF is an ␣/␤ hydrolase fold superfamily member with esterase activity toward palmitoyl coenzyme A (palmitoylCoA), malonyl-CoA, and p-nitrophenyl butyrate (pNP-C4) (19,25), it was not known to hydrolyze IPG esters nor known to exhibit enantioselectivity. Our results show that YbfF displays high activity toward IPG butyrate and IPG caprylate and prefers the R-enantiomer of these substrates, producing (S)-IPG with 72 to 94% enantiomeric excess (ee).…”

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confidence: 99%

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Discovery of an Escherichia coli Esterase with High Activity and Enantioselectivity toward 1,2- O -Isopropylideneglycerol Esters

Godinho

Reis

Tepper

et al. 2011

Appl Environ Microbiol

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Escherichia coli has been widely used as an expression host for the identification of desired biocatalysts through screening or selection assays. We have previously used E. coli in growth selection and screening assays for identification of Bacillus subtilis lipase variants (located in the periplasm) with improved activity and enantioselectivity toward 1,2-O-isopropylideneglycerol (IPG) esters. In the course of these studies, we discovered that E. coli itself exhibits significant cytoplasmic esterase activity toward IPG esters. In order to identify the enzyme (or enzymes) responsible for this esterase activity, we analyzed eight E. coli knockout strains, in which single esterase genes were deleted, for their ability to hydrolyze IPG butyrate. This approach led to the identification of esterase YbfF as the major E. coli enzyme responsible for the hydrolytic activity toward IPG esters. The gene coding for YbfF was cloned and overexpressed in E. coli, and the corresponding protein was purified and characterized for its biocatalytic performance. YbfF displays a high level of activity toward IPG butyrate and IPG caprylate and prefers the R-enantiomer of these substrates, producing the S-enantiomer of the IPG product with high enantiomeric excess (72 to 94% ee). The enantioselectivity of YbfF for IPG caprylate (E ‫؍‬ 40) could be significantly enhanced when using dimethylformamide (DMF) or dimethyl sulfoxide (DMSO) as cosolvents in kinetic resolution experiments. The enzyme also shows high enantioselectivity toward 1-phenylethyl acetate (E > 200), giving the chiral product (R)-1-phenylethanol with >99% ee. The high activity and enantioselectivity of YbfF make it an attractive enzyme for organic synthesis.Hydrolases (EC 3) represent a class of enzymes that catalyze the hydrolysis of a chemical bond and are widely employed in organic synthesis (7,8,29). Depending on the chemical bond that they act on, hydrolases are grouped into several subclasses. Esterases and other hydrolytic enzymes that belong to the broad group of carboxylic ester hydrolases (EC 3.1.1) catalyze the hydrolysis and synthesis of ester bonds (5). Their broad substrate acceptance and high stability and enantioselectivity and the fact that they do not require cofactors make them attractive biocatalysts for organic synthesis (5). For example, the carboxylesterase NP from Bacillus subtilis Thai I-8 shows very high levels of activity and stereoselectivity toward esters of nonsteroidal anti-inflammatory drugs (NSAID), such as the naproxen and ibuprofen methyl esters, demonstrating its exciting potential for selective drug synthesis (6).We are interested in developing a biocatalytic process for the kinetic resolution of racemic 1,2-O-isopropylideneglycerol (IPG) esters. In previous studies, we have used Escherichia coli in directed evolution experiments for the identification of Bacillus subtilis lipase variants (located in the periplasm) with improved activity and enantioselectivity toward IPG butyrate (substrate 1) and IPG caprylate (substrate 3) (Fig. 1) ...

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Section: Resultsmentioning

confidence: 83%

Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

mentioning

confidence: 99%

See 3 more Smart Citations

Discovery of an Escherichia coli Esterase with High Activity and Enantioselectivity toward 1,2- O -Isopropylideneglycerol Esters

Godinho

Reis

Tepper

et al. 2011

Appl Environ Microbiol

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An Esterase with Superior Activity and Enantioselectivity towards 1,2‐O‐Isopropylideneglycerol Esters Obtained by Protein Design

et al. 2012

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Abstract:The Escherichia coli esterase YbfF displays high activity towards 1,2-O-isopropylideneglycerol (IPG) butyrate and IPG caprylate, and prefers the R-enantiomer of these substrates, producing the Senantiomer of the IPG product in excess. To improve the potential of the enzyme for the kinetic resolution of racemic esters of IPG, an enhancement of the activity and enantioselectivity would be highly desirable. Molecular docking of the R-enantiomer of both IPG esters into the active site of YbfF allowed the identification of proximal YbfF active site residues. Four residues (25, 124, 185 and 235) were selected as targets for mutagenesis, in order to enhance YbfF activity and enantioselectivity towards IPG esters. Random mutagenesis at positions 25, 124, 185 and 235 yielded several best YbfF variants with enhanced activity and enantioselectivity towards IPG esters. The best YbfF mutant, W235I, exhibited a 2-fold higher enantioselectivity than wild-type YbfF, with an E = 38 for IPG butyrate and an E = 77 for IPG caprylate. Molecular docking experiments further support the enhanced enantioselectivity shown experimentally and the structural effects of this amino acid substitution on the active site of YbfF are provided. The engineered W235I mutant is an attractive catalyst for practical applications in the kinetic resolution of IPG esters.

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Decoupled Roles for the Atypical, Bifurcated Binding Pocket of the ybfF Hydrolase

et al. 2013

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Serine hydrolases have diverse intracellular substrates, biological functions, and structural plasticity, and are thus important for biocatalyst design. Amongst serine hydrolases, the recently described ybfF enzyme family are promising novel biocatalysts with an unusual bifurcated substrate-binding cleft and the ability to recognize commercially relevant substrates. We characterized in detail the substrate selectivity of a novel ybfF enzyme from Vibrio cholerae (Vc-ybfF) by using a 21-member library of fluorogenic ester substrates. We assigned the roles of the two substrate-binding clefts in controlling the substrate selectivity and folded stability of Vc-ybfF by comprehensive substitution analysis. The overall substrate preference of Vc-ybfF was for short polar chains, but it retained significant activity with a range of cyclic and extended esters. This broad substrate specificity combined with the substitutional analysis demonstrates that the larger binding cleft controls the substrate specificity of Vc-ybfF. Key selectivity residues (Tyr116, Arg120, Tyr209) are also located at the larger binding pocket and control the substrate specificity profile. In the structure of ybfF the narrower binding cleft contains water molecules prepositioned for hydrolysis, but based on substitution this cleft showed only minimal contribution to catalysis. Instead, the residues surrounding the narrow binding cleft and at the entrance to the binding pocket contributed significantly to the folded stability of Vc-ybfF. The relative contributions of each cleft of the binding pocket to the catalytic activity and folded stability of Vc-ybfF provide a valuable map for designing future biocatalysts based on the ybfF scaffold.

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High-resolution Structure of ybfF from Escherichia coli K12: A Unique Substrate-binding Crevice Generated by Domain Arrangement

Cited by 26 publications

References 37 publications

Discovery of an Escherichia coli Esterase with High Activity and Enantioselectivity toward 1,2- O -Isopropylideneglycerol Esters

Discovery of an Escherichia coli Esterase with High Activity and Enantioselectivity toward 1,2- O -Isopropylideneglycerol Esters

An Esterase with Superior Activity and Enantioselectivity towards 1,2‐O‐Isopropylideneglycerol Esters Obtained by Protein Design

Decoupled Roles for the Atypical, Bifurcated Binding Pocket of the ybfF Hydrolase

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