A Molecular Mechanism of Enantiorecognition of Tertiary Alcohols by Carboxylesterases

Henke, Erik; Bornscheuer, Uwe T.; Schmid, Rolf D.; Pleiss, Jürgen

doi:10.1002/cbic.200200518

Cited by 112 publications

(62 citation statements)

References 76 publications

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“…Escherichia coli strain Origami (Novagen) was routinely cultured overnight at 37°C in Luria-Bertani broth or on Luria-Bertani agar plates and was used as the host strain for cloning and expression of lipase-encoding genes. Plasmids pGaston (19) and pET101/ D-TOPO (Invitrogen) were used as expression vectors. Strain Burkholderia cenocepacia J2315 (kindly provided by H. Gomes Leitao) was cultured overnight at 37°C in Luria-Bertani broth or on Luria-Bertani agar plates.…”

Section: Methodsmentioning

confidence: 99%

Rhodococcus sp. Strain CR-53 LipR, the First Member of a New Bacterial Lipase Family (Family X) Displaying an Unusual Y-Type Oxyanion Hole, Similar to the Candida antarctica Lipase Clan

Bassegoda

Pastor

Dı́az

2012

Appl Environ Microbiol

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Bacterial lipases constitute the most important group of biocatalysts for synthetic organic chemistry. Accordingly, there is substantial interest in developing new valuable lipases. Considering the lack of information concerning the lipases of the genus Rhodococcus and taking into account the interest raised by the enzymes produced by actinomycetes, a search for putative lipaseencoding genes from Rhodococcus sp. strain CR-53 was performed. We isolated, cloned, purified, and characterized LipR, the first lipase described from the genus Rhodococcus. LipR is a mesophilic enzyme showing preference for medium-chain-length acyl groups without showing interfacial activation. It displays good long-term stability and high tolerance for the presence of ions and chemical agents in the reaction mixture. Amino acid sequence analysis of LipR revealed that it displays four unique amino acid sequence motifs that clearly separate it from any other previously described family of bacterial lipases. Using bioinformatics tools, LipR could be related only to several uncharacterized putative lipases from different bacterial origins, all of which display the four blocks of consensus amino acid sequence motifs that contribute to define a new family of bacterial lipases, namely, family X. Therefore, LipR is the first characterized member of the new bacterial lipase family X. Further confirmation of this new family of lipases was performed after cloning Burkholderia cenocepacia putative lipase, bearing the same conserved motifs and clustering in family X. Interestingly, all lipases grouping in the new bacterial lipase family X display a Y-type oxyanion hole, a motif conserved in the Candida antarctica lipase clan but never found among bacterial lipases. This observation contributes to confirm that LipR and its homologs belong to a new family of bacterial lipases. L ipases are glycerol ester hydrolases acting on acyl glycerols to liberate free fatty acids and glycerol. They catalyze reactions involving insoluble lipid substrates at the lipid-water interface and preserve their catalytic activity in organic solvents (23), acting as powerful tools for catalyzing not only hydrolysis but also various reverse reactions such as esterifications or transesterifications in anhydrous organic solvents (16, 23). Moreover, microbial lipases catalyze reactions with high specificity, regioselectivity, and enantioselectivity, constituting the most important group of biocatalysts for synthetic organic chemistry and other biotechnological applications (4,18,34,35). Accordingly, there is substantial interest in developing new lipases for use in food, biomedical, or chemical industries (18).Despite the large number of microbial lipases identified, cloned, and characterized in the last decades (3, 11, 12, 29-31, 36, 37, 39), there are still some cultivable microbial species which are promising sources of new lipases that have not yet been explored. In this respect, many rhodococci display the ability to degrade different alkanes or show tolerance to hydrocarbo...

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Section: Methodsmentioning

confidence: 99%

Rhodococcus sp. Strain CR-53 LipR, the First Member of a New Bacterial Lipase Family (Family X) Displaying an Unusual Y-Type Oxyanion Hole, Similar to the Candida antarctica Lipase Clan

Bassegoda

Pastor

Dı́az

2012

Appl Environ Microbiol

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“…However, tertiary alcohols and their esters are notoriously problematic substrates for hydrolases, the enzymes of choice for a kinetic resolution (Figure 1). [7][8][9][10][11][12] Only few examples of the successful hydrolysis of these esters are known, enantioselective conversions are even fewer and the application for a,a-disubstituted cyanohydrins is very limited. Only two non-commercial crude enzymes were described to perform this reaction with limited yields and ees [13][14][15] and just one successful example (a-trifluoromethyl-a-ethyl cyanohydrin acetate) for Candida rugosa lipase (CRL) is known.…”

mentioning

confidence: 99%

Hydrolase‐Catalysed Preparation of Chiral α,α‐Disubstituted Cyanohydrin Acetates

et al. 2007

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The enzymatic hydrolysis of esters of tertiary alcohols has long been a challenge. In particular their kinetic resolutions have virtually not been addressed. Here we describe the successful kinetic resolution of a,a-disubstituted cyanohydrin acetates, a type of protected tertiary alcohols that form particularly interesting building blocks in organic synthesis. Utilising Subtilisin A the hydrolysis reaction was (S)-selective, while Candida rugosa lipase was (R)-selective. With these commercially available enzymes both enantiomers of the a,a-disubstituted cyanohydrin acetates are now accessible.

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“…In contrast to Fae6, a recently reported esterase estCS2 from compost metagenomic library which also belongs to family VII lipolytic enzyme converted linalyl acetate to linalool (19). Interestingly, the estCS2 primary structure contained the G-G-A-F sequence, in contrast to a common G-G-G-F sequence which has been shown to provide enantiorecognition of tertiary alcohols (5,31).…”

Section: -O-[5-o-trans-feruloyl)-β-l-arabinofuranosyl]-d-xylopyranosementioning

confidence: 99%

A feruloyl esterase derived from a leachate metagenome library

Rashamuse¹,

Sanyika²,

Ronneburg³

et al. 2012

BMB Reports

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A Molecular Mechanism of Enantiorecognition of Tertiary Alcohols by Carboxylesterases

Abstract: Carboxylesterases containing the sequence motif GGGX

Cited by 112 publications

References 76 publications

Rhodococcus sp. Strain CR-53 LipR, the First Member of a New Bacterial Lipase Family (Family X) Displaying an Unusual Y-Type Oxyanion Hole, Similar to the Candida antarctica Lipase Clan

Rhodococcus sp. Strain CR-53 LipR, the First Member of a New Bacterial Lipase Family (Family X) Displaying an Unusual Y-Type Oxyanion Hole, Similar to the Candida antarctica Lipase Clan

Hydrolase‐Catalysed Preparation of Chiral α,α‐Disubstituted Cyanohydrin Acetates

A feruloyl esterase derived from a leachate metagenome library

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