Substrate Report for: Paranitrophenyl-palmitate

Cutinases are esterases that release fatty acids from the apoplastic layer in plants. As they accept bulky and hydrophobic substrates, cutinases could be used in many applications, ranging from valorization of bark-rich side streams to plastic recycling. Advancement of these applications with cutinases as biocatalysts, however, requires deeper knowledge of the enzymes' biodiversity and structure-function relationships. Here, we mined over 3000 members from Carbohydrate Esterase family 5 (CE5) for putative cutinases and condensed it to 151 genes from known or putative lignocellulose-targeting organisms. The 151 genes were subjected to a phylogenetic analysis. While cutinases with available crystal structures were phylogenetically closely related, we selected nine phylogenic diverse cutinases for characterization. The nine selected cutinases were recombinantly produced and their kinetic activity was characterized against para-nitrophenol substrates esterified with consecutively longer alkyl chains (pNP-C(2) to C(16)). The investigated cutinases each had a unique activity fingerprint against tested pNP-substrates. The five enzymes with the highest activity on pNP-C(12) and C(16), indicative of activity on bulky hydrophobic compounds, were selected for in-depth kinetic and structure-function analysis. All five enzymes showed a decrease in k(cat) values with increasing substrate chain length, while K(M) values and binding energies (calculated from in silico docking analysis) improved. Two cutinases from Fusarium solani and Cryptococcus sp. exhibited outstandingly low K(M) values, resulting in high catalytic efficiencies towards pNP-C(16). Docking analysis suggested that different clades of the phylogenetic tree may harbor enzymes with different modes of substrate interaction, involving a solvent-exposed catalytic triad, a lipase-like lid, or a clamshell-like active site possibly formed by flexible loops.

Malassezia globosa is a lipid-dependent yeast that is found on the human skin and is associated with various skin disorders, including dandruff and seborrhoeic dermatitis (SD). Despite its important role in skin diseases, the molecular basis for its pathogenicity is poorly understood. The current hypothesis is that dandruff and SD are linked to fatty acid metabolism and secretory lipolytic enzymes, which hydrolyse sebaceous lipids and release irritating free fatty acids. A previous genomic analysis of M. globosa identified a family of 13 homologous genes predicted to encode secreted lipases. We have also reported that M. globosa had significantly higher extracellular lipase activity compared with other species. To identify the major secretory lipases of this yeast during its growth, we successfully purified and characterized an extracellular lipase MgLIP2. Based on MALDI-TOF MS, the peptide mass fingerprint of a tryptically digested protein MgLIP2 corresponded to ORF MGL_4054 of M. globosa. This lipase showed high esterase activity against 4-nitrophenyl palmitate and 1-naphthyl palmitate but not 1-naphthyl acetate. This enzyme had optimal activity at 30 degreesC and pH 5.0. Furthermore, the activity significantly increased in the presence of Triton X-100 and was partially inhibited by PMSF but was unaffected by univalent and divalent metal ions.

A novel gene lipB, which encodes an extracellular lipolytic enzyme, was identified in the Bacillus subtilis genomic DNA sequence. We have cloned and overexpressed lipB in B. subtilis and Escherichia coli and have also purified the enzyme from a B. subtilis culture supernatant to electrophoretic homogeneity. Four different lipase assays were used to determine its catalytic activity: pH-stat, spectrophotometry, fluorimetry and the monomolecular film technique. LipB preferentially hydrolysed triacylglycerol-esters and p-nitrophenyl-esters of fatty acids with short chain lengths of <= 10 carbon atoms. Triolein, which is a typical substrate for true lipases, was not hydrolysed at all. These results led us to classify LipB as an esterase rather than a lipase. The catalytic triad of LipB consists of residues Ser78, Asp134, and His157 as demonstrated by amino-acid sequence alignments and site-directed mutagenesis. The nucleophile Ser78 is located in a lipase-specific consensus sequence, which is Ala-X-Ser-X-Gly for most Bacillus lipases. All other bacterial lipases contain a glycine residue instead of the alanine at position-2 with respect to the catalytic serine. We have investigated the role of this alanine residue by constructing LipB variant A76G, thereby restoring the lipase-specific consensus motif. When compared with LipB this variant showed a markedly reduced thermostability but an increased stability at pH 5-7. Determination of the specific activities of wild-type LipB and variant A76G using a monomolecular film of the substrate monoolein revealed an interesting result: the A76G substitution had converted the esterase LipB into a monoacylglycerol hydrolase.

Multiple sequence alignments of three lipase isoforms from the filamentous fungus, Cordyceps militaris, have revealed that the deduced protein from their common sequence belongs to the Candida rugosa lipase-like group. To express the protein in its active form, recombinant lipase from C. militaris (rCML) was extra cellularly expressed in Pichia pastoris X-33 after removing its signal peptide. Purified rCML was a stable monomeric protein with a molecular mass of 90 kDa, and was highly N-mannosylated compared to the native protein (69 kDa). The catalytic efficiency (k(cat)/K(m)) of rCML was greater than the native protein (1244.35 +/- 50.88 and 1067.17 +/- 29.07 mM(-1).min(-1), respectively), yet they had similar optimal pH values and temperatures (40 degreesC and pH 7.0-7.5), and showed preferences for Tween esters and short-chain triacylglycerols. Despite its monomeric conformation, interfacial activation was not observed for rCML, unlike the classical lipases. From the structural model of rCML, the binding pocket of rCML was predicted as a funnel-like structure consisting of a hollow space and an intramolecular tunnel, which is typical of C. rugosa lipase-like lipases. However, a blockage shortened the tunnel to 12-15 A, which endows strict short-chain selectivity towards triacylglycerols and a perfect match for tricaproin (C6:0). The limited depth of the tunnel may enable accommodation of triacylglycerols with medium-to-long-chain fatty acids, which differentiates rCML from other C. rugosa lipase-like lipases with broad substrate specificities.

Cutinases are esterases that release fatty acids from the apoplastic layer in plants. As they accept bulky and hydrophobic substrates, cutinases could be used in many applications, ranging from valorization of bark-rich side streams to plastic recycling. Advancement of these applications with cutinases as biocatalysts, however, requires deeper knowledge of the enzymes' biodiversity and structure-function relationships. Here, we mined over 3000 members from Carbohydrate Esterase family 5 (CE5) for putative cutinases and condensed it to 151 genes from known or putative lignocellulose-targeting organisms. The 151 genes were subjected to a phylogenetic analysis. While cutinases with available crystal structures were phylogenetically closely related, we selected nine phylogenic diverse cutinases for characterization. The nine selected cutinases were recombinantly produced and their kinetic activity was characterized against para-nitrophenol substrates esterified with consecutively longer alkyl chains (pNP-C(2) to C(16)). The investigated cutinases each had a unique activity fingerprint against tested pNP-substrates. The five enzymes with the highest activity on pNP-C(12) and C(16), indicative of activity on bulky hydrophobic compounds, were selected for in-depth kinetic and structure-function analysis. All five enzymes showed a decrease in k(cat) values with increasing substrate chain length, while K(M) values and binding energies (calculated from in silico docking analysis) improved. Two cutinases from Fusarium solani and Cryptococcus sp. exhibited outstandingly low K(M) values, resulting in high catalytic efficiencies towards pNP-C(16). Docking analysis suggested that different clades of the phylogenetic tree may harbor enzymes with different modes of substrate interaction, involving a solvent-exposed catalytic triad, a lipase-like lid, or a clamshell-like active site possibly formed by flexible loops.

Malassezia globosa is a lipid-dependent yeast that is found on the human skin and is associated with various skin disorders, including dandruff and seborrhoeic dermatitis (SD). Despite its important role in skin diseases, the molecular basis for its pathogenicity is poorly understood. The current hypothesis is that dandruff and SD are linked to fatty acid metabolism and secretory lipolytic enzymes, which hydrolyse sebaceous lipids and release irritating free fatty acids. A previous genomic analysis of M. globosa identified a family of 13 homologous genes predicted to encode secreted lipases. We have also reported that M. globosa had significantly higher extracellular lipase activity compared with other species. To identify the major secretory lipases of this yeast during its growth, we successfully purified and characterized an extracellular lipase MgLIP2. Based on MALDI-TOF MS, the peptide mass fingerprint of a tryptically digested protein MgLIP2 corresponded to ORF MGL_4054 of M. globosa. This lipase showed high esterase activity against 4-nitrophenyl palmitate and 1-naphthyl palmitate but not 1-naphthyl acetate. This enzyme had optimal activity at 30 degreesC and pH 5.0. Furthermore, the activity significantly increased in the presence of Triton X-100 and was partially inhibited by PMSF but was unaffected by univalent and divalent metal ions.

A novel gene lipB, which encodes an extracellular lipolytic enzyme, was identified in the Bacillus subtilis genomic DNA sequence. We have cloned and overexpressed lipB in B. subtilis and Escherichia coli and have also purified the enzyme from a B. subtilis culture supernatant to electrophoretic homogeneity. Four different lipase assays were used to determine its catalytic activity: pH-stat, spectrophotometry, fluorimetry and the monomolecular film technique. LipB preferentially hydrolysed triacylglycerol-esters and p-nitrophenyl-esters of fatty acids with short chain lengths of <= 10 carbon atoms. Triolein, which is a typical substrate for true lipases, was not hydrolysed at all. These results led us to classify LipB as an esterase rather than a lipase. The catalytic triad of LipB consists of residues Ser78, Asp134, and His157 as demonstrated by amino-acid sequence alignments and site-directed mutagenesis. The nucleophile Ser78 is located in a lipase-specific consensus sequence, which is Ala-X-Ser-X-Gly for most Bacillus lipases. All other bacterial lipases contain a glycine residue instead of the alanine at position-2 with respect to the catalytic serine. We have investigated the role of this alanine residue by constructing LipB variant A76G, thereby restoring the lipase-specific consensus motif. When compared with LipB this variant showed a markedly reduced thermostability but an increased stability at pH 5-7. Determination of the specific activities of wild-type LipB and variant A76G using a monomolecular film of the substrate monoolein revealed an interesting result: the A76G substitution had converted the esterase LipB into a monoacylglycerol hydrolase.