Gene_locus Report for: colgl-cutas

Cutinase catalyzes the hydrolysis of water-soluble esters and long-chain triglycerides and belongs to the family of serine hydrolases. The enzyme is thought to represent an evolutionary link between the esterase and lipase families and has potential applications in a wide range of industrial hydrolytic processes, for which an understanding of the molecular basis of its substrate specificity is critical. Glomerella cingulata cutinase has been cloned and the protein has been overexpressed in Escherichia coli, purified and subsequently crystallized in a wide range of different crystal forms in the presence and absence of inhibitors. The best crystals are those of the apo cutinase, which diffract to beyond 1.6 A resolution and belong to space group P4(1)2(1)2 or P4(3)2(1)2. Crystals of cutinase with the inhibitors PETFP or E600 belong to space groups P2(1)2(1)2(1) and P2(1), respectively, and diffract to approximately 2.5 A resolution. All of the crystals are suitable for structural studies, which are currently ongoing.

Colletotrichum kahawae is the causal agent of the coffee berry disease, infecting leaves and coffee berries at any stage of their development. Colletotrichum gloeosporioides is the causal agent of brown blight, infecting ripe berries only. Both fungi secrete the same pattern of carboxylesterases to the fermentation broth when cutin is used as carbon source. By using two different strategies composed of two precipitation steps (ammonium sulphate and acetic acid precipitation) and two chromatographic steps, two proteins displaying carboxylesterase activity were purified to electrophoretic homogeneity. One, with a molecular weight (MW) of 21 kDa, has a blocked N terminus and was identified as cutinase by peptide mass fingerprint and mass spectrometry/mass spectrometry data acquired after peptide derivatization with 4-sulphophenyl isothiocyanate. The second, with a MW of 40 kDa, displays significant carboxylesterase activity on tributyrin but low activity on p-nitrophenyl butyrate. N-terminal sequencing for this protein does not reveal any homology to other carboxylesterases. These two enzymes, which were secreted by both fungi, appear homologous.

Cutinase is an extracellular fungal enzyme that allows pathogenic fungi to penetrate through the cuticular barrier into the host plant during the initial stages of the fungal infection. mRNA isolated from glucose-grown Colletotrichum capsid, induced to produce cutinase by the addition of cutin hydrolysate, was used to prepare cDNA which was cloned in the expression vector Xgtl1. The primary structure of the cutinase from C.capsid was deduced from the nucleotide sequence of the cloned cutinase cDNA. Amino acid sequences of two tryptic peptides isolated from cutinase produced by C.capsid completely matched with two segments of the amino acid sequence deduced from the nucleotide sequence, strongly suggesting that the cloned cDNA was authentic cutinase cDNA. The cDNA clone was used as a probe to screen C.capsid and Colletotrichum gloeosporioides genomic libraries constructed in Charon35 and EMBL3, respectively. The nucleotide sequences of the cutinase structural genes from C.capsid and C.gloeosporioides were also determined. SI mapping was used to reveal the transcriptional start sites and polyadenylation site of the primary transcript from C.capsid. The primary sequences and gene structure of the enzymes from th eColletotrichum species were compared with the primary structure and gene structure of a cutinase from Fusarium solani f.sp. pisi. A comparison of the deduced primary structures of the enzymes showed that residues involved in the catalytic triad andd isulfide cross-linking of cutinase are strongly conserved. Yet, only 43% of the residues areconserved between all three enzymes. A comparison of the structure of the three genes revealed the location of the single intron has been conserved. The transcriptional start site of the C.capsid gene was centered on the sequence TCCAGACCA, the core of which (CAGAC) is found repeated after 21 nucleotides. The same core sequence, repeated after 11 nucleotides, was also identified in the 5' non translated regions of the C. gloeosporioides and F. solanigenes.

Cutinase belongs to a group of enzymes that catalyze the hydrolysis of esters and triglycerides. Structural studies on the enzyme from Fusarium solani have revealed the presence of a classic catalytic triad that has been implicated in the enzyme's mechanism. We have solved the crystal structure of Glomerella cingulata cutinase in the absence and in the presence of the inhibitors E600 (diethyl p-nitrophenyl phosphate) and PETFP (3-phenethylthio-1,1,1-trifluoropropan-2-one) to resolutions between 2.6 and 1.9 A. Analysis of these structures reveals that the catalytic triad (Ser136, Asp191, and His204) adopts an unusual configuration with the putative essential histidine His204 swung out of the active site into a position where it is unable to participate in catalysis, with the imidazole ring 11 A away from its expected position. Solution-state NMR experiments are consistent with the disrupted configuration of the triad observed crystallographically. H204N, a site-directed mutant, was shown to be catalytically inactive, confirming the importance of this residue in the enzyme mechanism. These findings suggest that, during its catalytic cycle, cutinase undergoes a significant conformational rearrangement converting the loop bearing the histidine from an inactive conformation, in which the histidine of the triad is solvent exposed, to an active conformation, in which the triad assumes a classic configuration.

Cutinase catalyzes the hydrolysis of water-soluble esters and long-chain triglycerides and belongs to the family of serine hydrolases. The enzyme is thought to represent an evolutionary link between the esterase and lipase families and has potential applications in a wide range of industrial hydrolytic processes, for which an understanding of the molecular basis of its substrate specificity is critical. Glomerella cingulata cutinase has been cloned and the protein has been overexpressed in Escherichia coli, purified and subsequently crystallized in a wide range of different crystal forms in the presence and absence of inhibitors. The best crystals are those of the apo cutinase, which diffract to beyond 1.6 A resolution and belong to space group P4(1)2(1)2 or P4(3)2(1)2. Crystals of cutinase with the inhibitors PETFP or E600 belong to space groups P2(1)2(1)2(1) and P2(1), respectively, and diffract to approximately 2.5 A resolution. All of the crystals are suitable for structural studies, which are currently ongoing.

Colletotrichum kahawae is the causal agent of the coffee berry disease, infecting leaves and coffee berries at any stage of their development. Colletotrichum gloeosporioides is the causal agent of brown blight, infecting ripe berries only. Both fungi secrete the same pattern of carboxylesterases to the fermentation broth when cutin is used as carbon source. By using two different strategies composed of two precipitation steps (ammonium sulphate and acetic acid precipitation) and two chromatographic steps, two proteins displaying carboxylesterase activity were purified to electrophoretic homogeneity. One, with a molecular weight (MW) of 21 kDa, has a blocked N terminus and was identified as cutinase by peptide mass fingerprint and mass spectrometry/mass spectrometry data acquired after peptide derivatization with 4-sulphophenyl isothiocyanate. The second, with a MW of 40 kDa, displays significant carboxylesterase activity on tributyrin but low activity on p-nitrophenyl butyrate. N-terminal sequencing for this protein does not reveal any homology to other carboxylesterases. These two enzymes, which were secreted by both fungi, appear homologous.

Cutinase is an extracellular fungal enzyme that allows pathogenic fungi to penetrate through the cuticular barrier into the host plant during the initial stages of the fungal infection. mRNA isolated from glucose-grown Colletotrichum capsid, induced to produce cutinase by the addition of cutin hydrolysate, was used to prepare cDNA which was cloned in the expression vector Xgtl1. The primary structure of the cutinase from C.capsid was deduced from the nucleotide sequence of the cloned cutinase cDNA. Amino acid sequences of two tryptic peptides isolated from cutinase produced by C.capsid completely matched with two segments of the amino acid sequence deduced from the nucleotide sequence, strongly suggesting that the cloned cDNA was authentic cutinase cDNA. The cDNA clone was used as a probe to screen C.capsid and Colletotrichum gloeosporioides genomic libraries constructed in Charon35 and EMBL3, respectively. The nucleotide sequences of the cutinase structural genes from C.capsid and C.gloeosporioides were also determined. SI mapping was used to reveal the transcriptional start sites and polyadenylation site of the primary transcript from C.capsid. The primary sequences and gene structure of the enzymes from th eColletotrichum species were compared with the primary structure and gene structure of a cutinase from Fusarium solani f.sp. pisi. A comparison of the deduced primary structures of the enzymes showed that residues involved in the catalytic triad andd isulfide cross-linking of cutinase are strongly conserved. Yet, only 43% of the residues areconserved between all three enzymes. A comparison of the structure of the three genes revealed the location of the single intron has been conserved. The transcriptional start site of the C.capsid gene was centered on the sequence TCCAGACCA, the core of which (CAGAC) is found repeated after 21 nucleotides. The same core sequence, repeated after 11 nucleotides, was also identified in the 5' non translated regions of the C. gloeosporioides and F. solanigenes.