2. LITERATURE REVIEW
2.6 Cutinase structure
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10.5 at pH 7 and 20 mL air min−1 within 15 min. Another widely used technique for purification of cutinase is ion-exchange chromatography. Chen et al., (2007) used a two-stage chromatography process for purifying cutinases from Colletotrichum kahawae and Colletotrichum gloeosporioides. They purified cutinases using a DEAE matrix (weak anionic exchanger) at pH 8.5 as the first chromatographic step devoid of proteins with no NPBase (p- Nitrophenyl butyrate) activity and also separated from the other 40 kDa proteins. An additional chromatographic step using an SP matrix (strong cationic exchanger) bound additional proteins with no activity to the matrix and allowed purification of cutinase to electrophoretic homogeneity. Cutinases purified in this method should have a pI within the range 7.5–8.5 so that they neither bound to DEAE at pH 8.5 nor to SP at pH 7.6. Sebastian and Kolattukudy (1988) purified cutinase from the culture filtrate by acetone precipitation followed by chromatography on DEAE-cellulose, QAE-Sephadex, Sepharose 6B, and Sephadex G-100. The enzyme was found to be a monomer by SDS-PAGE and gel filtration of the native enzyme obtained through a Sephadex G-100 column. Sebastian and Kolattukudy (1988) could obtain 200 fold purified protein with 3% yield. Chen et al., (2007) purified cutinases from C. kahawae and C. gloeosporioides in six steps and achieved purification fold of 15.5 and 16.5, respectively. Gindro and Pezet (1999) purified cutinase from Botrytis cinera in four steps with 2.9% yield and 66.5 purification fold.
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Fusarium solani f. pisi cutinase is a 196 amino acids enzyme with a molecular weight of 22 kDa. The three dimensional structure of F. solani f. pisi cutinase was solved at 1.6 Å resolution (Martinez et al., 1992) and the resolution was extended at 1.0 Å (Jelsch et al., 1998; Longhi et al., 1997). F. solani cutinase is a compact single domain molecule of 45×30×30 Å3 in size. Its structure includes a slightly twisted five-parallel-stranded β-sheet covered by four helices on either side of the sheet (Fig. 2.4 A). Two flexible loops including residues 80–87 and 180–187 with hydrophobic amino acids constitute the binding site of cutinase with catalytic triad Ser120, Asp175, and His188, accessible to the solvent. The stretch Gly-Tyr-Ser-Gln-Gly containing the active site Ser120 has even stronger homology with the consensus sequence Gly-(Tyr or His)-Ser-X-Gly commonly present in lipases. Two disulfide bridges exist in cutinase: one between Cys31 and Cys109 that helps to the stabilization of the global molecular folding and the other between Cys171 and Cys178.
Reduction of the disulfide bridges results in complete inactivation of the enzyme. The above mentioned loops 80-87 and 180-188, bearing hydrophobic amino acids (Leu81, Gly82, Ala85, Leu86, Pro87, Leu182, Ileu183 and Val184), may constitute the interfacial binding site (Martinez et al., 1992). Despite the existence of two side chain bridges of amino acids Leu81 and Val184, and Leu182 and Asn84, the catalytic serine of cutinase is not buried under surface loops, but is accessible to solvent and substrate.
Cutinases differ from classical lipases, as they do not exhibit interfacial activation. The absence of a flap, masking the active-site serine, as in other lipases, probably explains why cutinase is not activated by the presence of interfaces. The binding of cutinase to interfaces seems not to require a main-chain rearrangement, as in the case of lipases, but only the reorientation of few lipophilic side chains, for example Leu81 and Leu182, that play the role
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of a ‘mini’ flap. Another important feature on cutinase structure that differ from lipase is that the oxyanion hole is pre-formed in cutinase instead of being induced by ligand binding and seems to be stabilized by cutinase Ser42 side chain (Nicolas et al., 1996).
The crystal structure of A. oryzae resolved at 1.75 Å (Liu et al., 2009) revealed that it is a monomeric protein with an α/β fold hallmarked by a central β-sheet of 5 parallel strands surrounded by 10 α-helices. As a member of the α/β hydrolases, this enzyme possesses an active site composed of the catalytic triad residues Ser126, Asp181, and His194 (Fig. 2.4 B).
The catalytic site is surrounded by the two hydrophobic surfaces composed of residues 87-93 within helix 3, and residues 186-194 that represents the loop between helices 9 and 10 as well as the first 3 residues of the latter. A. oryzae cutinase bears an oxyanion hole composed of Ser48 and Gln127 backbone amides that are critical in polarizing the ester bond of the substrate and stabilizing the transition state of the formed substrate oxyanion (Fig. 2.4 B). The A. oryzae structure contains a unique disulfide bond between Cys63 and Cys76 that ties helix 2 to strand 2 of the central β-sheet. This disulfide bond has not been previously reported for any cutinase or hydrolase structures (Liu et al., 2009). The other two disulfide bonds, Cys37- Cys115 connecting the loop between helix 1 and strand 1 with the loop between helix 4 and strand 3 and Cys177-Cys184 linking the loop following strand 5 to helix 9, are well- conserved in cutinase structures, including that from F. solani. Sequence analysis suggests that this disulfide bond is unique for the cutinases from the Aspergillus family (sharing sequence identity of 50-77% with A. oryzae) and a few other filamentous fungi, such as Neosartorya fischeri (53% sequence identity) and E. nidulans (52% sequence identity). The cutinases from F. Solani and G. cingulata represent another group of filamentous fungi that do not have this special disulfide bond, although they share about 50% sequence identity with
TH-1214_KHEGDE
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the A. oryzae enzyme (Liu et al., 2009). Upon overlay, the A. oryzae and F. solani structures have a similar fold with an overall rms deviation of 1.02 Å, main chain deviation of 0.87 Å, and Cα deviation of 0.84 Å. However, comparison of the two sequences reveal that the A.
oryzea cutinase is shorter than that of F. solani as exhibited by smaller loops in the N and C- terminal regions, the N-terminal region (residues Thr26 to Asp30), the loops in between helix 1 and strand 1 (residues Gly35 to Pro49), as well as helix 2 and strand 2 (residues Ser71 to Asp73), helix 10 (residues Ser199 to Asp203), and C-terminal residues beyond helix 10 based on the A. oryzae structure deviate significantly from that of F. solani structure (Liu et al., 2009).
G. cingulata cutinase structure was solved at 1.9 Å by Nyon et al., (2009). Analysis of these structures reveals that it shares 50% fold and sequence identity with F. solani cutinase (Fig.
2.4 C). The catalytic triad (Ser136, Asp191, and His204) adopts an unusual configuration with the putative essential His204 swung out of the active site into a position where it is unable to participate in catalysis, with the imidazole ring 11 Å away from its expected position. Such a model implies that the 196–205 loop must undergo a significant conformational rearrangement in order to form the active state of the enzyme.
Recently crystal structure of a cutinase (Est119) from T. alba AHK119 was determined at a resolution of 1.76 Å by Kitadokoro et al., (2012). Overall structure of Est119 displays a typical α/β-hydrolase fold consisting of a central twisted β-sheet of nine β-strands that are flanked by nine α-helices. Est119 adopts a three-layer α/β sandwich fold, the central β-sheet being flanked by the α-helices α1, α2, α7, α8 and α9 on one side and α 3, α 4, α 5 and α 6 on the other. Catalytic site consists of Ser169, His247 and Asp215, which is located on the loops between the β-sheets and helices (Fig. 2.4 D). The oxyanion hole is formed by the main chain
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amides of Met170 and Tyr99. There is a disulfide bond present in the monomer (Cys280- Cys298) in the C-terminal region.The overall secondary structure of Est119 is very similar to that of S. exfoliatus lipase (SEL) (Kitadokoro et al., 2012) with highest Z-score of 46.2, RMSD of only 0.7 Å for 256 Cα atoms and 63% sequence identity.
The overall structural studies reviles that all the cutinases belong to the class of serine esterases and to the super family of the α/β hydrolases in which the nucleophilic serine is located at the center of an extremely sharp turn between a β-strand and an α-helix. They have one or more disulfide bridges among which one is essential for the catalytic activity. They show catalytic machinery similar to those present in serine proteases. They are characterized by the catalytic triad Ser, His, Asp residues, and by an oxyanion binding site that stabilizes the transition state via hydrogen bonds with two main chain amide groups (Nicolas et al., 1996).