2. LITERATURE REVIEW
2.5 Characterization of cutinase
2.5.1 Molecular properties
Molecular weight of Pseudomonas putida cutinase was reported to be 30 kDa (Sebastian and Kolattukudy, 1988), where as that of fungal cutinase was around 22-26 kDa (Koller and Parker, 1989). Four different fungal cutinases were identified by Chen et al., (2007) out of which two were of 21 kDa and other two were of 40 kDa (Chen et al., 2007). Cutinase is the smallest member of the α/β hydrolase fold family. The primary structure of cutinase has been determined from the nucleotide sequence of cloned cDNA by Soliday et al., (1984). The amino acid compositions of fungal cutinases from five different strains were compared and discussed in detail by Kollar and Parker (1989). The amino acid composition of bacterial cutinase has been studied in detail by Sebastian et al., (1987) and was reported to be distinctly different from that of fungal or plant cutinases. However, for any cutinase, this holds true that unlike most lipases; the catalytic serine is not buried under an amphipathic loop but is accessible to solvent and substrate. This explains why cutinase does not require interfacial activation (Carvalho et al., 1998a; Longhi et al., 1999). The cutinase isolated from P. putida and Thermobifida fusca was found to be 30 kDa and 29 kDa molecular weight, respectively.
The molecular weight of cutinases isolated from F. solani pisi and C. kahawae were found to be 21 kDa and 22.1 kDa, respectively (Chen et al., 2007). Sebastian and Kolattukudy (1988) reported that generally the molecular weight of bacterial cutinase lies between the molecular weight of fungal cutinase (22 kDa) and pollen cutinase (40 kDa). Although fungal cutinase has been extensively characterized, the molecular identity of bacterial cutinase has remained as a mystery. Chen et al., (2008) discussed in their study that giving the evidence indicating
Tfu 0882 and Tfu 0883 from the actinomycete T. fusca function as cutinases: (i) a 29-kDa protein was secreted into the T. fusca culture upon cutin induction and exhibited cutinase activity (ii) N-terminal amino acid sequencing of the 29-kDa protein matched two proteins, Tfu 0882 and Tfu 0883, which are 93% identical in sequence; (iii) both Tfu 0882 and Tfu 0883 are able to hydrolyse cutin, resulting in monomeric products typical of fungal cutinase.
Given their high sequence identity, it is not surprising that Tfu 0882 and Tfu 0883 have similar physical properties. Catalytically, both Tfu 0882 and Tfu 0883 are relatively versatile in utilizing both insoluble triglycerides (triolein) and soluble esters (pNPB) as substrate in addition to cutin. Moreover, the two enzymes share similar temperature and pH dependence profiles and thermostability. Therefore they may be defined as cutinase isoenzymes in T.
fusca. Although, Tfu 0882 and Tfu 0883 are sequential in the genome, likely due to a gene duplication event, they do not appear to be in an operon as determined by the operon prediction tool VIMSS (www.microbesonline.org/operons) (Price et al., 2005). Moreover, there is no synergism between the two enzymes in cutin degradation. Thus, further studies are needed to understand why there are two sequential genes for T. fusca cutinase. Sequence analysis suggested that T. fusca cutinases belong to the α/β-hydrolase fold superfamily (Nardini and Dijkstra, 1999). Enzymes in this superfamily exhibit a wide variety of hydrolytic activities as discussed previously. However, they all adopt a conserved three-dimensional fold and are believed to have evolved from a common ancestor (Nardini and Dijkstra, 1999). As expected, the homology model of Tfu 0883 displays a canonical α/β -hydrolase fold with a Ser170-His248-Asp216 triad and a preformed oxyanion hole, suggesting a classic serine hydrolase mechanism involving two tetrahedral transition states and an acyl-enzyme intermediate (Dodson and Wlodawer, 1998). Indeed this mechanism was supported by PMSF-
mediated irreversible inhibition of Tfu 0882 and Tfu 0883 as well as site-directed mutagenesis of the catalytic serine (to alanine) in both enzymes. A notable feature of the T.
fusca cutinase model was that the enzyme does not contain a lid insertion commonly observed in true lipases (Angkawidjaja et al., 2007; Brzozowski et al., 2000; Brzozowski et al., 1991;
Lang et al., 1996), and exposes its nucleophilic serine to the solvent. In lipases, the lid insertion was reported to be involved in the interfacial activation in which it undergoes conformational change in response to adsorption at the oil-water interface (Jaeger et al., 1994). The absence of such a lid insertion suggests that the T. fusca cutinases should belong to a class of α/β-hydrolases different from the classic lipases. On the other hand, the open active site of T. fusca cutinase readily explains the ability of the enzyme to accommodate large substrates like cutin. Further studies, especially x-ray crystallography studies of ligand bound cutinase may reveal the structural basis of substrate recognition. Comparative study on biochemical characterization of bacterial and fungal cutinases indicated that they have similar substrate specificity and catalytic properties except that T. fusca cutinases demonstrate remarkably greater thermo-stability. This unique feature may render T. fusca cutinases practically more amenable for industrial applications. Although both T. fusca and F. solani pisi cutinases belong to the α/β -hydrolases superfamily and contain an open active site, the bacterial enzymes have significantly longer sequences and demonstrate no sequence similarity to the fungal enzyme. Moreover, the fungal cutinase contains neither the two N-terminal β strands of the canonical α/β - hydrolase fold nor the unique C-terminal extension of Tfu 0883.
Thus, the bacterial and fungal enzymes must have undergone extensive evolutionary differentiation and are suggested to be classified into prokaryotic and eukaryotic cutinase subfamilies, respectively.