Purification and identification of cutinases from Colletotrichum kahawae and Colletotrichum gloeosporioides

Abstract

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.

The lytic cassette of mycobacteriophage Ms6 encodes an enzyme with lipolytic activity

“…However the amino acid sequence of LysB does not show characteristic features of any of the lipase families identified by Arpigny & Jaeger (1999). Cutinases, which are also lipolytic enzymes, can be considered as a link between esterases and lipases because they are able to efficiently hydrolyse soluble esters and emulsified triacylglycerols (Mannese et al, 1995;Chen et al, 2007;Longhi & Cambillau, 1999;Carvalho et al, 1999). Although the LysB pentapetide (Gly-Tyr-Ser-Gln-Gly) is exactly the same as that in most cutinases described so far, the amino acid sequence around the conserved motif does not match the PROSITE (www.expasy.org/prosite/) pattern of cutinases.…”

The DING family of proteins: ubiquitous in eukaryotes, but where are the genes?

“…(46,47) In the Ascomycetes Colletotrichum kahawae and C. gloeosporioides, DING proteins possessing a cutinase (carboxylesterase) activity were identified, but the significance of these for the infection process remains to be established. (48) Cutinases were previously found to be involved in spore attachment to the host, although it is not known whether they are related to DING proteins (see (49) , for example). Finally, a very recent report describes a thermostable poly(ADP-ribose)polymerase-like enzyme from an archaebacterium, Sulfolobus solfataricus, which has a characteristic DING N-terminal sequence.…”

Crystallization, diffraction data collection and preliminary crystallographic analysis of DING protein fromPseudomonas fluorescens

“…These proteins are named DING proteins owing to their conserved DINGGG N-terminal amino-acid sequence. Interestingly, DING proteins have been independently isolated from a wide range of eukaryotic sources: they have been identified in animals (Riah et al, 2000;Weebadda et al, 2001;Kumar et al, 2004), plants (Berna et al, 2002) and fungi (Chen et al, 2007). For example, a human synovial DING protein was described by Hain et al (1996) and a very similar protein has been characterized from human fibroblast and tumour cells (Adams et al, 2002).…”