Functions of a proteinase secreted by the sap-staining fungus Ophiostoma piceae
Creators: name: Abraham, Linda Deanne
Publisher: name: University of British Columbia
Year: 2009

description: Fungal colonisation that discolours wood decreases its market value, reducing profits on Canadian lumber products. Disrupting key enzymes involved in fungal metabolism could be a way of preventing such wood-inhabiting fungi from colonising lumber. Enzyme-targeted antisapstain formulations would be expected to have a low potential for adverse environmental impact. The production of proteinases by sapstaining fungi may be key to the acquisition of nitrogen required for growth since protein is considered to be the major source of organic nitrogen in wood. Proteolytic activity detected in wood powder and culture filtrates after growth of Ophiostoma piceae was inhibited by PMSF and EDTA. The major protein detected in culture filtrates, a proteinase with a p1 of 5.6 and a molecular weight of 33 kDa, was subsequently purified by hydrophobic interaction chromatography. The proteolytic activity of the purified proteinase was determined to be optimal at pH 7 to 9 and 40°C. The N-terminal sequence of the protein showed a high degree ofhomology with fungal alkaline serine proteinases classified as subtilisin class II enzymes. Agreements in inhibition patterns, electrophoretic and catalytic properties suggested the secretion of the same proteinase during growth on wood. Proteinase production was associated with active growth, suggesting a role in primary retrieval of nitrogen from wood proteins. Preliminary attempts to selectively inactivate the proteinase by application of chelators or serine proteinase inhibitors on wood prior to infection were inconclusive. Further efforts were hampered by the current lack of stable, non-toxic, specific proteinase inhibitors. The subtilisin-like serine proteinase was degraded by autoproteolysis under conditions of heating, altered pH or partial depletion of protein-bound ions by EDTA. The proteinase consisted of two major hydrolytic fragments, 19 kDa and 14 kDa, which had N-terminal sequences ofAla¹-Tyr²-Thr³-Thr⁴-G1n⁵-Thr⁶-G1y⁷-A1a⁸-Pro⁹- and Ser¹⁷⁰-Glu¹⁷¹-Pro¹⁷²-Ser¹⁷³- Val¹⁷⁴- X¹⁷⁵ -Thr¹⁷⁶ -Val¹⁷⁷ -Gly¹⁷⁸ -Ala¹⁷⁹ -, respectively. Since the former sequence was identical to the N-terminus of the native protein, the major autoproteolytic cleavage site for a class II subtilase appeared to be the N-side of Ser¹⁷⁰, consistent with a similar region identified for class I subtilases. The cleavage specificity of this subtilase was investigated on the insulin B-chain using electrospray ionisation mass spectrometry. Cleavage sites after hydrophobic, polar, and charged amino acids indicated a broad specificity. Degradation of proteins extracted from the xylem tissue of poplar was observed after incubation with the proteinase. Other proteins hydrolysed by the proteinase included gelatin, collagen, albumin, edestin, globulins and casein. This supports the conclusion that the proteinase has a broad specificity and is able to degrade physiological substrates. A thorough understanding of the nutritional requirements of staining fungi has important implications for preventing the growth of these fungi and other economically important ophiostomatoid fungi. The approach taken in this work - identifying key physiological enzymes as a strategy for controlling sapstaining fungi - has shown that these fungi require proteinases to breakdown wood proteins into assimilable nitrogen. Therefore, these enzymes are vital components of fungal physiology and their selective inactivation may be the target for future bioprotectants.