title: Proteomic and functional analysis of the cellulase system expressed by Postia placenta during brown rot of solid wood.
abstract: Brown rot basidiomycetes have an important ecological role in lignocellulose recycling and are notable for their rapid degradation of wood polymers via oxidative and hydrolytic mechanisms. However, most of these fungi apparently lack processive (exo-acting) cellulases, such as cellobiohydrolases, which are generally required for efficient cellulolysis. The recent sequencing of the Postia placenta genome now permits a proteomic approach to this longstanding conundrum. We grew P. placenta on solid aspen wood, extracted proteins from the biodegrading substrate, and analyzed tryptic digests by shotgun liquid chromatography-tandem mass spectrometry. Comparison of the data with the predicted P. placenta proteome revealed the presence of 34 likely glycoside hydrolases, but only four of these--two in glycoside hydrolase family 5, one in family 10, and one in family 12--have sequences that suggested possible activity on cellulose. We expressed these enzymes heterologously and determined that they all exhibited endoglucanase activity on phosphoric acid-swollen cellulose. They also slowly hydrolyzed filter paper, a more crystalline substrate, but the soluble/insoluble reducing sugar ratios they produced classify them as nonprocessive. Computer simulations indicated that these enzymes produced soluble/insoluble ratios on reduced phosphoric acid-swollen cellulose that were higher than expected for random hydrolysis, which suggests that they could possess limited exo activity, but they are at best 10-fold less processive than cellobiohydrolases. It appears likely that P. placenta employs a combination of oxidative mechanisms and endo-acting cellulases to degrade cellulose efficiently in the absence of a significant processive component.
authors.affiliation: Eco-Friendliness Research Department, Gyeongsangnam-do Agricultural Research and Extension Services, Jinju 660-360, Republic of Korea.
authors.forename: Jae San
authors.initials: JS
authors.lastname: Ryu
authors.forename: Semarjit
authors.initials: S
authors.lastname: Shary
authors.forename: Carl J
authors.initials: CJ
authors.lastname: Houtman
authors.forename: Ellen A
authors.initials: EA
authors.lastname: Panisko
authors.forename: Premsagar
authors.initials: P
authors.lastname: Korripally
authors.forename: Franz J
authors.initials: FJ
authors.lastname: St John
authors.forename: Casey
authors.initials: C
authors.lastname: Crooks
authors.forename: Matti
authors.initials: M
authors.lastname: Siika-Aho
authors.forename: Jon K
authors.initials: JK
authors.lastname: Magnuson
authors.forename: Kenneth E
authors.initials: KE
authors.lastname: Hammel
mesh_terms: D044602:Cellulases / Q000032:analysis*; D002482:Cellulose / Q000378:metabolism; D002853:Chromatography, Liquid; D003001:Cloning, Molecular; D055453:Coriolaceae / Q000737:chemistry / Q000201:enzymology* / Q000302:isolation & purification / Q000378:metabolism*; D004271:DNA, Fungal / Q000737:chemistry / Q000235:genetics; D015870:Gene Expression; D008969:Molecular Sequence Data; D020543:Proteome / Q000032:analysis*; D017422:Sequence Analysis, DNA; D053719:Tandem Mass Spectrometry; D014934:Wood / Q000378:metabolism* / Q000382:microbiology*
chemical_list: D004271:DNA, Fungal; D020543:Proteome; D002482:Cellulose; D044602:Cellulases