A number of oxidases have been proposed to play a role in hydrogen peroxide production in ligninolytic cultures of P. chrysosporium
, but until now, GLX was the only one that appeared to be secreted (27
). The temporal correlation of GLX, peroxidase, and oxidase substrate in liquid cultures is consistent with a close physiological connection between these components (24
). However, the nonspecificity of GLX, together with the possibility that the simple substrates may be derived from carbohydrate (24
), lignin (16
), and lipid (43
), suggests a complex role for GLX in metabolism. Alternatively, a family of related enzymes of similar but distinct specificities may function under various metabolic regimes.
In support of this possibility, here we demonstrate the existence of six sequences structurally related to GLX. All six contain conserved active site residues and predicted secretion signals, but they are diverse with regard to other structural features (Fig. ). The clustered genes cro3, cro4, and cro5 show remarkable conservation of exon/intron positions and form a subfamily of sequences that contain N-terminal WSC domains of unknown function. cro6 is most closely related to the GLX-encoding gene glx, but it also features a 200-amino-acid N-terminal region of unknown function.
The expression of cro genes is consistent with a role(s) in lignocellulose degradation. cDNAs of all six genes were identified in extensively decayed wood wafers. Transcript patterns determined by competitive RT-PCR showed differential regulation among the cro genes over the 120-day time course (Fig. ). The absence of cro4 and cro6 transcripts at 3 days of colonization suggests that these genes are not essential for hyphal penetration and early decay. Concentrated filtrates of P. chrysosporium grown in defined submerged medium were analyzed by LC-MS/MS and shown to contain a CRO2-specific peptide. The apparent absence of other CRO proteins in extracellular fluids may reflect low protein concentrations, compartmentalization, or, particularly in the case of WSC-containing genes, cell wall binding.
In addition to the cro
genes, complex gene families are well known in P. chrysosporium
, particularly among sequences encoding secreted proteins. Examples include LiPs (36
), certain glycosyl hydrolases (32
), and peptidases (34
). The role of such genetic multiplicity remains poorly understood. Structurally, the 10 LiP genes are highly conserved and all are believed to encode high-oxidation potential, nonspecific enzymes. However, slight differences in isozyme activities (14
) and differential transcriptional regulation (18
) suggest that the LiP genes are not merely redundant. Perhaps subtle differences in specificities enhance the efficiency of cell wall degradation under a broad range of environmental conditions. Targeted disruption or suppression would help clarify the role of individual genes, but suitable transformation systems are not yet available for P. chrysosporium
Beyond structural diversity and differential regulation of the cro genes, substrate preferences argue in favor of distinct biological roles, at least for glx and cro2. Our results show that heterologously produced CRO2 oxidizes glycolaldehyde dimer, but not methylglyoxal, the prototypical substrate for GLX. Therefore, the two oxidases are distinguished by catalytic differences. Clarification of their physiological roles, as well as those of the other CRO enzymes, will require comprehensive characterizations, including extensive substrate specificity screenings.