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Structure, computational and biochemical analysis of PcCel45A endoglucanase from Phanerochaete chrysosporium and catalytic mechanisms of GH45 subfamily C members.

ABSTRACT: The glycoside hydrolase family 45 (GH45) of carbohydrate modifying enzymes is mostly comprised of β-1,4-endoglucanases. Significant diversity between the GH45 members has prompted the division of this family into three subfamilies: A, B and C, which may differ in terms of the mechanism, general architecture, substrate binding and cleavage. Here, we use a combination of X-ray crystallography, bioinformatics, enzymatic assays, molecular dynamics simulations and site-directed mutagenesis experiments to characterize the structure, substrate binding and enzymatic specificity of the GH45 subfamily C endoglucanase from Phanerochaete chrysosporium (PcCel45A). We investigated the role played by different residues in the binding of the enzyme to cellulose oligomers of different lengths and examined the structural characteristics and dynamics of PcCel45A that make subfamily C so dissimilar to other members of the GH45 family. Due to the structural similarity shared between PcCel45A and domain I of expansins, comparative analysis of their substrate binding was also carried out. Our bioinformatics sequence analyses revealed that the hydrolysis mechanisms in GH45 subfamily C is not restricted to use of the imidic asparagine as a general base in the "Newton's cradle" catalytic mechanism recently proposed for this subfamily.

SUBMITTER: Godoy AS

PROVIDER: S-EPMC5829257 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:The activity of a self-sufficient cytochrome P450 enzyme, CYP505D6, from the lignin-degrading basidiomycete Phanerochaete chrysosporium was characterized. Recombinant CYP505D6 was produced in Escherichia coli and purified. In the presence of NADPH, CYP505D6 used a series of saturated fatty alcohols with C9-18 carbon chain lengths as the substrates. Hydroxylation occurred at the ω-1 to ω-6 positions of such substrates with C9-15 carbon chain lengths, except for 1-dodecanol, which was hydroxylated at the ω-1 to ω-7 positions. Fatty acids were also substrates of CYP505D6. Based on the sequence alignment, the corresponding amino acid of Tyr51, which is located at the entrance to the active-site pocket in CYP102A1, was Val51 in CYP505D6. To understand the diverse hydroxylation mechanism, wild-type CYP505D6 and its V51Y variant and wild-type CYP102A1 and its Y51V variant were generated, and the products of their reaction with dodecanoic acid were analyzed. Compared with wild-type CYP505D6, its V51Y variant generated few products hydroxylated at the ω-4 to ω-6 positions. The products generated by wild-type CYP102A1 were hydroxylated at the ω-1 to ω-4 positions, whereas its Y51V variant generated ω-1 to ω-7 hydroxydodecanoic acids. These observations indicated that Val51 plays an important role in determining the regiospecificity of fatty acid hydroxylation, at least that at the ω-4 to ω-6 positions. Aromatic compounds, such as naphthalene and 1-naphthol, were also hydroxylated by CYP505D6. These findings highlight a unique broad substrate spectrum of CYP505D6, rendering it an attractive candidate enzyme for the biotechnological industry.IMPORTANCEPhanerochaete chrysosporium is a white-rot fungus whose metabolism of lignin, aromatic pollutants, and lipids has been most extensively studied. This fungus harbors 154 cytochrome P450-encoding genes in the genome. As evidenced in this study, P. chrysosporium CYP505D6, a fused protein of P450 and its reductase, hydroxylates fatty alcohols (C9-15) and fatty acids (C9-15) at the ω-1 to ω-7 or ω-1 to ω-6 positions, respectively. Naphthalene and 1-naphthol were also hydroxylated, indicating that the substrate specificity of CYP505D6 is broader than those of the known fused proteins CYP102A1 and CYP505A1. The substrate versatility of CYP505D6 makes this enzyme an attractive candidate for biotechnological applications.

Dataset Information

Structure, computational and biochemical analysis of PcCel45A endoglucanase from Phanerochaete chrysosporium and catalytic mechanisms of GH45 subfamily C members.

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