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Atypical biosynthetic properties of a Delta 12/nu+3 desaturase from the model basidiomycete Phanerochaete chrysosporium.

ABSTRACT: The model white-rot basidiomycete Phanerochaete chrysosporium contains a single integral membrane Delta(12)-desaturase FAD2 related to the endoplasmic reticular plant FAD2 enzymes. The fungal fad2-like gene was cloned and distinguished itself from plant homologs by the presence of four introns and a significantly larger coding region. The coding sequence exhibits ca. 35% sequence identity to plant homologs, with the highest sequence conservation found in the putative catalytic and major structural domains. In vivo activity of the heterologously expressed enzyme favors C(18) substrates with nu+3 regioselectivity, where the site of desaturation is three carbons carboxy-distal to the reference position of a preexisting double bond (nu). Linoleate accumulated to levels in excess of 12% of the total fatty acids upon heterologous expression of P. chrysosporium FAD2 in Saccharomyces cerevisiae. In contrast to the behavior of the plant FAD2 enzymes, this oleate desaturase does not 12-hydroxylate lipids and is the first example whose activity increases at higher temperatures (30 degrees C versus 15 degrees C). Thus, while maintaining the hallmark activity of the fatty acyl Delta(12)-desaturase family, the basidiomycete fad2 genes appear to have evolved substantially from an ancestral desaturase.

SUBMITTER: Minto RE

PROVIDER: S-EPMC2643574 | biostudies-literature | 2009 Feb

REPOSITORIES: biostudies-literature

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Atypical biosynthetic properties of a Delta 12/nu+3 desaturase from the model basidiomycete Phanerochaete chrysosporium.

Minto Robert E RE Blacklock Brenda J BJ Younus Hina H Pratt Andrew C AC

Applied and environmental microbiology 20081216 4

The model white-rot basidiomycete Phanerochaete chrysosporium contains a single integral membrane Delta(12)-desaturase FAD2 related to the endoplasmic reticular plant FAD2 enzymes. The fungal fad2-like gene was cloned and distinguished itself from plant homologs by the presence of four introns and a significantly larger coding region. The coding sequence exhibits ca. 35% sequence identity to plant homologs, with the highest sequence conservation found in the putative catalytic and major structur ...[more]

PMID: 19088315

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Project description:A comprehensive analysis to survey heme-binding proteins produced by the white-rot fungus Phanerochaete chrysosporium was achieved using a biotinylated heme-streptavidin beads system. Mitochondrial citrate synthase (PcCS), glyceraldehyde 3-phosphate dehydrogenase (PcGAPDH), and 2-Cys thioredoxin peroxidase (mammalian HBP23 homolog) were identified as putative heme-binding proteins. Among these, PcCS and PcGAPDH were further characterized using heterologously expressed recombinant proteins. Difference spectra of PcCS titrated with hemin exhibited an increase in the Soret absorbance at 414 nm, suggesting that the axial ligand of the heme is a His residue. The activity of PcCS was strongly inhibited by hemin with Ki oxaloacetate of 8.7 μM and Ki acetyl-CoA of 5.8 μM. Since the final step of heme biosynthesis occurred at the mitochondrial inner membrane, the inhibition of PcCS by heme is thought to be a physiological event. The inhibitory mode of the heme was similar to that of CoA analogues, suggesting that heme binds to PcCS at His347 at the AcCoA-CoA binding site, which was supported by the homology model of PcCS. PcGAPDH was also inhibited by heme, with a lower concentration than that for PcCS. This might be caused by the different location of these enzymes. From the integration of these phenomena, it was concluded that metabolic regulations by heme in the central metabolic and heme synthetic pathways occurred in the mitochondria and cytosol. This novel pathway crosstalk between the central metabolic and heme biosynthetic pathways, via a heme molecule, is important in regulating the metabolic balance (heme synthesis, ATP synthesis, flux balance of the tricarboxylic acid (TCA) cycle and cellular redox balance (NADPH production) during fungal aromatic degradation. KEY POINTS: • A comprehensive survey of heme-binding proteins in P. chrysosporium was achieved. • Several heme-binding proteins including CS and GAPDH were identified. • A novel metabolic regulation by heme in the central metabolic pathways was found.

Dataset Information

Atypical biosynthetic properties of a Delta 12/nu+3 desaturase from the model basidiomycete Phanerochaete chrysosporium.

Publications

Atypical biosynthetic properties of a Delta 12/nu+3 desaturase from the model basidiomycete Phanerochaete chrysosporium.

Similar Datasets

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