No abstract

Understanding peroxidase function in plants is complicated by the lack of substrate specificity, the high number of genes, their diversity in structure and our limited knowledge of peroxidase gene transcription and translation. In the present study we sequenced expressed sequence tags (ESTs) encoding novel heme-containing class III peroxidases from Arabidopsis thaliana and annotated 73 full-length genes identified in the genome. In total, transcripts of 58 of these genes have now been observed. The expression of individual peroxidase genes was assessed in organ-specific EST libraries and compared to the expression of 33 peroxidase genes which we analyzed in whole plants 3, 6, 15, 35 and 59 days after sowing. Expression was assessed in root, rosette leaf, stem, cauline leaf, flower bud and cell culture tissues using the gene-specific and highly sensitive reverse transcriptasepolymerase chain reaction (RT-PCR).We predicted that 71 genes could yield stable proteins folded similarly to horseradish peroxidase (HRP). The putative mature peroxidases derived from these genes showed 28-94% amino acid sequence identity and were all targeted to the endoplasmic reticulum by N-terminal signal peptides. In 20 peroxidases these signal peptides were followed by various N-terminal extensions of unknown function which are not present in HRP. Ten peroxidases showed a C-terminal extension indicating vacuolar targeting. We found that the majority of peroxidase genes were expressed in root. In total, class III peroxidases accounted for an impressive 2.2% of root ESTs. Rather few peroxidases showed organ specificity. Most importantly, genes expressed constitutively in all organs and genes with a preference for root represented structurally diverse peroxidases (< 70% sequence identity). Furthermore, genes appearing in tandem showed distinct expression profiles. The alignment of 73 Arabidopsis peroxidase sequences provides an easy access to the identification of orthologous peroxidases in other plant species and will provide a common platform for combining knowledge of peroxidase structure and function relationships obtained in various species.

The three-dimensional structure of recombinant horseradish peroxidase in complex with BHA (benzhydroxamic acid) is the first structure of a peroxidase-substrate complex demonstrating the existence of an aromatic binding pocket. The crystal structure of the peroxidase-substrate complex has been determined to 2.0 A resolution with a crystallographic R-factor of 0.176 (R-free = 0. 192). A well-defined electron density for BHA is observed in the peroxidase active site, with a hydrophobic pocket surrounding the aromatic ring of the substrate. The hydrophobic pocket is provided by residues H42, F68, G69, A140, P141, and F179 and heme C18, C18-methyl, and C20, with the shortest distance (3.7 A) found between heme C18-methyl and BHA C63. Very little structural rearrangement is seen in the heme crevice in response to substrate binding. F68 moves to form a lid on the hydrophobic pocket, and the distal water molecule moves 0.6 A toward the heme iron. The bound BHA molecule forms an extensive hydrogen bonding network with H42, R38, P139, and the distal water molecule 2.6 A above the heme iron. This remarkably good match in hydrogen bond requirements between the catalytic residues of HRPC and BHA makes the extended interaction between BHA and the distal heme crevice of HRPC possible. Indeed, the ability of BHA to bind to peroxidases, which lack a peripheral hydrophobic pocket, suggests that BHA is a general counterpart for the conserved hydrogen bond donors and acceptors of the distal catalytic site. The closest aromatic residue to BHA is F179, which we predict provides an important hydrophobic interaction with more typical peroxidase substrates.