3VEF: Rhodococcus jostii RHA1 DypB N246H variant in complex with heme

Citation:
Abstract
DypB from Rhodococcus jostii RHA1 is a bacterial dye-decolorizing peroxidase (DyP) that oxidizes lignin and Mn(II). Three residues interact with the iron-bound solvent species in ferric DypB: Asn-246 and the conserved Asp-153 and Arg-244. Substitution of either Asp-153 or Asn-246 with alanine minimally affected the second order rate constant for Compound I formation (k(1) approximately 10(5) M(-1)s(-1)) and the specificity constant (k(cat)/K(m)) for H(2)O(2). Even in the D153A/N246A double variant, these values were reduced less than 30-fold. However, these substitutions dramatically reduced the stability of Compound I (t(1/2) approximately 0.13 s) as compared with the wild-type enzyme (540 s). By contrast, substitution of Arg-244 with leucine abolished the peroxidase activity, and heme iron of the variant showed a pH-dependent transition from high spin (pH 5) to low spin (pH 8.5). Two variants were designed to mimic the plant peroxidase active site: D153H, which was more than an order of magnitude less reactive with H(2)O(2), and N246H, which had no detectable peroxidase activity. X-ray crystallographic studies revealed that structural changes in the variants are confined to the distal heme environment. The data establish an essential role for Arg-244 in Compound I formation in DypB, possibly through charge stabilization and proton transfer. The principle roles of Asp-153 and Asn-246 appear to be in modulating the subsequent reactivity of Compound I. These results expand the range of residues known to catalyze Compound I formation in heme peroxidases.
PDB ID: 3VEFDownload
MMDB ID: 96591
PDB Deposition Date: 2012/1/7
Updated in MMDB: 2017/11
Experimental Method:
x-ray diffraction
Resolution: 2.64  Å
Source Organism:
Similar Structures:
Biological Unit for 3VEF: hexameric; determined by author and by software (PISA)
Molecular Components in 3VEF
Label Count Molecule
Proteins (6 molecules)
6
Dypb
Molecule annotation
Chemicals (12 molecules)
1
6
2
6
* Click molecule labels to explore molecular sequence information.

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