Abstract
Protoporphyrinogen IX oxidase, a monotopic membrane protein, which catalyzes the oxidation of protoporphyrinogen IX to protoporphyrin IX in the heme/chlorophyll biosynthetic pathway, is distributed widely throughout nature. Here we present the structure of protoporphyrinogen IX oxidase from Myxococcus xanthus, an enzyme with similar catalytic properties to human protoporphyrinogen IX oxidase that also binds the common plant herbicide, acifluorfen. In the native structure, the planar porphyrinogen substrate is mimicked by a Tween 20 molecule, tracing three sides of the macrocycle. In contrast, acifluorfen does not mimic the planarity of the substrate but is accommodated by the shape of the binding pocket and held in place by electrostatic and aromatic interactions. A hydrophobic patch surrounded by positively charged residues suggests the position of the membrane anchor, differing from the one proposed for the tobacco mitochondrial protoporphyrinogen oxidase. Interestingly, there is a discrepancy between the dimerization state of the protein in solution and in the crystal. Conserved structural features are discussed in relation to a number of South African variegate porphyria-causing mutations in the human enzyme.
Highlights
In eukaryotes, PPOX is functionally conserved despite a low sequence identity
The crystal structure of mitochondrial tobacco PPOX with a phenylpyrazol inhibitor has been solved to a 2.9-Å resolution [21]
We present the crystal structure of mxPPOX bound to AF, allowing visualization of this important complex for the first time
Summary
Expression and Purification of M. xanthus PPOX—The expression vector (pMx-PPO) containing M. xanthus cDNA [22] was kindly donated by Prof. A data set to 2.9 Å was collected at 100 K on station PX 9.6 of the Synchrotron Radiation Source, Daresbury, UK, using the well solution plus 25% glycerol as a cryoprotectant. Initial phases were determined by molecular replacement with PHASER [26] in P4 (4 molecules/asymmetric unit) using a truncated version of the mtPPOX structure (Protein Data Bank code 1SEZ) [21]. After initial model building using O [27] and refinement with crystallography NMR software using NCS restraints [28], a molecular replacement solution was found for the new model using MOLREP [29] in P4212 space group (2 molecules/asymmetric unit), and refinement with NCS restraints was performed. FAD, water, Tween 20, and glycerol molecules were added using COOT. The quality of the structure was validated with the program WHAT
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