Structural Basis for the Molecular Mechanism of Dehydration Reaction Catalyzed By a Novel Heme Protein
Authors: Aono Shigetoshi 
Published: February 2020

Oxd is a new heme-containing enzyme that works as a hydro-lyase. In this study, we determined the crystal structures of Oxd from Rhodococcus sp. N-771 (OxdRE) in the substrate-free and substrate-bound forms. The overall crystal structure of substrate-free OxdRE was solved by MAD and refined at a 1.8 Å resolution. OxdRE formed a homo-dimer with non-crystallographic two-fold symmetry, consistent with previous gel filtration analysis results. Each monomer contained one heme molecule. Each monomer of OxdRE has a α+β structure, consisting of an elliptic β-barrel flanked on both sides by α helices. The barrel was composed of eight β-strands with all strands exhibiting an anti-parallel pattern.

The structures of the BOx-bound OxdRE and PrOx-bound OxdRE were determined at 1.8 and 1.6 Å resolutions, respectively. The nitrogen atom of aldoxime was coordinated to the heme in these complexes, indicating that reduction by X-ray radiation has successfully reconstructed the coordination structure of the aldoxime-heme complex from the inactive to the active form, even in the crystal. The overall structure of the substrate-bound OxdRE was almost the same as that of the substrate-free OxdRE, but there were some local conformational changes upon substrate binding, as discussed below.

The OH group of the heme-bound substrate formed two hydrogen bonds with Ser219 and His320. In the distal heme pocket, the hydrogen bond network was retained among Glu143, Arg178, and His320, as was the case of the substrate-free form. Conformational differences between the substrate-free and substrate-bound forms were not observed in these distal residues. While no conformational differences were observed at the distal side of the heme among the determined structures, conformational variations were observed at the proximal side of the heme. The region 294-315, including the proximal α-helix (α10) and the following 310 helix (η4), displayed a conformational variation in each monomer in the asymmetric unit of the crystal. In the open form, the substrate binding cavity in the distal side of the heme was connected to the protein surface through a channel formed between the 310 helix (residues 309-312) and the loop. This cavity will serve as the substrate access and product release channel. However, the entrance of the channel on the protein surface was closed in the closed form. The conformational change from the open to the closed form resulted from the sliding of Phe306 by 3 Å vertically to the heme plane, accompanied by rotation of the proximal α-helix. This movement of Phe306 disconnected the substrate-binding cavity from the substrate access/product release channel. Thus, the 310 helix and side chain of Phe306 act as a gate for the channel, modulating substrate access and product release.

The crystal structures determined in this study reveal that hydrogen bonding between the OH group of aldoxime and the side chains of Ser219 and His320 controls the specific orientation of the heme-bound substrate suitable for the elimination of the OH group of aldoxime, and that these residues and the heme create a prefixed site for substrate recognition and binding. His320 also serves as a catalytic residue for the elimination of the substrate OH group to form H2O, consistent with previous mutagenesis studies. The H320A mutant lacked enzymatic activity. A hydrogen bond network existed among Glu143, Arg178, and His320 that fixed the proper orientation of His320 toward the heme-bound substrate. The hydrogen bond between Glu143 and His320 will play an important role not only for control of the proper orientation of His320, but also for stabilization of the imidazorium form of His320.