Crystal Structure of HugZ, a Novel Heme Oxygenase from Helicobacter pylori

Yonglin Hu,Fan Jiang,Ying Guo,Xihui Shen,Ying Zhang,Rui Zhang,Gang Guo,Xuhu Mao,Quanming Zou,Da-Cheng Wang

doi:10.1074/jbc.m110.172007

Yonglin Hu, Fan Jiang + Show 8 more

Open Access

https://doi.org/10.1074/jbc.m110.172007

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Abstract

The crystal structure of a heme oxygenase (HO) HugZ from Helicobacter pylori complexed with heme has been solved and refined at 1.8 Å resolution. HugZ is part of the iron acquisition mechanism of H. pylori, a major pathogen of human gastroenteric diseases. It is required for the adaptive colonization of H. pylori in hosts. Here, we report that HugZ is distinct from all other characterized HOs. It exists as a dimer in solution and in crystals, and the dimer adopts a split-barrel fold that is often found in FMN-binding proteins but has not been observed in hemoproteins. The heme is located at the intermonomer interface and is bound by both monomers. The heme iron is coordinated by the side chain of His(245) and an azide molecule when it is present in crystallization conditions. Experiments show that Arg(166), which is involved in azide binding, is essential for HugZ enzymatic activity, whereas His(245), surprisingly, is not, implying that HugZ has an enzymatic mechanism distinct from other HOs. The placement of the azide corroborates the observed γ-meso specificity for the heme degradation reaction, in contrast to most known HOs that have α-meso specificity. We demonstrate through sequence and structural comparisons that HugZ belongs to a new heme-binding protein family with a split-barrel fold. Members of this family are widespread in pathogenic bacteria and may play important roles in the iron acquisition of these bacteria.

Highlights

Microbe growth and to minimize the toxicity from superoxide anions and hydroxyl radicals generated by ferrous via Fenton reactions
Mutation of this protein renders H. pylori unable to grow on heme iron [6]
The difference between the overall folding of HugZ and that of other heme oxygenase (HO) is obvious, but some of the unique properties of HugZ, such as the ␦-meso specificity and the roles played by the heme iron-coordinating histidine residue, can be very well understood by localized structural features, i.e. the difference in the protein-heme interactions between HugZ and other HOs

Summary

EXPERIMENTAL PROCEDURES

Protein Purification and Crystallization—The purification and crystallization of HugZ with a His tag have been reported previously [6, 11]. The crystal structure of the HugZ-hemin complex was solved by the multiwavelength anomalous dispersion method using the 2.3 Å resolution datasets. Iterative cycles between CNS refinement and manual rebuilding were performed, during which 6 azide anions, 22 ethylene glycol molecules, and 1289 water molecules were added to the model, until the Rwork and Rfree reached 0.192 and 0.222, respectively. Desalted protein sample was loaded onto a HiTrap Q column (GE Healthcare) and was eluted in a 0 –150 mM NaCl gradient in 20 mM Tris-HCl, pH 8.9, buffer and used for heme binding and enzymatic activity assays. Protein and heme were added into 1 ml of solution containing 400 units of bovine liver catalase (Worthington), 20 mM Tris-HCl, pH 7.6, and 150 mM NaCl to a final concentration of 10 ␮M. All measurements were made at 298 K using a circulating water bath

RESULTS

A BLAST search identified two homologous proteins with similar molecular sizes

DISCUSSION