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Functional identification of HugZ, a heme oxygenase from Helicobacter pylori.

BMC microbiology (2008-12-19)
Ying Guo, Gang Guo, Xuhu Mao, Weijun Zhang, Jie Xiao, Wende Tong, Tao Liu, Bin Xiao, Xiaofei Liu, Youjun Feng, Quanming Zou
ABSTRACT

Iron is recognized as an important trace element, essential for most organisms including pathogenic bacteria. HugZ, a protein related to heme iron utilization, is involved in bacterial acquisition of iron from the host. We previously observed that a hugZ homologue is correlated with the adaptive colonization of Helicobacter pylori (H. pylori), a major gastro-enteric pathogen. However, its exact physiological role remains unclear. A gene homologous to hugZ, designated hp0318, identified in H. pylori ATCC 26695, exhibits 66% similarity to cj1613c of Campylobacter jejuni NCTC 11168. Soluble 6 x His fused-HugZ protein was expressed in vitro. Hemin-agrose affinity analysis indicated that the recombinant HugZ protein can bind to hemin. Absorption spectroscopy at 411 nm further revealed a heme:HugZ binding ratio of 1:1. Enzymatic assays showed that purified recombinant HugZ protein can degrade hemin into biliverdin and carbon monoxide in the presence of either ascorbic acid or NADPH and cytochrome P450 reductase. The biochemical and enzymatic characteristics agreed closely with those of Campylobacter jejuni Cj1613c protein, implying that hp0318 is a functional member of the HugZ family. A hugZ deletion mutant was obtained by homologous recombination. This mutant strain showed poor growth when hemoglobin was provided as the source of iron, partly because of its failure to utilize hemoglobin efficiently. Real-time quantitative PCR also confirmed that the expression of hugZ was regulated by iron levels. These findings provide biochemical and genetic evidence that hugZ (hp0318) encodes a heme oxygenase involved in iron release/uptake in H. pylori.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
Hemin−Agarose, Type I, saline suspension
Sigma-Aldrich
Myoglobin from human heart, ≥95% (SDS-PAGE), buffered aqueous glycerol solution
Sigma-Aldrich
Myoglobin from equine heart, ≥90% (SDS-PAGE), essentially salt-free, lyophilized powder
Supelco
Myoglobin from equine heart, Isoelectric focusing marker, pI (1) 6.8, (2) 7.2