In vitro heme biotransformation by the HupZ enzyme from Group A streptococcus

Abstract

In Group A streptococcus (GAS), the metallorepressor MtsR regulates iron homeostasis. Here we describe a new MtsR-repressed gene, which we named hupZ (heme utilization protein). A recombinant HupZ protein was purified bound to heme from Escherichia coli grown in the presence of 5-aminolevulinic acid and iron. HupZ specifically binds heme with stoichiometry of 1:1. The addition of NADPH to heme-bound HupZ (in the presence of cytochrome P450 reductase, NADPH-regeneration system and catalase) triggered progressive decrease of the HupZ Soret band and the appearance of an absorption peak at 660 nm that was resistance to hydrolytic conditions. No spectral changes were observed when ferredoxin and ferredoxin reductase were used as redox partners. Differential spectroscopy with myoglobin or with the ferrous chelator, ferrozine, confirmed that carbon monoxide and free iron are produced during the reaction. ApoHupZ was crystallized as a homodimer with a split β-barrel conformation in each monomer comprising six β strands and three α helices. This structure resembles the split β-barrel domain shared by the members of a recently described family of heme degrading enzymes. However, HupZ is smaller and lacks key residues found in the proteins of the latter group. Phylogenetic analysis places HupZ on a clade separated from those for previously described heme oxygenases. In summary, we have identified a new GAS enzyme-containing split β-barrel and capable of heme biotransformation in vitro; to the best of our knowledge, this is the first enzyme among Streptococcus species with such activity.

From Host Heme To Iron: The Expanding Spectrum of Heme Degrading Enzymes Used by Pathogenic Bacteria

“…Currently only apo-crystal structures exist for HutZ and HupZ, but analyses using single site substitution mutagenesis of HutZ have shown that His63 and Arg92 (corresponding to His245 and Arg166 in HugZ) interact with the propionate due to shifts in heme-bound spectra (Liu X. et al, 2012 ). HupZ, however, lacks His245 and contains a glycine instead of Arg166, making it difficult to determine the heme coordinating residues based on sequence analysis alone (Sachla et al, 2016 ). Interestingly, ChuZ crystallized not only with the expected heme binding site, but with an additional surface bound heme molecule (Zhang et al, 2011 ).…”

“…In vitro degradation of heme by HugZ, ChuZ, and HupZ was observed in the presence of catalase (Ridley et al, 2006 ; Guo et al, 2008 ; Sachla et al, 2016 ). All three proteins degrade heme to produce CO and a chromophore that absorbs at 660 nm (in the presence of ascorbate or CPR/NADH).…”

“…HPLC chromatography has confirmed that HugZ produce δ-biliverdin (Guo et al, 2008 ). While the chromophore produced by the ChuZ and HupZ reactions were not identified, the stability of HupZ's chromophore under acidic conditions suggests it is not verdoheme (Sachla et al, 2016 ). The iron-regulated expression of all three enzymes is consistent with a role in iron acquisition.…”

“…A DtxR-like protein named MtsR regulates the transcription of hupZ , a recently described enzyme in S. pyogenes (Bates et al, 2005 ; Sachla et al, 2016 ). MtsR binds to DNA in the presence of either iron or manganese.…”

“…MtsR binds to DNA in the presence of either iron or manganese. It was first recognized as the repressor of the sia operon ( shr/shp/siaABCDEFG ) which encodes a central heme import pathway and of the mtsABC metal transporter (Hanks et al, 2006 ; Sachla et al, 2016 ). Global transcription analysis of null mtsR mutants established MtsR as a global regulator that controls the expression of many S. pyogenes genes, including iron and heme uptake, metal homeostasis and important virulence factors (Toukoki et al, 2010 ).…”