Role of His63 in HutZ from<i>Vibrio cholerae</i>in the heme degradation reaction and heme binding

Uchida, Takeshi; Dojun, Nobuhiko; Sekine, Yukari; Ishimori, Koichiro

doi:10.1039/c9dt00926d

Cited by 2 publications

(6 citation statements)

References 45 publications

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“…Recent studies by our group further showed that subunit-subunit interactions play a key role in the heme degradation reaction [16]. The distance between heme and Trp109, which reflects subunit-subunit orientational changes, was increased from [16][17][18] Å for wild-type HutZ to 23-25 Å for R92A and R92L mutant enzymes, similar to data obtained for mutants of Ala31 [16], His63 [19] and Asp132. Based on the collective results, we propose an activation mechanism of HutZ involving conformational changes at Arg92, which are transmitted to the heme axial ligand His170 through Ala31, His63, and Asp132, with modulation of subunit-subunit interactions.…”

Section: Introductionsupporting

confidence: 80%

“…Changes of the subunit-subunit interactions also occurred in the His63 mutant [19]. His63 is presumed to be located close to heme and in the same subunit containing Asp132 but a different subunit from His170 and Arg92 (Supplemental Fig.…”

Section: Regulatory Mechanisms Of Heme Degradation By Hutzmentioning

confidence: 95%

“…The structure of apo-HutZ was solved and its docking structure of heme predicted that Arg92 and His63 coordinate heme [18]. However, the results of our mutational studies suggested that His170 is a sole heme ligand, which was not coincident with the docking model [19,20]. The crystal structure of a HutZ homologous protein HugZ from Helicobacter pylori suggests that His170 of HutZ is the proximal heme ligand, which is within hydrogen bonding distance of Asp132 (Fig.…”

Section: Introductionmentioning

confidence: 96%

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Role of conserved arginine in the heme distal site of HutZ from Vibrio cholerae in the heme degradation reaction

Uchida

Dojun

Ota

et al. 2019

Archives of Biochemistry and Biophysics

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HutZ from Vibrio cholerae is a dimeric enzyme that catalyzes degradation of heme. The highly conserved Arg92 residue in the HutZ family is proposed to interact with an iron-bound water molecule in the distal heme pocket. To clarify the specific role of Arg92 in the heme degradation reaction, the residue was substituted with alanine, leucine, histidine or lysine to modulate electrostatic interactions with iron-bound ligand. All four Arg92 mutants reacted with hydrogen peroxide to form verdoheme, a prominent intermediate in the heme degradation process. However, when ascorbic acid was used as an electron source, iron was not released even at pH 6.0 despite a decrease in the Soret band, indicating that non-enzymatic heme degradation occurred. Comparison of the rates of heme reduction, ligand binding and verdoheme formation suggested that proton transfer to the reduced oxyferrous heme, a potential rate-limiting step of heme degradation in HutZ, is hampered by mutation. In our previous study, we found that the increase in the distance between heme and Trp109 from 16 to 18 Å upon lowering the pH from 8.0 to 6.0 leads to activation of ascorbic acid-assisted heme degradation by HutZ. The distance in Arg92 mutants was >19 Å at pH 6.0, suggesting that subunit-subunit interactions at this pH are not suitable for heme degradation, similar to Asp132 and His63 mutants. These results suggest that interactions of Arg92 with heme-bound ligand induce alterations in the distance between subunits, which plays a key role in controlling the heme degradation activity of HutZ.

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Section: Introductionsupporting

confidence: 80%

Section: Regulatory Mechanisms Of Heme Degradation By Hutzmentioning

confidence: 95%

Section: Introductionmentioning

confidence: 96%

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Role of conserved arginine in the heme distal site of HutZ from Vibrio cholerae in the heme degradation reaction

Uchida

Dojun

Ota

et al. 2019

Archives of Biochemistry and Biophysics

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“…Many bacterial HOs have been characterized and the majority are structurally similar to mammalian HOs . Typically, HOs oxidatively cleave heme at the α‐ meso position to afford biliverdin IXα, CO, and Fe 2+ ; however, cleavage has been observed at all four meso positions (Figure A) . HOs as well as heme‐degrading/cytoplasmic heme‐binding (HemS) proteins with no sequence homology to canonical HOs have been reported in multiple species of bacteria.…”

Section: Figurementioning

confidence: 99%

“… A) Example of heme degradation into biliverdin IXα by a heme oxygenase . Cleavage has been observed at the β‐, δ‐, γ‐ meso positions across nature. B) Gene organization of heme/siderophore utilization operon from E. anophelis Ag1.…”

Section: Figurementioning

confidence: 99%

Coculturing of Mosquito‐Microbiome Bacteria Promotes Heme Degradation in Elizabethkingia anophelis

et al. 2020

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Anopheles mosquito microbiomes are intriguing ecological niches. Within the gut, microbes adapt to oxidative stress due to heme and iron after blood meals. Although metagenomic sequencing has illuminated spatial and temporal fluxes of microbiome populations, limited data exist on microbial growth dynamics. Here, we analyze growth interactions between a dominant microbiome species, Elizabethkingia anophelis, and other Anopheles‐associated bacteria. We find E. anophelis inhibits a Pseudomonas sp. via an antimicrobial‐independent mechanism and observe biliverdins, heme degradation products, upregulated in cocultures. Purification and characterization of E. anophelis HemS demonstrates heme degradation, and we observe hemS expression is upregulated when cocultured with Pseudomonas sp. This study reveals a competitive microbial interaction between mosquito‐associated bacteria and characterizes the stimulation of heme degradation in E. anophelis when grown with Pseudomonas sp.

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Role of His63 in HutZ fromVibrio choleraein the heme degradation reaction and heme binding

Abstract: His63 of HutZ from Vibrio cholerae does not contribute to regioselectivity of heme degradation but plays a key role in maintaining the orientation of subunits for HutZ to function in heme degradation.

Cited by 2 publications

References 45 publications

Role of conserved arginine in the heme distal site of HutZ from Vibrio cholerae in the heme degradation reaction

Role of conserved arginine in the heme distal site of HutZ from Vibrio cholerae in the heme degradation reaction

Coculturing of Mosquito‐Microbiome Bacteria Promotes Heme Degradation in Elizabethkingia anophelis

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