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Radical new paradigm for heme degradation in Escherichia coli O157:H7.

ABSTRACT: All of the heme-degrading enzymes that have been characterized to date require molecular oxygen as a cosubstrate. Escherichia coli O157:H7 has been shown to express heme uptake and transport proteins, as well as use heme as an iron source. This enteric pathogen colonizes the anaerobic space of the lower intestine in mammals, yet no mechanism for anaerobic heme degradation has been reported. Herein we provide evidence for an oxygen-independent heme-degradation pathway. Specifically, we demonstrate that ChuW is a radical S-adenosylmethionine methyltransferase that catalyzes a radical-mediated mechanism facilitating iron liberation and the production of the tetrapyrrole product we termed "anaerobilin." We further demonstrate that anaerobilin can be used as a substrate by ChuY, an enzyme that is coexpressed with ChuW in vivo along with the heme uptake machinery. Our findings are discussed in terms of the competitive advantage this system provides for enteric bacteria, particularly those that inhabit an anaerobic niche in the intestines.

SUBMITTER: LaMattina JW

PROVIDER: S-EPMC5087033 | biostudies-literature | 2016 Oct

REPOSITORIES: biostudies-literature

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Radical new paradigm for heme degradation in Escherichia coli O157:H7.

LaMattina Joseph W JW Nix David B DB Lanzilotta William Nicholas WN

Proceedings of the National Academy of Sciences of the United States of America 20161010 43

All of the heme-degrading enzymes that have been characterized to date require molecular oxygen as a cosubstrate. Escherichia coli O157:H7 has been shown to express heme uptake and transport proteins, as well as use heme as an iron source. This enteric pathogen colonizes the anaerobic space of the lower intestine in mammals, yet no mechanism for anaerobic heme degradation has been reported. Herein we provide evidence for an oxygen-independent heme-degradation pathway. Specifically, we demonstrat ...[more]

PMID: 27791000

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Project description:For many pathogenic microorganisms, iron acquisition from host heme sources stimulates growth, multiplication, ultimately enabling successful survival and colonization. In gram-negative Escherichia coli O157:H7, Shigella dysenteriae and Yersinia enterocolitica the genes encoded within the heme utilization operon enable the effective uptake and utilization of heme as an iron source. While the complement of proteins responsible for heme internalization has been determined in these organisms, the fate of heme once it has reached the cytoplasm has only recently begun to be resolved. Here we report the first crystal structure of ChuX, a member of the conserved heme utilization operon from pathogenic E. coli O157:H7 determined at 2.05 A resolution. ChuX forms a dimer which remarkably given low sequence homology, displays a very similar fold to the monomer structure of ChuS and HemS, two other heme utilization proteins. Absorption spectral analysis of heme reconstituted ChuX demonstrates that ChuX binds heme in a 1:1 manner implying that each ChuX homodimer has the potential to coordinate two heme molecules in contrast to ChuS and HemS where only one heme molecule is bound. Resonance Raman spectroscopy indicates that the heme of ferric ChuX is composed of a mixture of coordination states: 5-coordinate and high-spin, 6-coordinate and low-spin, and 6-coordinate and high-spin. In contrast, the reduced ferrous form displays mainly a 5-coordinate and high-spin state with a minor contribution from a 6-coordinate and low-spin state. The nu(Fe-CO) and nu(C-O) frequencies of ChuX-bound CO fall on the correlation line expected for histidine-coordinated hemoproteins indicating that the fifth axial ligand of the ferrous heme is the imidazole ring of a histidine residue. Based on sequence and structural comparisons, we designed a number of site-directed mutations in ChuX to probe the heme binding sites and dimer interface. Spectral analysis of ChuX and mutants suggests involvement of H65 and H98 in heme coordination as mutations of both residues were required to abolish the formation of the hexacoordination state of heme-bound ChuX.

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Radical new paradigm for heme degradation in Escherichia coli O157:H7.

Publications

Radical new paradigm for heme degradation in Escherichia coli O157:H7.

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