Beyond O2: Biochemistry of Chlorite Dismutases from Klebsiella and Staphylococcus

Doctoral Dissertation

Abstract

This thesis examines the relationships among members of the chlorite dismutase (Cld) heme protein family, focusing on sequence, structural, and functional characterization of two highly distinct chlorite dismutases from divergent phyla. The name “chlorite dismutase” refers to the heme-facilitated conversion of chlorite (ClO2-) to chloride (Cl-) and O2, a remarkable reaction in that Cld is the only well-characterized enzyme, aside from photosystem II, capable of O-O bond generation. The Cld from Dechloromonas aromatica (DaCld) is a homopentamer that very rapidly and efficiently generates oxygen from chlorite. Interestingly, Cld homologues are found among 10 diverse phyla, suggesting ancient origins for the family. The handful of Clds from known perchlorate respirers group closely in protein-based phylogenetic analysis, which could be indicative of an evolutionary response to a relatively recent pollutant, chlorite. The majority of Clds are not expected to respire perchlorate, leaving a vast amount of relevant bacterial genome unexplored with respect to gene product function. In order to explore Cld diversity, Cld homolog proteins from the Gram-positive Firmicutes Staphylococcus aureus (Sa) and the Gram-negative Proteobacterium Klebsiella pneumoniae (Kp), two very different types of bacteria, were functionally characterized. Both Sa and Kp ESKAPE pathogens, drug-resistant bacteria that increasingly escape the effect of antibiotics and account for about 2/3 of all hospital-acquired infections, so gene product characterization for these particular bacteria has the potential for eventual organism-specific antimicrobial applications. The active site of KpCld very closely resembles that of DaCld but the protein is a dimer, while SaCld is a homopentamer like DaCld but lacks the distal Arg critical for the Cld reaction. These two proteins were expressed, purified with bound heme, and studied through a wide array of bioinformatics and biochemical techniques. Furthermore, their corresponding gene knockouts were constructed and their phenotypes studied in parallel. Bioinformatic, biochemical, and genetic studies showed that SaCld, or SaHemQ, lacks Cld activity, instead playing a critical role in the heme biosynthesis pathway, particularly in the final few steps. Knockout growth curves, metabolite analyses, and reactivity studies, among others, provided evidence that SaCld is likely not a direct enzyme in the pathway, but rather serves as an iron, heme, or redox sensor that is able to influence the collaborative activity of protoporphyrinogen oxidase and ferrochelatase, the final two enzymes in the heme biosynthetic pathway. In stark contrast to this functionality is KpCld, a protein that performs the Cld reaction despite lacking both a perchlorate reductase and a biological need by the organism to metabolize perchorate. Instead, KpCld has been shown to be capable of reductant oxidation (specifically, ABTS) and chlorination upon chlorite addition, and the Cld reaction can be controlled by the presence (or absence) of O2 as well as by concentration of ABTS. Overall, this thesis provides insight into the functional diversity of the Cld family by examining two distinct representative Cld proteins. In order to look beyond only the sequence-based phylogeny of the Cld family, a more integrated approach has been taken that marries both sequence and structural information for functional study.

Attributes

Attribute NameValues
URN
  • etd-07222013-205644

Author Richard Charles Kurker, Jr.
Advisor Dr. Jennifer DuBois
Contributor Dr. Amanda Hummon, Committee Member
Contributor Dr. Jennifer DuBois, Committee Co-Chair
Contributor Dr. Anthony Serianni, Committee Member
Contributor Dr. Paul Huber, Committee Co-Chair
Degree Level Doctoral Dissertation
Degree Discipline Chemistry and Biochemistry
Degree Name PhD
Defense Date
  • 2013-07-11

Submission Date 2013-07-22
Country
  • United States of America

Subject
  • peroxidase

  • chlorite dismutase

  • protein biochemistry

  • bioinformatics

  • heme biosynthesis

  • catalase

Publisher
  • University of Notre Dame

Language
  • English

Record Visibility Public
Content License
  • All rights reserved

Departments and Units

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