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Novel Enzymes and Catalytic Mechanisms in Tetrapyrrole Metabolism: a Nitrogenase Homolog and a Noncanonical Heme Oxygenase




Li, Shuxin

Type of Degree

PhD Dissertation


Chemistry and Biochemistry


Methane is the major component of natural gas, which accounts for approximately one quarter of the world’s energy demands. There are many advantages to using methane as a fuel. Methane is an abundance resource. A huge number of methane hydrates are on the seafloor and microorganism break up organic matter to produce methane. Methane is the simplest organic compound and produces more heat per unit mass (55.7 kJ/g) than any other hydrocarbon. Methane is also a clean fuel. It releases less sulfur, carbon, and nitrogen than coal or oil, and leaves little ash. However, methane formations are usually distributed in remote areas, which require an extensive and complex pipeline network to transport natural gas to end users. Natural gas is highly flammable, so the transportation from wellheads to homes and businesses is dangerous. Meanwhile, methane is a greenhouse gas that has thirty times the global warming potential of carbon dioxide. Thus, there is great current interest in strategies to convert methane to liquid fuel or other more easily transported commodity chemicals. Methanogens are anaerobic archaea widely found in wetlands and the digestive tracts of animals, which produce around 1 billion tons of methane. Methanogenesis is balanced by aerobic or anaerobic methane oxidation. Anaerobic methanotrophic archaea (ANME) catalyze the anaerobic oxidation of methane (AOM), often with the aid of sulfate or nitrate reducing bacteria. Both methanogenesis and AOM use a key enzyme, methyl-coenzyme M reductase (MCR), which has a unique nickel-containing coenzyme F430 for activity. MCR catalyzes the interconversion of coenzyme B and methyl-coenzyme M to the mixed heterodisulfide CoB-S-S-CoM and methane. ANME have potential for use in natural gas-to-liquid fuel conversion strategies, but they have extremely slow growth rates, and they cannot be currently obtained in pure culture. A possible solution is to engineer the methane-conversion pathway and its key enzyme, MCR, into a more robust microorganism. Currently, MCR cannot be produced in an active form in a heterologous host, in part because the in vivo biosynthesis of coenzyme F430 is still a challenge. Our lab has identified the coenzyme F430 biosynthesis (cfb) genes and characterized the encoded enzymes in vitro. They include a sirohydrochlorin cobaltochelatase homolog, CfbA, which catalyzes the specific Ni-chelation of sirohydrochlorin; a cobyrinic acid a,c-diamide synthase homolog, CfbB, that catalyzes the adenosine triphosphate (ATP)-dependent amidation of the a- and c-acetate side chains of Ni-sirohydrochlorin using glutamine as an ammonia source; a nitrogenase homolog, CfbCD, which converts Ni-sirohydrochlorin a,c-diamide to 15,173-seco-F430-173-acid; and a Mur-ligase homolog, CfbE, that carries out the ATP-dependent cyclization of the g-proprionate side chain to generate the carbocyclic F ring. Chapter Two details the construction of compatible expression vectors containing the cfb genes and putative accessory factors from Methanosarcina acetivorans and experiments designed to produce coenzyme F430 in Escherichia coli. Key proteins in the coenzyme F430 biosynthesis pathway were extracted from the engineered cell line and tested for activity in vitro. Different physiological reducing systems, including ferredoxin (Fd)/ferredoxin:NADP+ reductase (FNR) and pyruvate:ferredoxin oxidoreductase (PFOR), were tested for their ability to support CfbCD catalysis in vivo. The effects of supplementation with an additional copy of the cfbCD genes from Methanosarcina thermophila (which encode a more stable/active variant of the CfbCD complex) and coexpression with 8 putative MCR maturation genes on the heterologous production of coenzyme F430 were also examined, as were various concentrations of supplementary chemicals and growth times/conditions. Coenzyme F430 is the most highly reduced tetrapyrrole in nature, and this is achieved by a single multi-electron redox reaction catalyzed by the nitrogenase homolog, CfbCD. The CfbCD-catalyzed conversion of Ni-sirohydrochlorin a,c-diamide to 15,173-seco-F430-173-acid involves a 6-electron reduction of the isobacteriochlorin ring system, cyclization of the c-acetamide side chain to form the γ-lactam E ring, and the formation of 7 stereocenters. The chemical mechanism of this unprecedented transformation is unknown, and there is debate as to whether γ-lactam ring formation is catalyzed by CfbCD or occurs non-enzymatically. In Chapter Three, mechanistic studies of the CfbCD reaction is presented. The CfbCD reaction was performed in deuterium oxide to label 15,173-seco-F430-173-acid and determine the stereochemistry of the reaction using nuclear magnetic resonance (NMR) spectroscopy. A 15N-labelled version of the immediate product of the CfbCD reaction was prepared and purified for structural characterization using NMR. Meanwhile, a novel tetrapyrrole, Ni-didecarboxysirohydrochlorin a,c-diamide, was synthesized for use as a mechanistic probe by combining the CfbA and CfbB reactions with an enzyme from the heme d1 biosynthetic pathway. Chapter Four describes a noncanonical heme oxygenase specific for the degradation of c-type heme. Heme oxygenases (HOs) play a critical role in recouping iron from the labile heme pool. The acquisition and liberation of heme iron is especially important for the survival of pathogenic bacteria. All characterized HOs, including those belonging to the HugZ superfamily, preferentially cleave free b-type heme. Another common form of heme found in nature is c-type heme, which is covalently linked to proteinaceous cysteine residues. However, mechanisms for direct iron acquisition from the c-type heme pool are unknown. We identified a HugZ homolog from the oligopeptide permease (opp) gene cluster of Paracoccus denitrificans that lacks any observable reactivity with heme b and show that it instead rapidly degrades c-type hemopeptides. This c-type heme oxygenase catalyzes the oxidative cleavage of the model substrate microperoxidase-11 at the - and/or -meso position(s), yielding the corresponding peptide-linked biliverdin, CO, and free iron. X-ray crystallographic analysis suggests that the switch in substrate specificity from b- to c-type heme involves loss of the N-terminal / domain and C-terminal loop containing the coordinating histidine residue characteristic of HugZ homologs, thereby accommodating a larger substrate that provides its own iron ligand. These structural features are also absent in certain heme utilization/storage proteins from human pathogens that exhibit low or no HO activity with free heme. This study thus expands the scope of known iron acquisition strategies to include direct oxidative cleavage of heme-containing proteolytic fragments of c-type cytochromes and helps to explain why certain oligopeptide permeases show specificity for the import of heme in addition to peptides.