Structure and function of class one non-symbiotic plant hemoglobins

Plants contain at least three kinds of hemoglobins. Those plants that carry out symbiotic nitrogen fixation use oxygen transport hemoglobins to deliver oxygen to the aerobic nitrogen fixing bacteria in their roots. The functions of other plant hemoglobins are not yet known with confidence, but are thought to also have roles in nitrogen metabolism. This dissertation examines plant hemoglobin structure and function in two distinct classes: oxygen transport hemoglobins and what we believe to be hemoglobins that function as dissimilatory nitrite reductases. The capacity for oxygen transport arose twice independently in two distinct phylogenetic classes of plant hemoglobins. From Class 2 hemoglobins arose the leghemoglobins common in many species of legumes including soybeans. From Class 1 hemoglobins arose an individual oxygen transport hemoglobin in the species Parasponia andersonii (ParaHb). ParaHb and leghemoglobins have convergently evolved the clear physical properties supporting oxygen transport. Hemogobin from a closely related species, Trema tomentosa, is not an oxygen transporter, in spite of \u3e 90% sequence identity to one another. The first part of this dissertation examines how such a small number of amino acid substitutions could result in the pronounced physical differences conferring a change in physiological function. The second part of the dissertation presents evidence establishing dissimilatory nitrogen reduction as a physiological function for Class 1 plant hemoglobins. These proteins are able to efficiently reduce nitrite and hydroxylamine in vitro, in contrast to most other hemoglobins. It is shown that the unique structure of Class 1 plant hemoglobins facilitates catalytic reduction of nitrite and hydroxylamine by providing a ligand binding site and enhancing intermolecular electron transfer in support of multi-electron reduction reactions

Studies on biosynthesis and activity of antibiotics thiomarinol from marine bacteria

Mupirocin (Pseudomonic acid A) has long been used against Methicillin Resistant Staphylococcus aureus MRSA, yet bacteria have developed resistance, threatening future use. Structurally similar to mupirocin is thiomarinol A, a natural compound produced by the marine bacterium Pseudoalteromonas spp, which possesses stronger antibacterial activities. However, it differs from mupirocin by four distinct differences and among these are extra 4-hydroxylation and joining to pyrrothine. Studying these differences should enhance our understanding of the molecular assembly and biosynthesis machinery. Complementation and mutagenesis studies identified the tmuB gene to be responsible for the 4-hydroxylation as a final tailoring step. In vivo and in vitro studies on purified TmuB revealed that it can hydroxylate diverse pseudomonic acids but is inhibited by molecules with an 8-hydroxyl group, which primarily affects catalysis rather than binding. Molecular modelling plus docking and mutagenesis provides increased understanding of both TmuB potential to modify other substrates and how mupirocin activity can be modulated by 4-hydroxylation. This study also expressed holA, purified its gene product, a non-ribosomal polypeptide synthetase (NRPS), and assayed its activity by pyrophosphate release. It presents a proposed pathway for pyrrothine biosynthesis catalysed by HolA, which exhibits the unusual ability to join two cysteine molecules by a single NRPS module