Copper-containing nitrite reductase (NiR) is a homotrimer of two cupredoxin domains and catalyzes the single electron reduction of NO2- to NO during dissimilatory denitrification. To investigate the evolution of NiR, methods of mutagenic library generation and high-throughput variant screening from E. coli colonies were developed. These methods allow for facile screening of 105 mutants for folding efficiency or substrate specificity. Initial proof of principle studies yielded several variants that oxidized the artificial substrate ο-dianisidine up to 8 times faster than wild type NiR, suggesting that this methodology has the potential to engineer NiR to acquire other reductase functions.
A crystal structure was solved for a putative multicopper oxidase (MCO) and NiR homologue from Arthrobacter sp. (AMMCO) to 1.8 Å resolution. The overall folds of AMMCO and NiR are very similar (r.m.s.d. of 2.0 Å over 250 Cα atoms); Like NiR, AMMCO is a trimer with type-1 Cu sites in the N-terminal domain of each monomer; however, the active site of AMMCO contains trinuclear Cu site characteristic of MCOs instead of a the mononuclear type-2 Cu site found in NiR. Detailed structural analysis supports the theory that two-domain MCOs similar to AMMCO were intermediaries in the evolution of NiR and the more common three-domain MCOs. The physiological function of AMMCO remains uncertain, but genomic, crystallographic and functional analysis suggests that the enzyme is involved in metal regulation.
Considering the extensive similarity between AMMCO and NiR, particularly at the active site, engineering a trinuclear cluster into NiR appears feasible with a modest number of alterations to the polypeptide chain. With the aid of my newly developed high-throughput screening technique and site-directed mutagenesis, the mononuclear NiR active site was remodelled into a trinuclear Cu site similar to that of MCO. A crystal structure of this variant was solved to 2.0 Å and the presence of three copper atoms at the engineered cluster was confirmed by Cu-edge anomalous diffraction data. Although the trinuclear copper cluster is present and catalyzes the reduction of oxygen, achieving rates of catalysis seen in native MCOs has proven more difficult. With the framework provided, further engineering NiR into a robust MCO is likely to provide further insights into the structural basis of oxygen reduction by trinuclear copper sites.
| Identifer | oai:union.ndltd.org:LACETR/oai:collectionscanada.gc.ca:BVAU.2429/259 |
| Date | 05 1900 |
| Creators | MacPherson, Iain |
| Publisher | University of British Columbia |
| Source Sets | Library and Archives Canada ETDs Repository / Centre d'archives des thèses électroniques de Bibliothèque et Archives Canada |
| Language | English |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
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