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Title: Biophysical analysis and nuclear magnetic resonance spectroscopy of CobR and vitamin B12 pathway intermediates
Author: Taylor , Samantha Louise
Awarding Body: University of Kent
Current Institution: University of Kent
Date of Award: 2012
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Abstract:
Vitamin B 12 (cobalamin) is of key medical and commercial importance. It is one of the eight B vitamins that are only produced by prokaryotes, but required in the diet by humans. It primarily affects DNA synthesis and regulation, but is also involved in fany acid synthesis and energy production. Deficiencies in cobalamin can lead to anaemia, sensory or motor deficiencies, fatigue, depression, dementia and other psychiatric problems. Commercially over 10 tonnes of cobalamin are produced every year, therefore understanding the pathway to make the process cost efficient is of significant importance. The de novo synthesis of cobalamin can proceed via two different routes, the aerobic and anaerobic pathway. and requires approximately thirty enzyme mediated steps. The aerobic pathway inserts the cobalt ion into the tetrapyrrole framework late in the pathway and CobR, the cobalt reductase, reduces cobalt to form a tighter complex within the molecule. CobR from Brucella melitensis was previously shown by NMR to be thermostable for two weeks at temperatures exceeding 60°C. Biophysical characterisation and NMR analysis were used to determine whether enhanced thermostability in CobR was required for function. A comparison of Brucella melitensis CobR with a homologue from Sinorhizobium melilol; as well as creating seven single point mutants of Brucella melitensis CobR provided important standpoints to test whether CobR thermostability underpinned function. The production of the Brucella melitensis CobR mutants revealed salt bridges and beta barrel components as important for enzyme stability. However the largest contributing factor was availability and binding of the cofactor, FAD. It was shown by differential scanning calorimetry there was an increased thermal stability when increasing the concentration of excess FAD. If the cofactor is stripped from the enzyme completely the protein aggregates, and it was also shown by production of a double mutant, that lowering the binding constant of the cofactor reduced the thennostability that produced a coordinated loss of catalytic activity that was only 10% of the wild type enzyme. The study of the pathway intermediates of tetrapyrrole biosynthesis have often proved challenging for several reasons including the complexity of synthesis, compound instability and the relatively low levels of each intermediate produced. The second focus of this thesis utilises a new method, devised in the Warren laboratory at Kent, that uses pathway enzymes as 'traps' to enable isolation, stabilization and analysis of these intennediates for the first time. The 'trap' method allows pathway intermediates to be produced that are stable for NMR analysis. Precorrin 7 (PC-7), precorrin-8 (PC·8), and hydrogenobyrinic acid (HBA), were produced without isotopic enrichment and characterised by NMR experiments that were acquired on a Broker AV3 using a QCI·F cryoprobe at 14.1 Tesla (600 MHz lH). Analysis of the data confirmed the seventh methylation position of PC-7 was at position CIS on the tetrapyrrole framework as well as highlighting major and minor forms that exist due to tautomeric interconversion. Assignment of PC·8 and HBA was also successfully completed. An additional study was completed to confirm binding of PC·6B (precorrin.6B). PC·7, PC·8 and HBA to Brucella melitensis CobE, a protein believed to playa crucial scavenging role in the vitamin Bu aerobic pathway.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.591917  DOI: Not available
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