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Title: Anaerobic degradation of long chain phenylalkane carboxylates by the phototrophic bacterium Rhodopseudomonas palustris
Author: Akbar, Abrar
ISNI:       0000 0004 5991 742X
Awarding Body: University of Sheffield
Current Institution: University of Sheffield
Date of Award: 2016
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Anaerobic bacteria typically degrade many types of aromatic monomers through conversion to benzoyl-CoA which is further degraded by ring reduction, ring cleavage and β-oxidation reactions. Rhodopseudomonas palustris has the ability to grow on different side-chain length aromatic compounds ranging from 3-phenylpropanoic acid to 8-phenyloctanoic acid. A β-oxidation or non β-oxidation metabolic pathway could be responsible for degradation of these compounds. To study the pathways and genes involved, a full cellular proteome analysis using mass spectrometry was carried out and the expression patterns for selected genes were measured by qRT-PCR. Two putative feruloyl-CoA synthetases, Fcs1 and Fcs2, in addition to a long chain fatty acid CoA-ligase (RPA1766) were overexpressed in E. coli as recombinant proteins and the purified enzymes were able to catalyse the formation of CoA thioesters of phenylcarboxylates with different chain lengths. The kinetic properties of Fcs1 enzyme showed a high kcat/Km value for 7-phenyl heptanoic acid. Activity was also recorded with 5-phenyl valeric acid, 6- phenyl hexanoic acid and 8- phenyl octanoic acid, but with lower kcat/Km values, while in contrast Fcs2 only utilised 3-phenyl propionic acid and the related hydroxycinnamic acid. RPA1766 was able to utilize only 8-phenyloctanoic acid with low affinity. These data along with protein structure modelling support the idea that Fcs1 catalyses the first step in a β-oxidation pathway for long chain phenylalkane carboxylates in R. palustris. However, other CoA ligases can contribute, as mutants lacking Fcs1 and Fcs2 genes were able to grow on long chain phenylalkane carboxylic acids. Evidence was obtained that the solute binding proteins of an ABC-transporter previously associated with pimelic acid and long chain fatty acid transportation (RPA3718-RPA3725) could bind certain phenylalkane carboxylates with a Kd value of 1.29 µM for RPA3723 with 7-phenylheptanoic acid.
Supervisor: Kelly, David Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available