Structural and biochemical analysis of HutD from Pseudomonas fluorescens SBW25 : a thesis submitted in fulfilment of the requirements for the degree of Master of Science in Molecular Biosciences at Massey University, Auckland, New Zealand
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Date
2009
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Massey University
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Abstract
Pseudomonas fluorescens SBW25 is a gram-negative soil bacterium capable of
growing on histidine as the sole source of carbon and nitrogen. Expression of histidine
utilization (hut) genes is controlled by the HutC repressor with urocanate, the first
intermediate of the histidine degradation pathway, as the direct inducer. Recent
genome sequencing of P. fluorescens SBW25 revealed the presence of hutD in the hut
locus, which encodes a highly conserved hypothetical protein. Previous genetic
analysis showed that hutD is involved in hut regulation, in such a way that it prevents
overproduction of the hut enzymes. Deletion of hutD resulted in a slow growth
phenotype in minimal medium with histidine as the sole carbon and nitrogen source.
While the genetic evidence supporting a role of hutD in hut regulation is strong,
nothing is known of the mechanism of HutD action.
Here I have cloned and expressed the P. fluorescens SBW25 hutD in E. coli. Purified
HutD was subjected to chemical and structural analysis. Analytic size-exclusion
chromatography indicated that HutD forms a dimer in the elution buffer. The crystal
structure of HutD was solved at 1.80 Å (R = 19.3% and Rfree = 22.3%) by using
molecular replacement based on HutD from P. aeruginosa PAO1. P. fluorescens
SBW25 HutD has two molecules in an asymmetric unit and each monomer consists of
one subdomain and two ß-barrel domains. Comparative structural analysis revealed a
conserved binding pocket. The interaction of formate with a highly conserved residue
Arg61 via salt-bridges in the pocket suggests HutD binds to small molecules with
carboxylic group(s) such as histidine, urocanate or formyl-glutamate.
The hypothesis that HutD functions via binding to urocanate, the hut inducer, was
tested. Experiments using a thermal shift assay and isothermal titration calorimetry
(ITC) analysis suggested that HutD binds to urocanate but not to histidine. However,
the signal of HutD-urocanate binding was very weak and detected only at high
urocanate concentration (53.23 mM), which is not physiologically relevant. The
current data thus does not support the hypothesis of HutD-urocanate binding in vivo.
Although the HutD-urocanate binding was not confirmed, this work has laid a solid
foundation for further testing of the many alternative hypotheses regarding HutD
function.
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Keywords
Soil bacteria, Histidine utilization, Urocanate