Structure, function and allostery of dihydrodipicolinate synthase from a psychrophilic bacterium
AffiliationBiochemistry and Molecular Biology
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Document TypePhD thesis
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© 2014 Dr. Jacinta Mary Wubben
Dihydrodipicolinate synthase (DHDPS) (EC 126.96.36.199) is the enzyme that catalyses the first step of the lysine biosynthesis pathway, namely the conversion of pyruvate and L-aspartate-β-semialdehyde to 4-hydroxy-2,3,4,5-tetrahydrodipicolinate. This enzyme has two key properties that are of considerable interest to scientists, which will be further explored within this thesis. Firstly, DHDPS has been shown to adopt different quaternary structures and architectures from different organisms, making it a model enzyme for exploring the role of oligomerisation in regards to protein stability. However, while DHDPS has been studied extensively from mesophilic and thermophilic species, it has yet to be characterised from a psychrophilic species. Hence, the first overall aim of this thesis was to explore the role of oligomerisation across the entire psychrophilic to thermophilic temperature range (which has not yet been studied to date) by characterising DHDPS from a psychrophilic species, namely from Shewanella benthica (Sb-DHDPS). Sb-DHDPS was resultantly cloned, expressed and purified to homogeneity. Analytical ultracentrifugation and small angle X-ray scattering studies determined that Sb-DHDPS was in a monomer-dimer equilibrium in solution, with a dimer dissociation constant (KD21) of 562 ± 46 nM. This KD21 was reduced 17-fold in comparison to the KD21 of DHDPS from the mesophilic methicillin-resistant Staphylococcus aureus (MRSA - Burgess et al., 2008). The difference between KD21 values in psychrophilic and mesophilic enzymes indicated a severe reduction in the quaternary structure affinity as a result of environmental temperature. Additionally, the crystal structure of Sb-DHDPS was determined. The number and type of contacts at the dimer interface were similar to the equivalent interface of known tetrameric DHDPS structures. In comparison, an increased number of contacts (and the introduction of unique electrostatic contacts) was observed across the MRSA DHDPS dimer interface. Therefore, these studies demonstrate that there is an increase in both the complexity and affinity of quaternary structure as a result of physiological temperature. The role of oligomerisation in protein stability was also explored by molecular dynamics simulations of Sb-DHDPS and MRSA DHDPS, in addition to a dimeric mutant of DHDPS from Escherichia coli that was previously shown to have attenuated function L197Y – Griffin et al., 2008). The active site geometry of the Sb-DHDPS and the L197Y mutant enzymes were comparable to that of the MRSA DHDPS enzyme at psychrophilic temperatures. This said, there were strikingly key contrasts in the active site geometry of both Sb-DHDPS and the L197Y mutant in comparison to MRSA DHDPS at mesophilic temperatures. These simulations therefore demonstrate that the active site geometry of the enzyme has also been optimised at the viable temperatures of the organism from which the enzyme originated. In addition, an increase in the conformational motions was evident across the dimer interface of all DHDPS enzymes studied at temperatures above their physiological norm. Hence the computational studies performed in this thesis further demonstrate that the complexity and affinity of the quaternary structure of the enzyme have been optimised for function at the temperatures required for physiological function, but deviations from these temperatures result in destabilisation of both the active site geometry and overall protein motions. Secondly, a number of DHDPS enzymes are allosterically feedback inhibited by its downstream product, lysine. However, allosteric inhibition varies amongst different species, with DHDPS from plants, Gram-negative bacteria and Gram-postive bacteria showing tight (10-100 μM), weak (0.2-1.0 mM) or no inhibition, respectively. The variation in the potency of lysine inhibition could potentially be explained by the chemical nature of the amino acid residue present at a key position that is known to alter conformation upon lysine binding in E. coli DHDPS, namely position 53. In this thesis, it is therefore hypothesised that the presence of an aromatic or hydrophobic residue at position 53 determines strong allosteric potency; whilst the presence of a histidine results in weak inhibition. The allosteric inhibition of Sb-DHDPS was henceforth investigated, as a hydrophobic isoleucine residue was present at position 53. Consistent with the above hypothesis, Sb-DHDPS binds tightly to lysine (Kd = 4.2 ± 0.59 μM) with a half maximal inhibitiory concentration (IC50) of 69 ± 1.9 μM. Furthermore, the I53 residue in Sb-DHDPS was additionally mutated to either a tryptophan or histidine residue in order to mimic the sequence of DHDPS from plants and E. coli, respectively. The I53W mutation resulted in a subtle, but significant increase in lysine inhibition (IC50 = 50 ± 0.95 μM); whilst the I53H mutation significantly reduced inhibition propensity (IC50 = 120 ± 6.3 μM). To gain insight into the importance of I53, the crystal structure of Sb-DHDPS bound to lysine was determined. The structure showed that lysine bound in the allosteric cleft, anchored by hydrogen bonding to H56 and E84. However, I53 does not change orientation or form hydrogen interactions with the bound lysine molecules. Given that mutations of this residue affect the potency of lysine inhibition, it is consequently proposed that the residue at position 53 has a role in directing lysine in and/or out of the allosteric site. Together, the functional, mutational and structural studies of Sb-DHDPS presented in this thesis demonstrate that the presence of a hydrophobic residue at position 53 is a key determinant of allosteric potency. Through the characterisation of the structure, function and allosteric regulation of Sb-DHDPS, insight was gained into both the allosteric feedback regulation of DHDPS by lysine and the molecular evolution of the quaternary structure of proteins in general. This could potentially lead to an improvement in protein engineering strategies for the improvement of lysine rich crops and the use of psychrophilic enzymes in important industrial processes.
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