Exploring the Evolution and Function of the first Enzyme in Histidine Biosynthesis
Authors:   , Khajeaian Parastoo 
Published: May 2022

<p><b>Adenosine triphosphate phosphoribosyl transferase (ATP-PRT) catalyses the first committed step of the histidine biosynthetic pathway in archaea, bacteria, fungi, and plants. The enzyme is allosterically inhibited by histidine, as a means of controlling the pathway in response to metabolic demand for this essential amino acid. Two molecular architectures including a homo-hexameric long form (HisGL) and a hetero-octameric short form (HisGs) have been described for ATP-PRT. A single chain of HisGL comprises domains I and II that house the active site while domain III, or the regulatory domain provides the binding site for histidine as an allosteric inhibitor. HisGS shares a highly similar catalytic core to HisGL, but is associated with a second protein, HisZ, to form its active, regulated complex. Recently, a possible third architecture, the “super-long” form, has been revealed. Resembling the non-covalent hetero-octameric complex of the short form, this protein has been studied from Leuconostoc mesenteroides. It appears likely that this protein has evolved via the fusion of the hisG and hisZ genes encoding the short form complex. This thesis characterises a new member of the ATP-PRT short form from the hyperthermophile Aquifex aeolicus (AaeHisZGS), the super-long form from Leuconostoc mesenteroides (LmeHisZGSL) and explores the functional and evolutionary relationship between the long and short form enzymes.</b></p>
<p>Chapter 2 reports the characteristics of the hyperthermophilic AaeHisZGS complex. HisZ regulatory domains of short form ATP-PRTs and histidyl-tRNA synthetases (HisRS) are derived from the same ancestral gene. Specifically, the regulatory AaeHisZ domain of AaeHisZGS shows high similarity to AaeHisRS due to the presence of the C-terminal anti-codon binding domain, not found for all HisZ sequences. The role of this C-terminal anti-codon binding domain in ligand binding is discussed and a comparison between the wild-type enzyme and the truncated AaeHisZ-Y303term is presented. Kinetic studies confirmed that ADP can act as an alternative substrate equivalent to ATP for both AaeHisZGS wild-type and AaeHisZ-Y303term complexes. Furthermore, the truncation of the C-terminal domain of AaeHisZ caused a change in the histidine inhibition characteristics. Firstly, a two-fold increase in the KiHis of the AaeHisZG-Y303term complex was noted, closely matching the value reported for the mesophilic short form HisZGS from Lactococcus lactis, which naturally lacks the C-terminal HisZ domain. Secondly, changes in the thermodynamic behaviour of the complexes upon histidine binding were observed by isothermal titration calorimetry (ITC), which surprisingly suggests a significant involvement of the AaeHisZ C-terminal domain in the inhibitor binding process.</p>
<p>Chapter 3 describes the investigation of the thermophilic short form enzyme from Thermotoga maritima (TmaHisZGS) as well as the modularity of short form ATP-PRT complexes. Several mix-and-match combinations of HisG and HisZ proteins from different sources were assessed for complex formation and functionality. Additionally, the successful design and construction of a chimeric protein — by covalent fusion of the regulatory domain of the long form enzyme to the C-terminus of a short form enzyme — was carried out to probe the ability of the regulatory domain to confer both feedback regulation and enzyme function into a contemporary short form HisGS.</p>
<p>Chapter 4 reports the characterisation of the novel ATP-PRT structure, the super-long LmeHisZGSL, that combines HisZ and HisGS in a single open reading frame. Several attempts were made to achieve a functional LmeHisZGSL enzyme with ATP-PRT activity, with no success. Also, the covalently linked LmeHisZGSL was split into two separate units LmeHisG and LmeHisZ. The non-covalent complex between the two subunits of the enzyme was purified successfully but the complex was still devoid of ATP-PRT activity.</p>
<p>Chapter 5 describes studies undertaken to give insight into the evolution of the ATP-PRT family using directed evolution methods such as error-prone PCR (epPCR). The generation of gene variant libraries for four different inactive/marginally active ATP-PRT constructs using epPCR is detailed, with a heavy focus on introducing mutations in an unbiased fashion, while achieving a reasonable number of mutated copies in each library. To identify and select variants which attained or improved activity, an E. coli ΔhisG knockout strain, auxotrophic to histidine, was used. A mutant (mutant 50) was successfully identified and isolated from the chimeric LlaHisGS-McubACTChimera random mutation library, displaying three substitutions and exhibiting measurable catalytic activity and histidine sensitivity.</p>