Structural studies on histidine metabolism in microorganisms

Lohkamp, Bernhard (2003) Structural studies on histidine metabolism in microorganisms. PhD thesis, University of Glasgow.

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Printed Thesis Information: https://eleanor.lib.gla.ac.uk/record=b2180551

Abstract

Histidine is an important amino acid involved in enzyme catalysis and metal binding in many proteins. Bacteria, fungi and plants synthesise histidine via the 10 step histidine pathway, whereas histidine is an essential amino acid required in the diet of animals. The synthesis of histidine is energetically expensive with about 41 ATP needed to produce one histidine. As a result the histidine biosynthetic pathway is tightly regulated and some microorganisms can consume excess histidine as a nitrogen and carbon source via a histidine utilisation (hut) pathway. The absence of a histidine biosynthetic pathway in mammals makes it a good target for the development of antimicrobial and antifungal drugs as well as herbicides. In addition to an overall regulation of the histidine synthesis by charged histidyl-tRNA and histidyl-tKNA synthetase, the histidine pathway is regulated in various ways by the first enzyme of the pathway ATP-phosphoribosyltransferase (HisG). The enzyme catalyses the condensation of ATP and phosphoribosylpyrophosphate (PRPP) to form phosphoribosyl-ATP (PR-ATP) and is feedback regulated by histidine, the end product of the pathway. Furthermore, HisG is inhibited by its product PR-ATP and also by AMP and ppGpp, reflecting the overall energy status of the cell. Inhibition of HisG coincides with a change in aggregation state, in which the active dimers form inactive hexamers. Some bacterial genomes contain a hisG gene, which lacks about 80 residues at the C-terminus, but have an additional gene, hisZ. This short form of HisG itself is inactive and forms an active complex with HisZ. This thesis describes the solution of the 3-dimensional structures of the hexameric forms of bacterial HisGs by X-ray crystallography. The structure of the Escherichia coli HisG was determined in the presence of the inhibitors AMP and PR- ATP. Additionally, a histidine-bound structure of HisG from the thermophilic bacterium Methanobacteriim thermoautotrophicum has been obtained. The monomer of HisG comprises of three a/p domains. The first two domains show a periplasmic protein-like fold with the active site located in a cleft between the two domains. The C-terminal domain, similar to Pn signal transduction proteins, binds histidine and is mainly involved in the formation of the hexamers. Additionally the structure of HisG represents a new type of phosphoribosyltransferase. The structure of the binary complex with AMP has allowed the identification of the PRPP binding site and explains the competitive inhibition by AMP with respect to both substrates. Furthermore the AMP binary structure and the PR-ATP-complexed structure identified the binding site of the ATP purine ring. The histidine-bound structure explains the histidine inhibition by disruption of the PRPP binding site on the dimer interface by elongation of the dimer and hence the hexanier. A mode of inhibition common to all the inhibitors investigated is the formation of hexamers upon inhibitor binding which buries the active sites in the inside of the hexamer and makes the sites less accessible to the substrates. Mutational and kinetic analysis showed that the extra C-terminal domain in the long form of HisG is not essential for activity. Subsequently, it was demonstrated that the naturally short form of HisG can be activated by thermal energy. This suggests a tense conformation for the short form of the enzyme which can be relaxed and activated by forming an oligomeric complex with HisZ. The structure of an unidentified protein in the histidine utilisation (hut) pathway from Pseudomonas aeruginosa, PAS104, was determined to better than 1 A resolution. Solution of the structure was accomplished by a unique approach combining SIRAS phasing with direct methods (Sayre equation). PAS104 is an all-beta protein with a bicupin fold. One of the P-barrels is open and accommodates a small molecule binding site. A different conformation of PAS 104 was obtained from a second structure solved using a different crystal form. Comparison of the structures and interactions in the active site identifies glutamate or derivatives (intermediates in the hut pathway) as possible substrates, which can induce a conformational change and may give PAS104 a regulatory function.

Item Type: Thesis (PhD)
Qualification Level: Doctoral
Keywords: Microbiology, physiology.
Subjects: Q Science > QP Physiology
Q Science > QR Microbiology
Colleges/Schools: College of Medical Veterinary and Life Sciences
Supervisor's Name: Coggins, Prof. John and Lapthorn, Dr. Adrian
Date of Award: 2003
Depositing User: Mrs Marie Cairney
Unique ID: glathesis:2003-9065
Copyright: Copyright of this thesis is held by the author.
Date Deposited: 01 Apr 2019 09:55
Last Modified: 01 Apr 2019 09:55
URI: http://theses.gla.ac.uk/id/eprint/9065

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