Crystallographic studies of folypolyglutamate synthetase and recombinant human lactoferrin : a dissertation submitted in partial fulfilment of the requirements for the degree of Doctor of Philosophy in the Department of Biochemistry, Institute of Molecular Biosciences at Massey University, New Zealand
This thesis is written in two parts. In the first (chapters 1-4), crystallographic studies on the enzyme folylpolyglutamate synthetase from Lactobacillus casei, both in complex with MgATP2- and in its apo form, are presented. In the second part (chapters 5-8), a structural analysis of recombinant diferric human lactoferrin is reported. Folylpolyglutamate synthetase (FPGS) is an ATP-dependent enzyme from eukaryotic and bacterial sources. It catalyzes the addition of glutamate residues to folate to produce folylpolyglutamates which are required for effective intracellular retention of folate and are the preferred substrates for the enzymes of one-carbon metabolism. The crystal structures of L. casei FPGS in both the MgATP2- -bound and apo forms have been determined by the methods of multiple isomorphous replacement and molecular replacement, and refined by restrained least squares method using data to 2.4 Å resolution. The structural analysis of MgATP-FPGS reveals that folylpolyglutamate synthetase is a modular protein consisting of two domains, one with a typical mononucleotide-binding fold and the other strikingly similar to the folate-binding enzyme, dihydrofolate reductase (DHFR). The ATP-binding site is located in a interdomain cleft and a presumed mode of folate-binding has also been suggested for FPGS by analogy to the structure of DHFR. An unexpected structural similarity has been discovered between FPGS and the UDP-N-acetylmuramoyl-L-alanine:D-glutamate ligase (MurD). It is proposed that FPGS and MurD might carry out their biological functions in a very similar way and the structural comparison suggests that a possible domain movement could be involved in the catalytic reaction of FPGS. Two disordered loop regions in the MgATP-FPGS structure are well defined in apo-FPGS, allowing analysis of the interactions between these loops and surrounding structures of the protein. Human lactoferrin (hLf) has considerable potential as a therapeutic agent. Over-expression of hLf in the fungus Aspergillus awamori has resulted in the availability of large quantities of this protein. Here the crystal structure of the recombinant human lactoferrin (rhLf) has been determined by X-ray crystallography at 2.2 Å resolution. Superposition of the rhLf structure on to the native milk hLf shows a very high level of correspondence, and their dynamic properties, as indicated by the B factor distribution, also agree closely. This demonstrates that the structure of the protein is not affected by the mode of expression or the use of strain improvement procedures.