Mechanistic and structural insights into the specificity and biological functions of e. Coli had superfamily phosphatase had4/yihx

Zappala, Davide 2023. Mechanistic and structural insights into the specificity and biological functions of e. Coli had superfamily phosphatase had4/yihx. PhD Thesis, Cardiff University. Item availability restricted.

Preview	PDF - Accepted Post-Print Version Download (7MB) | Preview
	PDF (Cardiff University Electronic Publication Form) - Supplemental Material Restricted to Repository staff only Download (149kB)

Abstract

The HAD superfamily has been extensively studied in literature, representing a major class of enzymes which are responsible mainly for phosphoryl transfer in prokaryotes and eukaryotes. The characteristic structural motifs are conserved throughout the whole superfamily and reaction mechanisms for many HAD enzymes have been validated. In this work, the spotlight is focused on YihX, encoded in the E. coli genome. This peculiar case is of interest since this enzyme features an uncommon DxG signature that still enables the catalysis of phosphoryl transfer while lacking key structural and catalytic features needed for the currently known mechanism. The first part will focus on the exploration of the possibility of YihX being part of the sulfoglycolytic pathway, and the D-tyrosine metabolism operon due to its collocation in the genome, located directly downstream of the sulfoglycolysis and upstream of the D-tyrosine metabolism operon. In the second part, new experimental evidence is produced in the form of crystal structures, kinetic studies and mechanistic observations. The only structure present in the PDB shows some substantial flaws. Metal fluorides as transition state analogues led to two new crystal structures, apo conformation and a transition state analogue (TSA) complex of a rarely observed octahedral MgF3(H2O)- entity. The formation of the TSA complex and chemical environment around it is supported by 19F NMR studies. A new reaction mechanism has been proposed for phosphoryl transfer catalysed by HAD phosphatases containing the DXG motif. Serendipitous discovery of a new phosphoryl transfer activity for YihX, ATP synthesis from ADP, showed that it is a dual-activity enzyme. Finally, a new expression system was created and tested for recombinant human Thymidine Kinase 2 to produce high quantities of soluble protein for crystallisation trials to hopefully obtain a long overdue crystal structure to boost the therapeutics field for mitochondrial DNA depletion syndromes.

Item Type:	Thesis (PhD)
Date Type:	Completion
Status:	Unpublished
Schools:	Chemistry
Date of First Compliant Deposit:	30 November 2023
Last Modified:	30 Nov 2024 02:30
URI:	https://orca.cardiff.ac.uk/id/eprint/164460

Actions (repository staff only)

Edit Item