Biology subjects

Cliff, M.

Publications and source records attributed to Cliff, M..

2 recordsLinked to original sources

bioRxiv2024

Metal fluorides - multi-functional tools for the study of phosphoryl transfer enzymes

Enzymes facilitating the transfer of phosphate groups constitute the most extensive protein group across all kingdoms of life, making up approximately 10% of the proteins found in the human genome. Understanding the mechanisms by which enzymes catalyse these reactions is essential in characterising the processes they regulate. Metal fluorides can be used as multifunctional tools for the study of these enzymes. These ionic species bear the same charge as phosphate and the transferring phosphoryl group and, in addition, allow the enzyme to be trapped in catalytically important states with spectroscopically sensitive atoms interacting directly with active site residues. The ionic nature of these phosphate surrogates also allows their removal and replacement with other analogues. Here, we describe the best practices to obtain these complexes, their use in NMR, X-ray crystallography, cryoEM and SAXS and describe a new metal fluoride, scandium tetrafluoride, which has significant anomalous signal with soft X-rays. Highlights Enzymes that catalyse phosphoryl transfer are the largest family of enzymes and are involved in the storage and transmission of genetic information, energy transfer, signalling and cellular differentiation Metal fluorides form a comprehensive tool kit to study the mechanisms of these enzymes by stabilizing the active conformation; mimicking both the transition state and ground state; and placing spectroscopically sensitive atoms into the active site A guide is presented to the optimal formation of these complexes and their use in a wide variety of techniques in structural biology

molecular biology↗

bioRxiv2021

Spinning sugars in antigen biosynthesis: a direct study of the Coxiella burnetii and Streptomyces griseus TDP-sugar epimerases

The sugars streptose and dihydrohydroxystreptose (DHHS) are unique to the bacteria Streptomyces griseus and Coxiella burnetii respectively. Streptose forms the central moiety of the antibiotic streptomycin, whilst DHHS is found in the O-antigen of the zoonotic pathogen C. burnetii. Biosynthesis of these sugars has been proposed to follow a similar path to that of TDP-rhamnose, catalysed by the enzymes RmlA/RmlB/RmlC/RmlD. Streptose and DHHS biosynthesis unusually require a ring contraction step that might be performed by the orthologues of RmlC or RmlD. Genome sequencing of S. griseus and C. burnetii proposed the StrM and CBU1838 proteins respectively as RmlC orthologues. Here, we demonstrate through both coupled and direct observation studies that both enzymes can perform the RmlC 3,5 double epimerisation activity; and that this activity supports TDP-rhamnose biosynthesis in vivo. We demonstrate that proton exchange is faster at the 3 position than the 5 position, in contrast to a previously studied orthologue. We solved the crystal structures of CBU1838 and StrM in complex with TDP and show that they form an active site highly similar to previously characterised enzymes. These results further support the hypothesis that streptose and DHHS are biosynthesised using the TDP pathway and are consistent with the ring contraction step being performed on a double epimerised substrate, most likely by the RmlD paralogue. This work will support the determination of the full pathways for streptose and DHHS biosynthesis.

biochemistry↗