Mechanistic studies of the pyridoxal 5'- phosphate-dependent enzyme serine palmitoyltransferase; substrates, cofactor and inhibitors.

Sphingolipids (SL) are essential structural components of membranes found&#13;\nin all eukaryotes and have also been identified in some bacteria. &#13;\n&#13;\nThe first step of the&#13;\nSL biosynthetic pathway across all species is catalysed by serine&#13;\npalmitoyltransferase (SPT), a member of the alpha-oxoamine synthase (AOS) family&#13;\nof pyridoxal 5’- phosphate (PLP)-dependent enzymes. AOS enzymes are involved in&#13;\nthe biosynthesis of a range of important natural products such as heme, vitamins and&#13;\nantibiotics where they catalyse the reaction between amino acid and acyl-thioester&#13;\nsubstrates. Substrate specificity across the family is of great importance, as human&#13;\nmutant SPTs shift the substrate specificity from L-serine to glycine or L-alanine that&#13;\nlead to production of deoxy-sphingolipids that are toxic to mammalian cells. PLP, a&#13;\nform of vitamin B6, is one of nature’s most versatile catalysts and is involved in over&#13;\n160 enzymes that carry out diverse reactions on amine-containing substrates. This&#13;\nwork probes the functional role of the phosphate group of PLP, usually housed in a&#13;\nphosphate binding cup (PBC) and investigates the need for a novel and unexpected&#13;\nH-bond between the hydroxyl group of the L-serine substrate and the 5’-phosphate&#13;\ngroup of PLP in SPT. In this study, the PLP cofactor was removed from SPT with&#13;\namino-thiol substrates which act as mechanism-based inhibitors of SPT via&#13;\nproduction of a thiazolidine adduct. Replacement of natural PLP with the&#13;\ndephosphorylated form of the cofactor, pyridoxal, allowed a study on the importance&#13;\nof the PLP phosphate:L-serine H-bond on substrate specificity and optimal SPT&#13;\nactivity. Furthermore, analysis of the phosphate binding cup of the&#13;\nALAS:PLP:glycine external aldimine, a related AOS family member; revealed an&#13;\nimportant residue that could possibly be involved in determining substrate specificity&#13;\nof different members of the AOS family. PBC analysis also expanded, with a&#13;\ndetailed and interesting study of a novel SPT:PLP:myriocin inhibitor complex.&#13;\n&#13;\nHuman SPT is a heterodimeric, membrane-bound enzyme composed of two&#13;\nsubunits (hLCB1/hLCB2) which is thought to contain a single PLP-containing active&#13;\nsite. Mutations in human hLCB1 have been linked to the rare sphingolipid metabolic&#13;\ndisease hereditary sensory neuropathy I (HSAN1). Recent studies identified three&#13;\nheterozygous missense mutations in the second human SPT subunit hLCB2 that&#13;\nshow a significant loss in SPT activity. The three human SPT mutations V359M,&#13;\nG385V and I504F were mapped onto the bacterial S. paucimobilis SPT as V246M,&#13;\nG268V and G385F. These bacterial SPT mutant mimics reveal that the amino acid&#13;\nchanges have varying impacts; they perturb the PLP cofactor binding, reduce the&#13;\naffinity for both substrates, decrease the enzyme activity, and, in the most severe&#13;\ncase, cause the protein to be expressed in an insoluble form.&#13;\n&#13;\nSPTs and most of the other members of the AOS family utilise an acyl-CoA&#13;\nthioester substrate. In contrast, a sphingolipid-producing bacterium, S. wittichii, is&#13;\nthought to use a small type II acyl carrier protein (ACP) to deliver the acyl chain to&#13;\nits homodimeric SPT target. Converting the unmodified apo-ACP to the activated&#13;\n“substrate” acyl-ACP, has proven difficult and amino acid sequence alignment,&#13;\ncombined with modelling studies revealed an unusual tryptophan residue that could&#13;\nprevent modification to the acyl-ACP form. In this study a double mutant ACP&#13;\nE36G/W37A has been prepared and characterised. Both wild-type and mutant S.&#13;\nwittichii ACP are expressed in the recombinant E. coli host in their inactive apoform.&#13;\n&#13;\nThe transfer of a phosphopantethiene (4’PP) linker by a specific PPTase (also&#13;\nknown as an acyl carrier protein synthase (AcpS)) has been successful in modifying&#13;\nthe mutant form of ACP to its holo-form but could not transfer a palmitoyl group&#13;\n(C16).&#13;\n&#13;\nE.coli ACP has been successfully expressed, purified and characterised in this&#13;\nstudy. For the first time, ion mobility mass spectromerty (IM-MS) has been used on&#13;\nthis protein to gain structural insight into the different forms of ACP. Collisional&#13;\ncross section (CCS) distributions have been calculated for different acylated states of&#13;\nthe ACP concluding that the protein exists in equilibrium between two states: a&#13;\ncompact and an extended conformation.

Topic: Enzyme Structure and Function
Subfield: Materials Chemistry
Field: Materials Science
Domain: Physical Sciences