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

Field: Biochemistry, Genetics and Molecular Biology (Life Sciences)

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