Gotowa bibliografia na temat „SAICAR Synthetase”

The bifunctional human enzyme phosphoribosylaminoimidazole carboxylase and phosphoribosylaminoimidazolesuccinocarboxamide synthetase (PAICS) catalyzes two essential steps in the de novo purine biosynthesis pathway. PAICS is overexpressed in many cancers and could be a promising target for the development of cancer therapeutics. Here, using gene knockdowns and clonogenic survival and cell viability assays, we demonstrate that PAICS is required for growth and survival of prostate cancer cells. PAICS catalyzes the carboxylation of aminoimidazole ribonucleotide (AIR) and the subsequent conversion of carboxyaminoimidazole ribonucleotide (CAIR) and l-aspartate to N-succinylcarboxamide-5-aminoimidazole ribonucleotide (SAICAR). Of note, we present the first structures of human octameric PAICS in complexes with native ligands. In particular, we report the structure of PAICS with CAIR bound in the active sites of both domains and SAICAR bound in one of the SAICAR synthetase domains. Moreover, we report the PAICS structure with SAICAR and an ATP analog occupying the SAICAR synthetase active site. These structures provide insight into substrate and product binding and the architecture of the active sites, disclosing important structural information for rational design of PAICS inhibitors as potential anticancer drugs.

Streptococcus pneumoniaeis a multidrug-resistant pathogen that is a target of considerable interest for antibacterial drug development. One strategy for drug discovery is to inhibit an essential metabolic enzyme. The seventh step of thede novopurine-biosynthesis pathway converts carboxyaminoimidazoleribonucleotide (CAIR) and L-aspartic acid (Asp) to 4-(N-succino)-5-aminoimidazole-4-carboxamide ribonucleotide (SAICAR) in the presence of adenosine 5′-triphosphate (ATP) using the enzyme PurC. PurC has been shown to be conditionally essential for bacterial replication. Two crystal structures of this essential enzyme fromStreptococcus pneumoniae(spPurC) in the presence of adenosine 5′-diphosphate (ADP), Mg2+, aminoimidazoleribonucleotide (AIR) and/or Asp have been obtained. This is the first structural study ofspPurC, as well as the first of PurC from any species with Asp in the active site. Based on these findings, two model structures are proposed for the active site with all of the essential ligands (ATP, Mg2+, Asp and CAIR) present, and a relay mechanism for the formation of the product SAICAR is suggested.

Coformycin and pentostatin are structurally related N-nucleoside inhibitors of adenosine deaminase characterized by an unusual 1,3-diazepine nucleobase. Herein, the cof gene cluster responsible for coformycin biosynthesis is identified. Reconstitution of the coformycin biosynthetic pathway in vitro demonstrates that it overlaps significantly with the early stages of l-histidine biosynthesis. Committed entry into the coformycin pathway takes place via conversion of a shared branch point intermediate to 8-ketocoformycin-5′-monophosphate catalyzed by CofB, which is a homolog of succinylaminoimidazolecarboxamide ribotide (SAICAR) synthetase. This reaction appears to proceed via a Dieckmann cyclization and a retro-aldol elimination, releasing ammonia and D-erythronate-4-phosphate as coproducts. Completion of coformycin biosynthesis involves reduction and dephosphorylation of the CofB product, with the former reaction being catalyzed by the NADPH-dependent dehydrogenase CofA. CofB also shows activation by adenosine triphosphate (ATP) despite the reaction requiring neither a phosphorylated nor an adenylated intermediate. This may serve to help regulate metabolic partitioning between the l-histidine and coformycin pathways.

Understanding the structural details of a biological macromolecule is highly essential for a clear interpretation of its functional roles. Structural biology evolved over a period of time has been able to explain various cellular processes which has consequently lead to the discovery of several effective drugs. X-ray crystallography is one of the powerful tools in structural biology, contributing to ∼88% of the three-dimensional structures deposited in the Protein Data Bank (PDB). A single experiment can however provide only one of the numerous conformational states a macromolecule can exhibit. This setback is overcome by classical mechanics based molecular dynamics simulation technique developed based on various force fields that can generate many possible conformational states a macromolecule can take up. This thesis describes the use of both X-ray crystallography and molecular dynamics simulation techniques to elucidate the structural and dynamic aspects of a thermostable enzyme from a hyperthermophilic organism focusing on its thermostability and catalytic mechanism. Nucleotides have significantly numerous roles in the cell, forming the basis of an organ-ism’s genome, providing cellular energy, assisting as coenzymes in many enzyme catalyzed reactions, acting as secondary messengers etc. These biologically important chemical entities are classified into purines and pyrimidines. Biochemical synthesis of either purine or pyrimidine nucleotides are accomplished either by de novo or salvage pathway. SAICAR synthetase is one of the enzymes involved in de novo purine biosynthesis pathway. It catalyzes the seventh (in humans and higher eukaryotes) or eighth step (in bacteria and fungi) of de novo purine biosynthesis pathway. The enzyme is an ATP dependent ligase which forms C-N bond between 5-Amino-1-(5-phospho-D-ribosyl)imidazole-4-carboxylate (CAIR) and L-aspartate (ASP), in the presence of magnesium, resulting in the formation of 5-Amino-4-imidazole-N-succinocarboxamide ribonucleotide (SAICAR). An overview of the structural and biochemical aspects of SAICAR synthetase along with an introduction to the structural and dynamic basis of protein thermostability is described in chapter-1. The work described in this thesis starts with the clone of SAICAR synthetase. A series of procedures involving protein expression and its subsequent purification results in a pure protein which is crystallized by underoil microbatch crystallization method using appropriate conditions. X-ray diffraction data of the protein crystals are collected using Cu-Kα radiation (1.5417 Å) as a source on MAR Research image plate detectors. Diffraction data are processed using IMOSFLM, scaled using SCALA. Molecular replacement method is used for solving the structures using PHASER. Refinement of the structures are performed using REFMAC. Model building of the structures are carried out using COOT. Structures are validated using RCSB validation tools. Simulations of the structures are carried out using a popular open source program, GROMACS. A detailed account of all the programs and tools used to carry out the work is described in chapter-2. Chapter-3 elaborates on the structure of the enzyme SAICAR synthetase from Pyro-coccus horikoshii along with the details on its thermal stability in comparison with the structures from other sources. Crystals in two space groups - H3 (Type-1) and C2221 (Type-2) are obtained. These are the first apo structures of SAICAR synthetase from a hyperthermophilic archaea reported. Some of the structural deviations between two forms are due to the presence of Cd2+ ion used in the crystallization condition. Structurally they closely resembled the enzyme from other organisms. Probing the amino acid compo-sitions of mesophilic, thermophilic and hyperthermophilic SAICAR synthetase structures reveal the prevalence of certain amino acids in each temperature class. Certain type of non-bonded interactions are prominent in hyperthermophilic structures. As an extension of structural studies, simulations using molecular dynamics of SAICAR synthetase structures, revealed yet another facet of thermostability of proteins. The observations made from the study are in consensus with the Somero’s corresponding states hypothesis according to which the proteins show similar degrees of flexibility at their respective growth temperatures. Helices are observed to be relatively more susceptible to unfolding among the secondary structures. Increased flexibility of the mesophilic proteins at higher temperatures manifests by forming more number of short lived contacts. Fur-ther, hyperthermophilic proteins tend to reduce the hydrogen bonding interactions with water at higher temperatures compared to the mesophilic proteins. Of many non-bonded interactions in the protein, salt-bridge and hydrophobic interactions play a key role in providing thermostability to the hyperthermophilic proteins. The finer points are presented in chapter-4. Co-crystallization experiments and simulations of the ligand bound SAICAR syn-thetase structures from Pyrococcus horikoshii are elucidated in chapter-5. All eight crystal structures described in this chapter contain an adenine nucleotide. With no major struc-tural deviations detected in the ligand bound structures compared to their apo forms, these structures however confirmed the position of one of the substrates - aspartate. Out of other two substrates binding sites, CAIR site harbors any nucleotide, while, ATP site binds only to an adenosine nucleotide. One of the structures, explains the possibility of a phosphorylation occurring prior to aspartate attack on CAIR. Simulation studies are able to demonstrate the structural roles of the magnesium ions. The stability and antagonism among the substrates are also expounded in the chapter. Few other crystal structures of nucleotide complexes with SAICAR synthetase are described in chapter-6. Six structures provide answers to the mode of inhibition of some of the nucleotides observed in yeast SAICAR synthetase. One of the structures also provides a possible path taken for the entry of the nucleotide into the CAIR site. However, further experiments are being planned to explore the activity of SAICAR synthetase in detail.