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Enzymes Required for the Biosynthesis of N-Formylated Sugars

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Enzymes Required for the Biosynthesis of N-Formylated Sugars Available online at www.sciencedirect.com ScienceDirect Enzymes required for the biosynthesis of N-formylated sugars 1 1 2 Hazel M Holden , James B Thoden and Michel Gilbert The N-formyltransferases, also known as transformylases, play This region, also referred to as the O-antigen, consists key roles in de novo purine biosynthesis where they catalyze of repeating units, which typically contain three to five the transfer of formyl groups to primary amine acceptors. sugars. The O-antigens are thought to play a role in the 10 These enzymes require N -formyltetrahydrofolate for activity. virulence of a bacterium and also in its ability to evade Due to their biological importance they have been extensively antibacterial agents [3]. investigated for many years, and they are still serving as targets for antifolate drug design. Most of our understanding of the For more than 30 years it has been known that some O- N-formyltransferases has been derived from these previous antigens contain quite unusual deoxysugars. Due to the studies. It is now becoming increasingly apparent, however, increased sensitivities of such techniques as NMR, how- that N-formylation also occurs on some amino sugars found on ever, it is becoming apparent that the O-antigens are far the O-antigens of pathogenic bacteria. This review focuses on more complicated than originally thought. Recent re- recent developments in the biochemical and structural search has demonstrated, for example, that the O-anti- characterization of the sugar N-formyltransferases. gens of some Gram-negative bacteria contain quite Addresses remarkable formylated dideoxysugars including 3-forma- 1 Department of Biochemistry, University of Wisconsin, Madison, WI mido-3,6-dideoxy-D-glucose (Qui3NFo), 3-formamido- 53706, United States 2 3,6-dideoxy-D-galactose (Fuc3NFo), 4-formamido-4,6- Human Health Therapeutics, National Research Council Canada, dideoxy-D-glucose (Qui4NFo), and 4-formyl-D-perosa- Ottawa, Ontario K1A OR6, Canada mine as depicted in Figure 1b [4]. These unusual sugars Corresponding author: Holden, Hazel M have been found on such organisms as Brucella abortus [5], ([email protected]) Salmonella enterica O60 [6], Providencia alcalifaciens O40 [7], Francisella tularensis [8], and Campylobacter jejuni [9 ]. Current Opinion in Structural Biology 2016, 41:1–9 Strikingly, all of the above organisms are extremely pathogenic. B. abortus, for example, is the causative agent This review comes from a themed issue on Catalysis and regulation of brucellosis [10]. S. enterica is a notorious human patho- Edited by David Christianson and Nigel Scrutton gen known to be a leading cause of hospitalizations and deaths due to the consumption of contaminated food [11]. P. alcalifaciens is an opportunistic organism associated http://dx.doi.org/10.1016/j.sbi.2016.04.003 with enteric diseases and was implicated in the 1996 food poisoning outbreak in Fukui, Japan [12]. F. tularensis is 0959-440/# 2016 Elsevier Ltd. All rights reserved. the causative agent of tularemia or ‘rabbit fever,’ and because it can be produced as a highly infectious aerosol, it is classified as a select agent by the Centers of Disease Control in the United States [13]. Finally, C. jejuni is a major cause of gastroenteritis worldwide and, important- Introduction ly, is now considered a triggering agent for the develop- ´ The lipopolysaccharide or LPS is the major structural ment of Guillain–Barre syndrome, a devastating acquired component of the outer membrane of Gram-negative autoimmune peripheral neuropathy leading to severe bacteria where it has been estimated to occupy 75% muscle weakness and in some cases paralysis [14]. of the total surface area [1]. It is a complex glycoconju- gate, which varies from species to species (and within The genes encoding the enzymes required for the bio- species) in specific content, but in all cases, is thought to synthesis of such formylated sugars are typically located provide a permeability barrier to hydrophobic or nega- within clusters. The source of the formyl group is the 10 tively charged molecules. Conceptually, the LPS can be cofactor N -formyltetrahydrofolate (Figure 1c). On the thought of in terms of three specific regions: the lipid A basis of bioinformatics, a pathway for the synthesis of one component, the core oligosaccharide, and the O-specific of these sugars, Qui4Fo, has been proposed as shown in polysaccharide as highlighted in Figure 1a [2]. It is the O- Figure 2 [15]. Like most pathways for the biosynthesis of specific polysaccharide region, which extends farthest unusual sugars, the starting ligand, which in this case is away from the bacterium, that displays the most variation glucose-1-phosphate, is activated by its attachment to a from species to species (and between serotypes of nucleoside monophosphate. The enzyme required for the same species), and it is highly immunogenic [3]. this reaction is a nucleotidylyltransferase. The second www.sciencedirect.com Current Opinion in Structural Biology 2016, 41:1–9 2 Catalysis and regulation Figure 1 (a) O-specific polysaccharideCore polysaccharide Lipid A KDO P P Hep P KDO GlcN Hep Hep KDO GlcN P n P (b) HO O O HO H H HCN HC N OH OH O O OH OH Qui3NFo Fuc3NFo OH O O O O HCN HCN H H HO HO OH OH OH Qui4NFo 4-formyl-D-perosamine (c) O OH O OH O O OH O N OH H 10 O O H N HN N N O N H HN N O 5 H H2N N N O H H2N N N H N5-formyltetrahydrofolate N10-formyltetrahydrofolate Current Opinion in Structural Biology Gram-negative bacteria contain on their outermost surface a complex glycoconjugate referred to as the lipopolysaccharide or LPS. As schematically shown in (a), it is composed of a lipid A molecule, the core polysaccharide, and the O-specific polysaccharide. It is the O-specific polysaccharide or O-antigen that contributes to the wide species variations seen in nature. Quite unusual dideoxysugar sugars are sometimes found in the O-antigens including the formylated sugars depicted in (b). The N-formyltransferases that are involved in the biosynthesis of these 10 5 formylated sugars employ N -formyltetrahydrofolate as the carbon source (c). In many structural analyses the N -formyltetrahydrofolate ligand is used because of its stability, but it is not catalytically competent. Current Opinion in Structural Biology 2016, 41:1–9 www.sciencedirect.com N-formyl sugar biosynthesis Holden, Thoden and Gilbert 3 Figure 2 OH OH O O dTTP PPi H2O O O HO NAD+ HO HO HO HO OH Step1 OH Step 2 OH OPO2– 3 O dTDP O dTDP glucose-1-phosphat e dTDP-D-glucose dTDP-4-keto-6-deoxyglucose L-Glu Step 3 PLP 10 α-ketoglutarate O THF N -formyl-THF O HCN O H H2N HO HO OH Step 4 OH O dTDP O dTDP dTDP-4-formamido-4,6-dideoxy- D-glucose dTDP-4-amino-4,6-dideoxy-D-glucose Current Opinion in Structural Biology A possible biosynthetic pathway for the production of dTDP-4-formamido-4,6-dideoxy-D-glucose is shown. Steps 1, 2, 3, and 4 require dTTP, + 10 + NAD , PLP and L-glutamate, and N -formyltetrahydrofolate, respectively. In step 2, the NAD is transiently reduced to NADH in the first step of the reaction. The hydride from NADH is subsequently transferred to the substrate in a subsequent step. 0 step involves an oxidation of the C-4 carbon and removal negative charge of the LPS is reduced thereby leading 0 + of the C-6 hydroxyl group by an NAD -dependent 4,6- to CAMPS resistance [18]. dehydratase. There is a subsequent amination of the 0 sugar via a pyridoxal 5 -phosphate (PLP) dependent A key enzyme involved in the production of 4-amino-4- aminotransferase. The final step is the N-formylation of deoxy-L-arabinose (L-Ara4N) is ArnA [19]. It is a bifunc- 0 10 the C-4 amino moiety by an enzyme requiring N - tional enzyme with its N-terminal domain functioning as formyltetrahydrofolate for activity. Although the exis- an N-formyltransferase and its C-terminal domain cata- tence of N-formylated sugars was first reported in lyzing an oxidative decarboxylation reaction. Formylation 1985 [16], it is only within the last several years that of the sugar is thought to be an obligatory step in the the overall architectures of the sugar N-formyltransferases ultimate production of L-Ara4N-modified lipid A [19]. have been defined in detail. This review focuses on our current understanding of the structures and functions of In 2005 the structure of the N-terminal domain of ArnA these intriguing enzymes. was reported by two independent research groups [20 ,21]. These initial models defined the overall archi- tecture of the N-formyltransferase domain of ArnA and First structure of a sugar N-formyltransferase provided details concerning the manner in which UMP 5 As indicated in Figure 1a, the lipid A component of the and N -formyltetrahydrofolate (shown in Figure 1c) are LPS contains phosphorylated sugars. These sugars, along accommodated within the active site region. In addition, a with other phosphate moieties in the LPS, result in a characteristic signature sequence of HxSLLPKxxG motif negatively charged outer surface of the bacterium. As a was identified with the proline adopting a cis peptide first line of defense against pathogenic bacteria, host conformation and the histidine residue functioning in epithelial cells as well as circulating neutrophils and catalysis [20 ]. Although informative, these structures macrophages produce cationic antimicrobial peptides did not reveal the manner in which ArnA binds a nucleo- (CAMPS) that interact with the bacterial LPS ultimately tide-linked sugar in its N-formyltransferase domain. resulting in cell death [17]. Strikingly, some human pathogens, such as Salmonella typhimurium and Pseudomo- Structure of an N-formyltransferase from nas aeruginosa, have been shown to alter their LPS com- C.
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