Most gram-negative organisms require lipopolysaccharide and its membrane anchor, lipid A, for growth and survival. Also known as endotoxin, lipid A is synthesized via a nine-step enzymatic process, culminating in a conserved hexa-acylated, bis-phosphorylated disaccharide of glucosamine. This framework is often altered by condition- or species-specific lipid A modifications, which change the biochemical properties of the molecule in response to and to defend against environmental stress signals. Here, we expound on two stories in different gram-negative organisms, both involving novel or unanticipated biochemistry that impacts lipid A structure. First, the missing acyltransferase in the Epsilonproteobacterium Helicobacter pylori lipid A biosynthesis pathway is identified. This enzyme transfers a secondary acyl chain to the 3'-linked primary acyl chain of lipid A like E. coli LpxM, but shares almost no sequence similarity with the E. coli acyltransferase. It is reannotated as LpxJ and demonstrated to possess an unprecedented ability to act before the 2'-secondary acyltransferase, LpxL, as well as the 3-deoxy-D-manno-octulosonic acid transferase, KdtA. LpxJ is one member of a large class of acyltransferases found in a diverse range of organisms that lack an E. coli LpxM homolog, suggesting that LpxJ participates in lipid A biosynthesis in place of an LpxM homolog. The second story focuses on regulation of modifications to endotoxin structure that occur after the conserved biosynthesis pathway. E. coli pmrD is shown to be required for PmrAB-dependent lipid A modifications in conditions that exclusively activate PhoPQ; this result proves that PmrD connects PhoPQ and PmrAB despite previous reports that it is an inactive connector in this organism. Further, RNA sequencing and polymyxin B survival assays solidify the role of E. coli pmrD in influencing expression of pmrA and its target genes and promoting survival during exposure to cationic antimicrobial peptides. Notably, the presence of an unknown factor or system capable of activating pmrD to promote lipid A modification in the absence of the PhoPQ system is also revealed. In all, the findings presented here expand our understanding of alternative approaches to lipid A biosynthesis and the complex systems that regulate modifications of this dynamic molecule.

<p>Gram-negative bacteria possess an asymmetric outer membrane in which the inner leaflet is composed primarily of phospholipids while the outer leaflet contains both phospholipids and lipopolysaccharide (LPS). LPS forms a structural barrier that protects Gram-negative bacteria from antibiotics and other environmental stressors. The lipid A anchor of LPS is a glucosamine-based saccharolipid that is further modified with core and O-antigen sugars. In addition to serving a structural role as the hydrophobic anchor of LPS, lipid A is recognized by the innate immune system in animal cells and macrophages. The enzymes of Lipid A biosynthesis are conserved in Gram-negative bacteria; in most species, a single copy of each bio-synthetic gene is present. The exception is lpxH, which is an essential gene encoding a membrane-associated UDP-2,3-diacylglucosamine hydrolase, which catalyzed the attack of water upon the alpha-phosphate of its substrate and the leaving of UMP, resulting in the formation of lipid X. Many Gram-negatives lack an lpxH orthologue, yet these species must possess an activity analogous to that of LpxH. We used bioinformatics approaches to identify a candidate gene, designated lpxI, encoding this activity in the model organism Caulobacter crescentus. We then demonstrated that lpxI can rescue Escherichia coli deficient in lpxH. Moreover, we have shown that LpxI possesses robust and specific UDP-2,3-diacylglucosamine hydrolase activity in vitro. We have developed high-yield purification schema for Caulobacter crescentus LpxI (CcLpxI) heterologously expressed in E. coli. We crystallized CcLpxI and determined its 2.6 Å x-ray crystal structure in complex with lipid X. CcLpxI, which has no known homologues, consists of two novel domains connected by a linker. Moreover, we have identified a point mutant of CcLpxI which co-purifies with its substrate in a 0.85:1 molar ratio. We have solved the x-ray crystal structure of this mutant to 3.0 Å; preliminary comparison with the product-complexed model reveals striking differences. The findings described herein set the stage for further mechanistic and structural characterization of this novel enzyme.</p> <p></p> <p>In this work, we also isolate and characterize LpxB, an essential lipid A biosynthetic gene which is conserved among all Gram-negative bacteria. We purify E. coli and Hemophilus influeznea LpxB to near-homogeneity on a 10 mg scale, and we determine that E. coli LpxB activity is dependent upon the bulk surface concentration of its substrates in a mixed micellar assay system, suggesting that catalysis occurs at the lipid interface. E. coli LpxB partitions with membranes, but this interaction is partially abolished in high-salt conditions, suggesting that a significant component of LpxB's membrane association is ionic in nature. E. coli LpxB (Mr ~ 43 kDa) is a peripheral membrane protein, and we demonstrate that it co-purifies with phospholipids. We estimate, by autoradiography and mass-spectrometry, molar ratios of phospholipids to purified enzyme of 1.6-3.5:1. Transmission electron microscopy reveals the accumulation of intra-cellular membranes when LpxB is massively over-expressed. Alanine-scanning mutagenesis of selected conserved LpxB residues identified two, D89A and R201A, for which no residual catalytic activity is detected. Our data support the hypothesis that LpxB performs catalysis at the cytoplasmic surface of the inner membrane, and provide a rational starting-point for structural studies. This work contributes to knowledge of the small but growing set of structurally and mechanistically characterized enzymes which perform chemistry upon lipids.</p> / Dissertation

This thesis entitled ‘ Unusual Acylation Properties of Type II Fatty Acid Biosynthesis Acyl Carrier Proteins’ describes the discovery of self-acylation and malonyl transferase activity in acyl carrier proteins involved in type II fatty acid biosynthesis and assigns a physiological role to these processes inside the cellular milieu. Acyl carrier protein (ACP) is one of the most abundant proteins present inside the cell and almost 4% enzymes require it as a cofactor. Acyl carrier proteins can exist either as discrete proteins or as domains of large functional proteins. They function in a variety of synthases as central molecules to which growing acyl intermediates and nascent product molecules are covalently tethered during the elongation and modification steps required to produce the final product. A prototypical bacterial ACP is composed of 70-80 amino acids and is generally expressed in the apo form. It is post-translationally modified to active holo form by the addition of 4'-phosphopantetheine moiety to an absolutely conserved serine residue in a reaction catalyzed by holo-ACP synthase or 4'-phosphopantetheine transferase. Chapter 1 surveys literature related to carrier proteins inside the cell and describes the thesis objective. It also presents an overview of the acyl carrier proteins and their involvement in various metabolic pathways inside the cell. The chapter details the structural organization of acyl carrier proteins from various sources revealing the conservation in their structure and also details the molecular basis of interaction of ACP with other enzymes inside the cell. The discovery of unusual self-acylation property in acyl carrier proteins involved in polyketide biosynthesis and its absence in acyl carrier proteins involved in fatty acid biosynthesis prompted me to investigate the reasons for this selective behavior. Discovery of self-acylation property in acyl carrier proteins Plasmodium falciparum and chloroplast targeted Brassica napus acyl carrier proteins involved in type II fatty acid biosynthesis and the mechanism of this reaction forms the basis of Chapter 2. In this chapter it has been shown that self-acylation property is intrinsic to a given acyl carrier protein and is not dependent on the pathway in which it is involved. Based on primary sequence analysis and site directed mutagenesis studies presence of an aspartate/glutamate has been identified to be critical for the self-acylation event. Furthermore, it has also been shown that the self-acylation event in type II fatty acid biosynthesis acyl carrier proteins is highly specific in nature employing only dicarboxylic acid –CoAs as substrates unlike the polyketide biosynthesis acyl carrier proteins which utilize both dicarboxylic acid and β-keto acid thiol ester -CoAs as substrates. The detailed kinetics of these reactions has also been worked out. Combining all the results a plausible mechanism for the self-acylation reaction has been proposed. Chapter 3 describes the discovery of a novel malonyl transferase behavior in acyl carrier proteins involved in type II fatty acid biosynthesis. Malonyl transferase property in ACPs of type II FAS from a bacterium (Escherichia coli), a plant (Brassica napus) and a parasitic protozoon (Plasmodium falciparum) were investigated to present a unifying paradigm for the mechanism of malonyl transferase behavior in ACPs. Identification of malonyl transferase property in Plasmodium falciparum ACP and Escherichia coli ACP (EcACP) and the absence of this property in Brassica napus ACP has been described in this chapter. Detailed investigations demonstrated that presence of an arginine or a lysine in loop II and an arginine or glutamine at the start of helix III as the residues that are critical for the transferase activity. In order to assign a physiologic function to these unusual acylation properties, fabD(Ts) mutant strain of Escherichia coli was utilized for heterologous complementation by the various wild type and mutant ACPs that are able to catalyze either or both of the activities. Growth of the mutant strain at non-permissive temperature, when complemented with ACPs catalyzing both the reactions confirmed that these properties have a physiologic relevance. Extensive mutagenesis experiments in conjunction with complementation studies allowed me to propose a plausible mechanism on how the self-malonylation and malonyl transferase properties operate in tandem. Chapter 4 describes the thermodynamic characterization of self-acylation process using Isothermal Titration Calorimetry. Isothermal Titration Calorimetric studies on the binding of malonyl, succinyl, butyryl and methylmalonyl –CoA to Plasmodium falciparum and Brassica napus acyl carrier proteins were performed to investigate the role of thermodynamic parameters in the specificity of self-acylation reaction. Calculation of the parameters showed that the thermodynamics does not control the self-acylation reaction. The evolution of self-acylation property in various acyl carrier proteins and its possible significance in the evolution of various metabolic events is described in Chapter 5. Extensive bioinformatics search was performed and phylogenetic analysis on acyl carrier proteins from 60 different taxa was done using the MEGA4 program. Analysis showed that this property was first found in cyanobacterium. Later, during the course of evolution this property was lost in most acyl carrier proteins, and was retained either in acyl carrier proteins that are targeted to organelles of cyanobaterial orgin viz. apicoplast in apicomplexans and chlorplasts in plants or in acyl carrier proteins involved in secondary metabolic events such as polyketide biosynthesis. Chapter 6 summarizes the findings of the thesis. Acyl carrier protein from Plasmodium falciparum, Brassica napus and Escherichia coli were characterized for their self-acylation and malonyl transferase properties and a combined mechanism for these two properties is proposed. The work done also provides an in vivo rationale to these in vitro processes. Furthermore, the evolutionary significance of the self-acylation behavior is also discussed in the thesis. The thesis also probes into the thermodynamics of the self-acylation reaction in Plasmodium falciparum and Brassica napus acyl carrier proteins. Thus, the thesis adds a new dimension to the much unexplored ACP biology and paves the way to study in vivo roles of these processes in detail. Appendix I describes the Isothermal Titration calorimetric characterization of binding of various acyl-PO4 molecules to Escherichia coli PlsX (Acyl-phosphate acyltransferase). PlsX, the first enzyme of phosphatidic acid biosynthesis pathway catalyzes the conversion of acyl-ACP into acyl-PO4, which is further used by other enzymes leading to the formation of phosphatidic acid. ITC results presented in this section show that longer chain length acyl-PO4 molecules show better binding to PlsX, as compared to the smaller ones demonstrating that long chain acyl molecules serve as better substrates for phosphatidic acid synthesis.