Browsing by Author "Raetz, Christian R H"

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Open Access
Biochemical Characterization of Lipid A Modification Enzymes From Rhizobium leguminosarum and Rhizobium etli
(2010) Ingram, Brian O'Neal
The lipid A component of lipopolysaccharide (LPS) in the nitrogen-fixing plant endosymbionts Rhizobium leguminosarum and Rhizobium etli is strikingly different when compared to that of enteric bacteria such as Escherichia coli. The Rhizobium species produce several unique enzymes that process the lipid A biosynthetic intermediate Kdo₂-lipid IV_A. These enzymes include a 1-phosphatase (LpxE), a 4´-phosphatase (LpxF), a 3-O-deacylase (PagL), and a lipid A oxidase (LpxQ). The biological functions and enzymological properties of many of the modification enzymes have remained unconfirmed and/or unknown. The purpose of these studies was to confirm the activities of these enzymes and to explore the functional significance of the resulting lipid A modifications.

To confirm the proposed biological functions of the enzymes in vivo, homologs of the lipid A phosphatases, LpxE and LpxF, from Francisella novicida and the lipid A oxidase LpxQ, were expressed heterologously in combination in E. coli. The resulting novel lipid A hybrids were analyzed by thin-layer chromatography (TLC) and electrospray ionization-mass spectrometry (ESI-MS).

The lipid A oxidase LpxQ, was characterized further biochemically. Two new purification procedures and a new in vitro assay were developed to analyze the properties of the enzyme. Purified LpxQ was shown to be dependent on oxygen and divalent cations for activity. Hydrogen peroxide was found to be a product of lipid A oxidation. A new fluorescence-based assay based on the detection of hydrogen peroxide was developed to monitor oxidation. LpxQ did not co-purifiy with any discernable cofactors, suggesting that it may employ a unique mechanism for the oxidation of lipid A.

The biological roles of LpxE and LpxF in plant nodulation were analyzed. Deletion mutants of the two phosphatases were generated in R. etli. The mutant strains accumulated the expected structures, confirming the specificity of the enzymes. Single and double phosphatase mutants were able to fix nitrogen in planta. Antimicrobial susceptibility testing indicated that dephosphorylation of lipid A increases resistance to cationic antimicrobials.

The biological role of the 3-O-deacylase, PagL, was also investigated. The pagL gene was identified using systematic homology searches. PagL was shown to be stimulated by calcium. A deletion mutant of the enzyme in R. etli was constructed and analyzed. The deletion mutant was found to be viable and unaltered in its ability to fix nitrogen. In conclusion, these studies have confirmed the roles of LpxE, LpxF, PagL, and LpxQ in Rhizobium lipid A biosynthesis and contributed new knowledge regarding the biochemical properties of LpxQ.
Open Access
Characterization of Peripheral-Membrane Enzymes Required for Lipid A Biosynthesis in Gram-Negative Bacteria
(2010) Metzger, Louis Eugene
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.

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.
Open Access
Development of new approaches to NMR data collection for protein structure determination
(2007-05-10T16:02:04Z) Coggins, Brian E.
Multidimensional nuclear magnetic resonance (NMR) spectroscopy has become one of the most important techniques available for studying the structure and function of biological macromolecules at atomic resolution. The conventional approach to multidimensional NMR involves the sampling of the time domain on a Cartesian grid followed by a multidimensional Fourier transform (FT). While this approach yields high quality spectra, as the number of dimensions is increased the time needed for sampling on a Cartesian grid increases exponentially, making it impractical to record 4-D spectra at high resolution and impossible to record 5-D spectra at all. This thesis describes new approaches to data collection and processing that make it possible to obtain spectra at higher resolution and/or with a higher dimensionality than was previously feasible with the conventional method. The central focus of this work has been the sampling of the time domain along radial spokes, which was recently introduced into the NMR community. If each radial spoke is processed by an FT with respect to radius, a set of projections of the higher-dimensional spectrum are obtained. Full spectra at high resolution can be generated from these projections via tomographic reconstruction. We generalized the lower-value reconstruction algorithm from the literature, and later integrated it with the backprojection algorithm in a hybrid reconstruction method. These methods permit the reconstruction of accurate 4-D and 5- D spectra at very high resolution, from only a small number of projections, as we demonstrated in the reconstruction of 4-D and 5-D sequential assignment spectra on small and large proteins. For nuclear Overhauser spectroscopy (NOESY), used to measure interproton distances in proteins, one requires quantitative reconstructions. We have successfully obtained these using filtered backprojection, which we found was equivalent to processing the radially sampled data by a polar FT. All of these methods represent significant gains in data collection efficiency over conventional approaches. The polar FT interpretation suggested that the problem could be analyzed using FT theory, to design even more efficient methods. We have developed a new approach to sampling, using concentric rings of sampling points, which represents a further improvement in efficiency and sensitivity over radial sampling.
Open Access
Exploring the structurial diversity and engineering potential of thermophilic periplasmic binding proteins
(2007-05-02T17:37:41Z) Cuneo, Matthew Joseph
The periplasmic binding protein (PBP) superfamily is found throughout the genosphere of both prokaryotic and eukaryotic organisms. PBPs function as receptors in bacterial solute transport and chemotaxis systems; however the same fold is also used in transcriptional regulators, enzymes, and eukaryotic neurotransmitter receptors. This versatility has been exploited for structure-based computational protein design experiments where PBPs have been engineered to bind novel ligands and serve as biosensors for the detection of small-molecule ligands relevant to biomedical or defense-related interests. In order to further understand functional adaptation from a structural biology perspective, and to provide a set of robust starting points for engineering novel biosensors by structure-based design, I have characterized the ligand-binding properties and solved the structure of nine PBPs from various thermophilic bacteria. Analysis of these structures reveals a variety of mechanisms by which diverse function can be encoded in a common fold. It is observed that re-modeling of secondary structure elements (such as insertions, deletions, and loop movements), and re-decoration of amino acid side-chains are common diversification mechanisms in PBPs. Furthermore, the relationship between hinge-bending motion and ligand binding is critical to understanding the function of natural or engineered adaptations in PBPs. Three of these proteins were solved in both the presence and absence of ligand which allowed for the first time the observation and analysis of ligand-induced structural rearrangements in thermophilic PBPs. This work revealed that the magnitude and transduction of local and global ligand-induced motions are diverse throughout the PBP superfamily. Through the analysis of the open-to-closed transition, and the identification of natural structural adaptations in thermophilic members of the PBP superfamily, I reveal strategies which can be applied to computational protein design to significantly improve current strategies.