Characterization of the ß-KDO Transferase KpsS, the Initiating Enzyme in the Biosynthesis of the Lipid Acceptor for Escherichia coli Polysialic Acid.

Antiphagocytic capsular polysaccharides are key components of effective vaccines against pathogenic bacteria. Neisseria meningitidis groups B and C, as well as Escherichia coli serogroups K1 and K92, are coated with polysialic acid capsules. Although the chemical structure of these polysaccharides and the organization of the associated gene clusters have been described for many years, only recently have the details of the biosynthetic pathways been discovered. The polysialic acid chains are synthesized by polysialyltransferases on a proposed phosphatidylglycerol lipid acceptor with a poly keto-deoxyoctulosonate (KDO) linker. Synthesis of this acceptor requires at least three enzymes in E. coli K1: KpsS, KpsC, and NeuE. In this report, we have characterized the ß-KDO glycosyltransferase KpsS, the first enzyme in the pathway for lipid acceptor synthesis. After purification of KpsS in a soluble active form, we investigated its function and substrate specificity and showed that KpsS can transfer a KDO residue to a fluorescently labeled phosphatidylglycerol lipid. The enzyme tolerated various lengths of fatty acid acyl chains on the phosphatidylglycerol, including fluorescent tags, but exhibited a preference for phosphatidylglycerol diacylated with longer fatty acid chains as indicated by the smaller Kd and Km values for substrates with chains with more than 14 members. Additional structural analysis of the KpsS product confirmed that KpsS transfers KDO from CMP-KDO to the 1-hydroxyl of phosphatidylglycerol to form a ß-KDO linkage.

Effect of Acceptor Chain Length and Hydrophobicity on Polymerization Kinetics of the Neisseria meningitidis Group C Polysialyltransferase.

Polysialic acids (PSA) are important extracellular virulence factors of the human pathogens Neisseria meningitidis and Escherichia coli. The importance of these polysaccharides in virulence make the polysialyltransferases (PST) targets for therapeutic drugs and protein engineering to facilitate efficient vaccine production. Here, we have generated recombinant bovine nucleotide monophosphate kinase to facilitate steady state kinetic assays of the PST. We have characterized the N. meningitidis group C (NmC) PST kinetically, using substrate analogues to describe the polymerization reaction. We observed a decrease in Km as the length of the oligo-sialic acid acceptor was increased, indicating a tighter binding of longer oligomers. In addition, we observed a biphasic relationship between kcat and chain length, which can be attributed to a switch in the mechanism of transfer of sialic acid from distributive to processive as the chain length increased above six sialic acid units. Substitution of donor substrate with the analogue CMP-9-F-sialic acid had minimal effect on acceptor Km, but it decreased kcat 6-fold. We propose that this decrease in kcat is caused by a destabilization of the transition state and/or an increase affinity of the product due to presence of the fluoro substituent. The acceptor's hydrophobicity also plays a role in catalysis. The kinetic analysis of the NmC PST with hydrophobic aglycon acceptor substrates indicated that they bind tighter and are turned over at a faster rate than the α-2,9 polysialic acid substrates lacking the hydrophobic end. This finding suggests the presence of a secondary ligand binding site that tethers the acceptor substrate to the enzyme active site.

Interaction of Neisseria meningitidis Group X N-acetylglucosamine-1-phosphotransferase with its donor substrate.

Neisseria meningitidis Group X is an emerging cause of bacterial meningitis in Sub-Saharan Africa. The capsular polysaccharide of Group X is a homopolymer of N-acetylglucosamine α(1-4) phosphate and is a vaccine target for prevention of disease associated with this meningococcal serogroup. We have demonstrated previously that the formation of the polymer is catalyzed by a phosphotransferase which transfers N-acetylglucosamine-1-phosphate from UDP-N-acetylglucosamine to the 4-hydroxyl of the N-acetylglucosamine on the nonreducing end of the growing chain. In this study, we use substrate analogs of UDP-GlcNAc to define the enzyme/donor substrate interactions critical for catalysis. Our kinetic analysis of the phosphotransferase reaction is consistent with a sequential mechanism of substrate addition and product release. The use of novel uracil modified analogs designed by Wagner et al. enabled us to assess whether the CsxA-catalyzed reaction is consistent with a donor dependent conformational change. As expected with this model for glycosyltransferases, UDP-GlcNAc analogs with bulky uracil modifications are not substrates but are inhibitors. An analog with a smaller iodo uracil substitution is a substrate and a less potent inhibitor. Moreover, our survey of analogs with modifications on the N-acetylglucosamine residue of the sugar nucleotide donor highlights the importance of substituents at C2 and C4 of the sugar residue. The hydroxyl group at C4 and the structure of the acyl group at C2 are very important for specificity and substrate interactions during the polymerization reaction. While most analogs modified at C2 were inhibitors, acetamido analogs were also substrates suggesting the importance of the carbonyl group.

Orotidine 5'-monophosphate decarboxylase: transition state stabilization from remote protein-phosphodianion interactions.

Mutants of orotidine 5'-monophosphate decarboxylase containing all possible single (Q215A, Y217F, and R235A), double, and triple substitutions of the side chains that interact with the phosphodianion group of the substrate orotidine 5'-monophosphate have been prepared. Essentially the entire effect of these mutations on the decarboxylation of the truncated neutral substrate 1-(ß-d-erythrofuranosyl)orotic acid that lacks a phosphodianion group is expressed as a decrease in the third-order rate constant for activation by phosphite dianion. The results are consistent with a model in which phosphodianion binding interactions are utilized to stabilize a rare closed enzyme form that exhibits a high catalytic activity for decarboxylation.

Azalysine analogues as probes for protein lysine deacetylation and demethylation.

Reversible lysine acetylation and methylation regulate the function of a wide variety of proteins, including histones. Here, we have synthesized azalysine-containing peptides in acetylated and unacetylated forms as chemical probes of the histone deacetylases (HDAC8, Sir2Tm, and SIRT1) and the histone demethylase, LSD1. We have shown that the acetyl-azalysine modification is a fairly efficient substrate for the sirtuins, but a weaker substrate for HDAC8, a classical HDAC. In addition to deacetylation by sirtuins, the acetyl-azalysine analogue generates a novel ADP-ribose adduct that was characterized by mass spectrometry, Western blot analysis, and nuclear magnetic resonance spectroscopy. This peptide-ADP-ribose adduct is proposed to correspond to a derailed reaction intermediate, providing unique evidence for the direct 2'-hydroxyl attack on the O-alkylimidate intermediate that is formed in the course of sirtuin catalyzed deacetylation. An unacetylated azalysine-containing H3 peptide proved to be a potent inhibitor of the LSD1 demethylase, forming an FAD adduct characteristic of previously reported related structures, providing a new chemical probe for mechanistic analysis.

A selective phenelzine analogue inhibitor of histone demethylase LSD1.

Lysine-specific demethylase 1 (LSD1) is an epigenetic enzyme that oxidatively cleaves methyl groups from monomethyl and dimethyl Lys4 of histone H3 (H3K4Me1, H3K4Me2) and can contribute to gene silencing. This study describes the design and synthesis of analogues of a monoamine oxidase antidepressant, phenelzine, and their LSD1 inhibitory properties. A novel phenelzine analogue (bizine) containing a phenyl-butyrylamide appendage was shown to be a potent LSD1 inhibitor in vitro and was selective versus monoamine oxidases A/B and the LSD1 homologue, LSD2. Bizine was found to be effective at modulating bulk histone methylation in cancer cells, and ChIP-seq experiments revealed a statistically significant overlap in the H3K4 methylation pattern of genes affected by bizine and those altered in LSD1-/- cells. Treatment of two cancer cell lines, LNCaP and H460, with bizine conferred a reduction in proliferation rate, and bizine showed additive to synergistic effects on cell growth when used in combination with two out of five HDAC inhibitors tested. Moreover, neurons exposed to oxidative stress were protected by the presence of bizine, suggesting potential applications in neurodegenerative disease.