Characterisation and engineering of a thermophilic RNA ligase from Palaeococcus pacificus.

RNA ligases are important enzymes in molecular biology and are highly useful for the manipulation and analysis of nucleic acids, including adapter ligation in next-generation sequencing of microRNAs. Thermophilic RNA ligases belonging to the RNA ligase 3 family are gaining attention for their use in molecular biology, for example a thermophilic RNA ligase from Methanobacterium thermoautotrophicum is commercially available for the adenylation of nucleic acids. Here we extensively characterise a newly identified RNA ligase from the thermophilic archaeon Palaeococcus pacificus (PpaRnl). PpaRnl exhibited significant substrate adenylation activity but low ligation activity across a range of oligonucleotide substrates. Mutation of Lys92 in motif I to alanine, resulted in an enzyme that lacked adenylation activity, but demonstrated improved ligation activity with pre-adenylated substrates (ATP-independent ligation). Subsequent structural characterisation revealed that in this mutant enzyme Lys238 was found in two alternate positions for coordination of the phosphate tail of ATP. In contrast mutation of Lys238 in motif V to glycine via structure-guided engineering enhanced ATP-dependent ligation activity via an arginine residue compensating for the absence of Lys238. Ligation activity for both mutations was higher than the wild-type, with activity observed across a range of oligonucleotide substrates with varying sequence and secondary structure.

A role for the ATP-dependent DNA ligase lig E of Neisseria gonorrhoeae in biofilm formation.

BACKGROUND: The ATP-dependent DNA ligase Lig E is present as an accessory DNA ligase in numerous proteobacterial genomes, including many disease-causing species. Here we have constructed a genomic Lig E knock-out in the obligate human pathogen Neisseria gonorrhoeae and characterised its growth and infection phenotype. RESULTS: This demonstrates that N. gonorrhoeae Lig E is a non-essential gene and its deletion does not cause defects in replication or survival of DNA-damaging stressors. Knock-out strains were partially defective in biofilm formation on an artificial surface as well as adhesion to epithelial cells. In addition to in vivo characterisation, we have recombinantly expressed and assayed N. gonorrhoeae Lig E and determined the crystal structure of the enzyme-adenylate engaged with DNA substrate in an open non-catalytic conformation. CONCLUSIONS: These findings, coupled with the predicted extracellular/ periplasmic location of Lig E indicates a role in extracellular DNA joining as well as providing insight into the binding dynamics of these minimal DNA ligases.

Mechanisms of host manipulation by Neisseria gonorrhoeae.

Neisseria gonorrhoeae (also known as gonococcus) has been causing gonorrhoea in humans since ancient Egyptian times. Today, global gonorrhoea infections are rising at an alarming rate, in concert with an increasing number of antimicrobial-resistant strains. The gonococcus has concurrently evolved several intricate mechanisms that promote pathogenesis by evading both host immunity and defeating common therapeutic interventions. Central to these adaptations is the ability of the gonococcus to manipulate various host microenvironments upon infection. For example, the gonococcus can survive within neutrophils through direct regulation of both the oxidative burst response and maturation of the phagosome; a concerning trait given the important role neutrophils have in defending against invading pathogens. Hence, a detailed understanding of how N. gonorrhoeae exploits the human host to establish and maintain infection is crucial for combating this pathogen. This review summarizes the mechanisms behind host manipulation, with a central focus on the exploitation of host epithelial cell signaling to promote colonization and invasion of the epithelial lining, the modulation of the host immune response to evade both innate and adaptive defenses, and the manipulation of host cell death pathways to both assist colonization and combat antimicrobial activities of innate immune cells. Collectively, these pathways act in concert to enable N. gonorrhoeae to colonize and invade a wide array of host tissues, both establishing and disseminating gonococcal infection.

Combatting antimicrobial resistance via the cysteine biosynthesis pathway in bacterial pathogens.

Antibiotics are the cornerstone of modern medicine and agriculture, and rising antibiotic resistance is one the biggest threats to global health and food security. Identifying new and different druggable targets for the development of new antibiotics is absolutely crucial to overcome resistance. Adjuvant strategies that either enhance the activity of existing antibiotics or improve clearance by the host immune system provide another mechanism to combat antibiotic resistance. Targeting a combination of essential and non-essential enzymes that play key roles in bacterial metabolism is a promising strategy to develop new antimicrobials and adjuvants, respectively. The enzymatic synthesis of L-cysteine is one such strategy. Cysteine plays a key role in proteins and is crucial for the synthesis of many biomolecules important for defense against the host immune system. Cysteine synthesis is a two-step process, catalyzed by two enzymes. Serine acetyltransferase (CysE) catalyzes the first step to synthesize the pathway intermediate O-acetylserine, and O-acetylserine sulfhydrylase (CysK/CysM) catalyzes the second step using sulfide or thiosulfate to produce cysteine. Disruption of the cysteine biosynthesis pathway results in dysregulated sulfur metabolism, altering the redox state of the cell leading to decreased fitness, enhanced susceptibility to oxidative stress and increased sensitivity to antibiotics. In this review, we summarize the structure and mechanism of characterized CysE and CysK/CysM enzymes from a variety of bacterial pathogens, and the evidence that support targeting these enzymes for the development of new antimicrobials or antibiotic adjuvants. In addition, we explore and compare compounds identified thus far that target these enzymes.

Serine acetyltransferase from Neisseria gonorrhoeae; structural and biochemical basis of inhibition.

Serine acetyltransferase (SAT) catalyzes the first step in the two-step pathway to synthesize l-cysteine in bacteria and plants. SAT synthesizes O-acetylserine from substrates l-serine and acetyl coenzyme A and is a key enzyme for regulating cellular cysteine levels by feedback inhibition of l-cysteine, and its involvement in the cysteine synthase complex. We have performed extensive structural and kinetic characterization of the SAT enzyme from the antibiotic-resistant pathogen Neisseria gonorrhoeae. Using X-ray crystallography, we have solved the structures of NgSAT with the non-natural ligand, l-malate (present in the crystallization screen) to 2.01 Šand with the natural substrate l-serine (2.80 Å) bound. Both structures are hexamers, with each monomer displaying the characteristic left-handed parallel ß-helix domain of the acyltransferase superfamily of enzymes. Each structure displays both extended and closed conformations of the C-terminal tail. l-malate bound in the active site results in an interesting mix of open and closed active site conformations, exhibiting a structural change mimicking the conformation of cysteine (inhibitor) bound structures from other organisms. Kinetic characterization shows competitive inhibition of l-cysteine with substrates l-serine and acetyl coenzyme A. The SAT reaction represents a key point for the regulation of cysteine biosynthesis and controlling cellular sulfur due to feedback inhibition by l-cysteine and formation of the cysteine synthase complex. Data presented here provide the structural and mechanistic basis for inhibitor design and given this enzyme is not present in humans could be explored to combat the rise of extensively antimicrobial resistant N. gonorrhoeae.

The Inflection Point Hypothesis: The Relationship between the Temperature Dependence of Enzyme-Catalyzed Reaction Rates and Microbial Growth Rates.

The temperature dependence of biological rates at different scales (from individual enzymes to isolated organisms to ecosystem processes such as soil respiration and photosynthesis) is the subject of much historical and contemporary research. The precise relationship between the temperature dependence of enzyme rates and those at larger scales is not well understood. We have developed macromolecular rate theory (MMRT) to describe the temperature dependence of biological processes at all scales. Here we formalize the scaling relationship by investigating MMRT both at the molecular scale (constituent enzymes) and for growth of the parent organism. We demonstrate that the inflection point (Tinf) for the temperature dependence of individual metabolic enzymes coincides with the optimal growth temperature for the parent organism, and we rationalize this concordance in terms of the necessity for linearly correlated rates for metabolic enzymes over fluctuating environmental temperatures to maintain homeostasis. Indeed, Tinf is likely to be under strong selection pressure to maintain coordinated rates across environmental temperature ranges. At temperatures at which rates become uncorrelated, we postulate a regulatory catastrophe and organism growth rates precipitously decline at temperatures where this occurs. We show that the curvature in the plots of the natural log of the rate versus temperature for individual enzymes determines the curvature for the metabolic process overall and the curvature for the temperature dependence of the growth of the organism. We have called this "the inflection point hypothesis", and this hypothesis suggests many avenues for future investigation, including avenues for engineering the thermal tolerance of organisms.

An essential pentatricopeptide repeat protein in the apicomplexan remnant chloroplast.

The malaria parasite Plasmodium and other apicomplexans such as Toxoplasma evolved from photosynthetic organisms and contain an essential, remnant plastid termed the apicoplast. Transcription of the apicoplast genome is polycistronic with extensive RNA processing. Yet little is known about the mechanism of apicoplast RNA processing. In plants, chloroplast RNA processing is controlled by multiple pentatricopeptide repeat (PPR) proteins. Here, we identify the single apicoplast PPR protein, PPR1. We show that the protein is essential and that it binds to RNA motifs corresponding with previously characterized processing sites. Additionally, PPR1 shields RNA transcripts from ribonuclease degradation. This is the first characterization of a PPR protein from a nonphotosynthetic plastid.

Cysteine biosynthesis in Neisseria species.

The principal mechanism of reducing sulfur into organic compounds is via the synthesis of l-cysteine. Cysteine is used for protein and glutathione synthesis, as well as being the primary sulfur source for a variety of other molecules, such as biotin, coenzyme A, lipoic acid and more. Glutathione and other cysteine derivatives are important for protection against the oxidative stress that pathogenic bacteria such as Neisseria gonorrhoeae and Neisseria meningitidis encounter during infection. With the alarming rise of antibiotic-resistant strains of N. gonorrhoeae, the development of inhibitors for the future treatment of this disease is critical, and targeting cysteine biosynthesis enzymes could be a promising approach for this. Little is known about the transport of sulfate and thiosulfate and subsequent sulfate reduction and incorporation into cysteine in Neisseria species. In this review we investigate cysteine biosynthesis within Neisseria species and examine the differences between species and with other bacteria. Neisseria species exhibit different arrangements of cysteine biosynthesis genes and have slight differences in how they assimilate sulfate and synthesize cysteine, while, most interestingly, N. gonorrhoeae by virtue of a genome deletion, lacks the ability to reduce sulfate to bisulfide for incorporation into cysteine, and as such uses the thiosulfate uptake pathway for the synthesis of cysteine.

VapC proteins from Mycobacterium tuberculosis share ribonuclease sequence specificity but differ in regulation and toxicity.

The chromosome of Mycobacterium tuberculosis (Mtb) contains a large number of Type II toxin-antitoxin (TA) systems. The majority of these belong to the VapBC TA family, characterised by the VapC protein consisting of a PIN domain with four conserved acidic residues, and proposed ribonuclease activity. Characterisation of five VapC (VapC1, 19, 27, 29 and 39) proteins from various regions of the Mtb chromosome using a combination of pentaprobe RNA sequences and mass spectrometry revealed a shared ribonuclease sequence-specificity with a preference for UAGG sequences. The TA complex VapBC29 is auto-regulatory and interacts with inverted repeat sequences in the vapBC29 promoter, whereas complexes VapBC1 and VapBC27 display no auto-regulatory properties. The difference in regulation could be due to the different properties of the VapB proteins, all of which belong to different VapB protein families. Regulation of the vapBC29 operon is specific, no cross-talk among Type II TA systems was observed. VapC29 is bacteriostatic when expressed in Mycobacterium smegmatis, whereas VapC1 and VapC27 displayed no toxicity upon expression in M. smegmatis. The shared sequence specificity of the five VapC proteins characterised is intriguing, we propose that the differences observed in regulation and toxicity is the key to understanding the role of these TA systems in the growth and persistence of Mtb.