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Bacterial nitroreductase enzymes capable of activating imaging probes and prodrugs are valuable tools for gene-directed enzyme prodrug therapies and targeted cell ablation models. We recently engineered a nitroreductase (E. coli NfsB F70A/F108Y) for the substantially enhanced reduction of the 5-nitroimidazole PET-capable probe, SN33623, which permits the theranostic imaging of vectors labeled with oxygen-insensitive bacterial nitroreductases. This mutant enzyme also shows improved activation of the DNA-alkylation prodrugs CB1954 and metronidazole. To elucidate the mechanism behind these enhancements, we resolved the crystal structure of the mutant enzyme to 1.98 Å and compared it to the wild-type enzyme. Structural analysis revealed an expanded substrate access channel and new hydrogen bonding interactions. Additionally, computational modeling of SN33623, CB1954, and metronidazole binding in the active sites of both the mutant and wild-type enzymes revealed key differences in substrate orientations and interactions, with improvements in activity being mirrored by reduced distances between the N5-H of isoalloxazine and the substrate nitro group oxygen in the mutant models. These findings deepen our understanding of nitroreductase substrate specificity and catalytic mechanisms and have potential implications for developing more effective theranostic imaging strategies in cancer treatment.
Assuntos
Metronidazol , Nitroimidazóis , Nitrorredutases , Nitrorredutases/metabolismo , Nitrorredutases/química , Nitrorredutases/genética , Nitroimidazóis/química , Nitroimidazóis/metabolismo , Metronidazol/química , Metronidazol/metabolismo , Metronidazol/farmacologia , Pró-Fármacos/metabolismo , Pró-Fármacos/química , Proteínas de Escherichia coli/metabolismo , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Tomografia por Emissão de Pósitrons/métodos , Escherichia coli/genética , Escherichia coli/metabolismo , Domínio Catalítico , Engenharia de Proteínas , Modelos Moleculares , Aziridinas/química , Aziridinas/metabolismoRESUMO
Many enzymes display non-Arrhenius behavior with curved Arrhenius plots in the absence of denaturation. There has been significant debate about the origin of this behavior and recently the role of the activation heat capacity (ΔCP⧧) has been widely discussed. If enzyme-catalyzed reactions occur with appreciable negative values of ΔCP⧧ (arising from narrowing of the conformational space along the reaction coordinate), then curved Arrhenius plots are a consequence. To investigate these phenomena in detail, we have collected high precision temperature-rate data over a wide temperature interval for a model glycosidase enzyme MalL, and a series of mutants that change the temperature-dependence of the enzyme-catalyzed rate. We use these data to test a range of models including macromolecular rate theory (MMRT) and an equilibrium model. In addition, we have performed extensive molecular dynamics (MD) simulations to characterize the conformational landscape traversed by MalL in the enzyme-substrate complex and an enzyme-transition state complex. We have crystallized the enzyme in a transition state-like conformation in the absence of a ligand and determined an X-ray crystal structure at very high resolution (1.10 Å). We show (using simulation) that this enzyme-transition state conformation has a more restricted conformational landscape than the wildtype enzyme. We coin the term "transition state-like conformation (TLC)" to apply to this state of the enzyme. Together, these results imply a cooperative conformational transition between an enzyme-substrate conformation (ES) and a transition-state-like conformation (TLC) that precedes the chemical step. We present a two-state model as an extension of MMRT (MMRT-2S) that describes the data along with a convenient approximation with linear temperature dependence of the activation heat capacity (MMRT-1L) that can be used where fewer data points are available. Our model rationalizes disparate behavior seen for MalL and previous results for a thermophilic alcohol dehydrogenase and is consistent with a raft of data for other enzymes. Our model can be used to characterize the conformational changes required for enzyme catalysis and provides insights into the role of cooperative conformational changes in transition state stabilization that are accompanied by changes in heat capacity for the system along the reaction coordinate. TLCs are likely to be of wide importance in understanding the temperature dependence of enzyme activity and other aspects of enzyme catalysis.
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Red edge excitation shift (REES) spectroscopy relies on the unique emission profiles of fluorophore-solvent interactions to profile protein molecular dynamics. Recently, we reported the use of REES to compare the stability of 32 polymorphic IgG antibodies natively containing tryptophan reporter fluorophores. Here, we expand on this work to investigate the sensitivity of REES to variations in tryptophan content using a subset of IgG3 antibodies containing arginine to tryptophan polymorphisms. Structural analysis revealed that the additional tryptophan residues were situated in highly solvated environments. Subsequently, REES showed clear differences in fluorescence emission profiles when compared with the unmutated variants, thereby limiting direct comparison of their structural dynamics. These findings highlight the exquisite sensitivity of REES to minor variations in protein structure and tryptophan composition.
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Proteínas , Triptofano , Triptofano/química , Espectrometria de Fluorescência/métodosRESUMO
Quantifying the rate of thermal adaptation of soil microbial respiration is essential in determining potential for carbon cycle feedbacks under a warming climate. Uncertainty surrounding this topic stems in part from persistent methodological issues and difficulties isolating the interacting effects of changes in microbial community responses from changes in soil carbon availability. Here, we constructed a series of temperature response curves of microbial respiration (given unlimited substrate) using soils sampled from around New Zealand, including from a natural geothermal gradient, as a proxy for global warming. We estimated the temperature optima ([Formula: see text]) and inflection point ([Formula: see text]) of each curve and found that adaptation of microbial respiration occurred at a rate of 0.29 °C ± 0.04 1SE for [Formula: see text] and 0.27 °C ± 0.05 1SE for [Formula: see text] per degree of warming. Our results bolster previous findings indicating thermal adaptation is demonstrably offset from warming, and may help quantifying the potential for both limitation and acceleration of soil C losses depending on specific soil temperatures.
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Aclimatação , Microbiologia do Solo , Clima , Aceleração , SoloRESUMO
Bacterial nitroreductase enzymes that convert prodrugs to cytotoxins are valuable tools for creating transgenic targeted ablation models to study cellular function and cell-specific regeneration paradigms. We recently engineered a nitroreductase ("NTR 2.0") for substantially enhanced reduction of the prodrug metronidazole, which permits faster cell ablation kinetics, cleaner interrogations of cell function, ablation of previously recalcitrant cell types, and extended ablation paradigms useful for modelling chronic diseases. To provide insight into the enhanced enzymatic mechanism of NTR 2.0, we have solved the X-ray crystal structure at 1.85 Angstroms resolution and compared it to the parental enzyme, NfsB from Vibrio vulnificus. We additionally present a survey of reductive activity with eight alternative nitroaromatic substrates, to provide access to alternative ablation prodrugs, and explore applications such as remediation of dinitrotoluene pollutants. The predicted binding modes of four key substrates were investigated using molecular modelling.
Assuntos
Pró-Fármacos , Animais , Especificidade por Substrato , Pró-Fármacos/química , Metronidazol , Animais Geneticamente Modificados , Nitrorredutases/metabolismoRESUMO
The constant regions of clinical monoclonal antibodies are derived from a select number of allotypes found in IgG subclasses. Despite a long-term acknowledgment that this diversity may impact both antibody function and developability, there is a lack of data on the stability of variants carrying these mutations. Here, we generated a panel of IgG1, IgG2, and IgG3 antibodies with 32 unique constant region alleles and performed a systematic comparison of stability using red edge excitation shift (REES). This technique exploits the fluorescent properties of tryptophan residues to measure antibody structural dynamics which predict flexibility and the propensity to unfold. Our REES measurements revealed broad stability differences between subclasses with IgG3 possessing the poorest overall stability. Further interrogation of differences between variants within each subclass enabled the high-resolution profiling of individual allotype stabilities. Crucially, these observed differences were not found to be linked to N297-linked glycan heterogeneity. Our work demonstrates diverse stabilities (and dynamics) for a range of naturally occurring constant domain alleles and the utility of REES as a method for rapid and sensitive antibody stability profiling, requiring only laboratory spectrophotometry equipment.
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Anticorpos Monoclonais , Imunoglobulina G , Imunoglobulina G/químicaRESUMO
DNA ligases are widely used in molecular biology to generate recombinant DNA. However, having evolved for nick-sealing, they are inefficient at catalysing the blunt-ended ligations that are critical to many biotechnological applications, including next-generation sequencing. To facilitate engineering of superior blunt-ended DNA ligases, we have developed and validated a compartmentalised self-replication protocol that can select for the most effective ligases from a library of variants. Parallel cultures of Escherichia coli cells expressing different plasmid-encoded variants act as both a source of template DNA for discrete whole-plasmid PCR reactions, and a source of expressed ligase to circularise the corresponding PCR amplicons. The most efficient ligases generate the greatest number of self-encoding plasmids, and are thereby selected over successive rounds of transformation, amplification and ligation. By individually optimising critical steps, we arrived at a coherent protocol that, over five rounds of selection, consistently enriched for cells expressing the more efficient of two recombinant DNA ligases.
Assuntos
DNA Ligases , DNA Recombinante , DNA Ligases/genética , Plasmídeos/genética , Reação em Cadeia da Polimerase , Escherichia coli/genética , Ligases/genéticaRESUMO
Climate warming can reduce global soil carbon stocks by enhancing microbial decomposition. However, the magnitude of this loss remains uncertain because the temperature sensitivity of the decomposition of the major fraction of soil carbon, namely resistant carbon, is not fully known. It is now believed that the resistance of soil carbon mostly depends on microbial accessibility of soil carbon with physical protection being the primary control of the decomposition of protected carbon, which is insensitive to temperature changes. However, it is still unclear whether the temperature sensitivity of the decomposition of unprotected carbon, for example, carbon that is not protected by the soil mineral matrix, may depend on the chemical recalcitrance of carbon compounds. In particular, the carbon-quality temperature (CQT) hypothesis asserts that recalcitrant low-quality carbon is more temperature-sensitive to decomposition than labile high-quality carbon. If the hypothesis is correct, climate warming could amplify the loss of unprotected, but chemically recalcitrant, carbon and the resultant CO2 release from soils to the atmosphere. Previous research has supported this hypothesis based on reported negative relationships between temperature sensitivity and carbon quality, defined as the decomposition rate at a reference temperature. Here we show that negative relationships can arise simply from the arbitrary choice of reference temperature, inherently invalidating those tests. To avoid this artefact, we defined the carbon quality of different compounds as their uncatalysed reaction rates in the absence of enzymes. Taking the uncatalysed rate as the carbon quality index, we found that the CQT hypothesis is not supported for enzyme-catalysed reactions, which showed no relationship between carbon quality and temperature sensitivity. The lack of correlation in enzyme-catalysed reactions implies similar temperature sensitivity for microbial decomposition of soil carbon, regardless of its quality, thereby allaying concerns of acceleration of warming-induced decomposition of recalcitrant carbon.
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Artefatos , Carbono , Temperatura , Carbono/química , Microbiologia do Solo , Solo/químicaRESUMO
We report here the complete genome sequence of Mycobacterium tuberculosis strain Colonial S-type 1 (CS1), which has been responsible for ongoing outbreaks of tuberculosis in New Zealand over the past 30 years. CS1 appears to be highly transmissible, with greater rates of progression to active disease, compared to other circulating M. tuberculosis strains; therefore, comparison of its genomic content is of interest.
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Uncovering the role of global protein dynamics in enzyme turnover is needed to fully understand enzyme catalysis. Recently, we have demonstrated that the heat capacity of catalysis, ΔC P , can reveal links between the protein free energy landscape, global protein dynamics, and enzyme turnover, suggesting that subtle changes in molecular interactions at the active site can affect long-range protein dynamics and link to enzyme temperature activity. Here, we use a model promiscuous enzyme (glucose dehydrogenase from Sulfolobus solfataricus) to chemically map how individual substrate interactions affect the temperature dependence of enzyme activity and the network of motions throughout the protein. Utilizing a combination of kinetics, red edge excitation shift (REES) spectroscopy, and computational simulation, we explore the complex relationship between enzyme-substrate interactions and the global dynamics of the protein. We find that changes in ΔC P and protein dynamics can be mapped to specific substrate-enzyme interactions. Our study reveals how subtle changes in substrate binding affect global changes in motion and flexibility extending throughout the protein.
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Activation heat capacity is emerging as a crucial factor in enzyme thermoadaptation, as shown by the non-Arrhenius behaviour of many natural enzymes. However, its physical origin and relationship to the evolution of catalytic activity remain uncertain. Here we show that directed evolution of a computationally designed Kemp eliminase reshapes protein dynamics, which gives rise to an activation heat capacity absent in the original design. These changes buttress transition-state stabilization. Extensive molecular dynamics simulations show that evolution results in the closure of solvent-exposed loops and a better packing of the active site. Remarkably, this gives rise to a correlated dynamical network that involves the transition state and large parts of the protein. This network tightens the transition-state ensemble, which induces a negative activation heat capacity and non-linearity in the activity-temperature dependence. Our results have implications for understanding enzyme evolution and suggest that selectively targeting the conformational dynamics of the transition-state ensemble by design and evolution will expedite the creation of novel enzymes.
Assuntos
Enzimas/metabolismo , Evolução Química , Catálise , Enzimas/química , Simulação de Dinâmica Molecular , Conformação Proteica , TermodinâmicaRESUMO
Here, we report a label-free gold nanoparticle-based single-molecule optical platform to study the immobilization, activity, and thermodynamics of single enzymes. The sensor uses plasmonic gold nanoparticles coupled to optical whispering gallery modes (WGMs) to probe enzyme conformational dynamics during turnover at a microsecond time resolution. Using a glucosidase enzyme as the model system, we explore the temperature dependence of the enzyme turnover at the single-molecule (SM) level. A recent physical model for understanding enzyme temperature dependencies (macromolecular rate theory; MMRT) has emerged as a powerful tool to study the relationship between enzyme turnover and thermodynamics. Using WGMs, SM enzyme measurements enable us to accurately track turnover as a function of conformational changes and therefore to quantitatively probe the key feature of the MMRT model, the activation heat capacity, at the ultimate level of SM. Our data shows that WGMs are extraordinarily sensitive to protein conformational change and can discern both multiple steps with turnover as well as microscopic conformational substates within those steps. The temperature dependence studies show that the MMRT model can be applied to a range of steps within turnover at the SM scale that is associated with conformational change. Our study validates the notion that MMRT captures differences in dynamics between states. The WGM sensors provide a platform for the quantitative analysis of SM activation heat capacity, applying MMRT to the label-free sensing of microsecond substates of active enzymes.
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Enzyme-catalyzed reactions sometimes display curvature in their Eyring plots in the absence of denaturation, indicative of a change in activation heat capacity. However, the effects of pH and (de)protonation on this phenomenon have remained unexplored. Herein, we report a kinetic characterization of the thermophilic pyrimidine nucleoside phosphorylase from Geobacillus thermoglucosidasius across a two-dimensional working space covering 35 °C and 3 pH units with two substrates displaying different pKa values. Our analysis revealed the presence of a measurable activation heat capacity change ΔCp⧧ in this reaction system, which showed no significant dependence on medium pH or substrate charge. Our results further describe the remarkable effects of a single halide substitution that has a minor influence on ΔCp⧧ but conveys a significant kinetic effect by decreasing the activation enthalpy, causing a >10-fold rate increase. Collectively, our results present an important piece in the understanding of enzymatic systems across multidimensional working spaces where the choice of reaction conditions can affect the rate, affinity, and thermodynamic phenomena independently of one another.
Assuntos
Bacillaceae/metabolismo , Fosforilases/metabolismo , Purina-Núcleosídeo Fosforilase/química , Catálise , Temperatura Alta , Concentração de Íons de Hidrogênio , Cinética , Pentosiltransferases/química , Fosforilases/fisiologia , Pirimidina Fosforilases/química , Especificidade por Substrato , Condutividade Térmica , TermodinâmicaRESUMO
The temperature dependence of global photosynthesis and respiration determine land carbon sink strength. While the land sink currently mitigates ~30% of anthropogenic carbon emissions, it is unclear whether this ecosystem service will persist and, more specifically, what hard temperature limits, if any, regulate carbon uptake. Here, we use the largest continuous carbon flux monitoring network to construct the first observationally derived temperature response curves for global land carbon uptake. We show that the mean temperature of the warmest quarter (3-month period) passed the thermal maximum for photosynthesis during the past decade. At higher temperatures, respiration rates continue to rise in contrast to sharply declining rates of photosynthesis. Under business-as-usual emissions, this divergence elicits a near halving of the land sink strength by as early as 2040.
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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.
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Enzimas/metabolismo , Escherichia coli/crescimento & desenvolvimento , Ensaios Enzimáticos , Enzimas/química , Escherichia coli/enzimologia , Glicólise/fisiologia , Cinética , Modelos Biológicos , TemperaturaRESUMO
Experiments and biomolecular simulations are revealing new and unexpected details of how enzymes are adapted to specific temperatures. These findings are elucidating enzyme evolutionary trajectories and offer great promise for design and engineering of natural and artificial enzymes. They also have implications for understanding responses of larger scale biological temperature dependence, relevant for understanding the effects of climate change on ecosystems. We review recent work on the temperature dependence of enzyme-catalysed reaction rates and the implications for enzyme evolution. Evidence from kinetic isotope effects, temperature dependent reaction rates, molecular dynamics simulations and thermodynamics provides new insights into enzyme thermoadaptation and evolution.
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Enzimas , Ecossistema , Enzimas/química , Enzimas/metabolismo , Evolução Molecular , Cinética , TermodinâmicaRESUMO
PhoH2 proteins are highly conserved across bacteria and archaea yet their biological function is poorly characterised. We examined the growth profiles of Mycobacterium smegmatis strains mc2155 and mc2155 ΔphoH2 and observed the same growth profile and growth rate in a variety of conditions. In light of the comparable growth, we used RNAseq to provide a snapshot of the differences between the transcriptomes of M. smegmatis mc2155 and M. smegmatis mc2155 ΔphoH2 during normal growth. At 48 hours, elevated expression of the sigF regulon was observed in ΔphoH2 relative to wild type. In biochemical assays, PhoH2 showed activity toward sigF mRNA insinuating a role of PhoH2 in modulating the pool of sigF mRNA in the cell during normal growth, adding further complexity to the repertoire of reported mechanisms of post-translational regulation. Multiple copies of the preferred target site of PhoH2 were identified in loops of the sigF mRNA structure, leading us to propose a mechanism for the activity of PhoH2 that is initiated after assembly on specific single-stranded loops of RNA. We hypothesise that PhoH2 is a toxin-antitoxin that contributes to the regulation of SigF at a post-transcriptional level through targeted activity on sigF mRNA. This work presents the first evidence for post-transcriptional regulation of SigF along with the biological function of PhoH2 from M. smegmatis. This has implications for the highly conserved PhoH2 toxin-antitoxin module across the mycobacteria including the important human pathogen M. tuberculosis.
Assuntos
Proteínas de Bactérias/genética , Toxinas Bacterianas/genética , Mycobacterium smegmatis/metabolismo , Fator sigma/metabolismo , Proteínas de Bactérias/metabolismo , Toxinas Bacterianas/metabolismo , Regulação Bacteriana da Expressão Gênica , Mycobacterium smegmatis/crescimento & desenvolvimento , Processamento de Proteína Pós-Traducional , RNA Mensageiro/metabolismo , Fator sigma/genéticaRESUMO
Most biomolecular interactions are typically thought to increase the (local) rigidity of a complex, for example, in drug-target binding. However, detailed analysis of specific biomolecular complexes can reveal a more subtle interplay between binding and rigidity. Here, we focussed on the human leucocyte antigen (HLA), which plays a crucial role in the adaptive immune system by presenting peptides for recognition by the αß T-cell receptor (TCR). The role that the peptide plays in tuning HLA flexibility during TCR recognition is potentially crucial in determining the functional outcome of an immune response, with obvious relevance to the growing list of immunotherapies that target the T-cell compartment. We have applied high-pressure/temperature perturbation experiments, combined with molecular dynamics simulations, to explore the drivers that affect molecular flexibility for a series of different peptide-HLA complexes. We find that different peptide sequences affect peptide-HLA flexibility in different ways, with the peptide cargo tuning a network of correlated motions throughout the pHLA complex, including in areas remote from the peptide-binding interface, in a manner that could influence T-cell antigen discrimination.
Assuntos
Antígeno HLA-A2/química , Peptídeos/química , Receptores de Antígenos de Linfócitos T alfa-beta/química , Regulação Alostérica , Sítio Alostérico , Sequência de Aminoácidos , Sítios de Ligação , Cristalografia por Raios X , Antígeno HLA-A2/metabolismo , Humanos , Insulina/química , Modelos Moleculares , Simulação de Dinâmica Molecular , Movimento (Física) , Peptídeos/metabolismo , Pressão , Ligação Proteica , Conformação Proteica , Precursores de Proteínas/química , Receptores de Antígenos de Linfócitos T alfa-beta/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Temperatura , Microglobulina beta-2/química , Microglobulina beta-2/metabolismoRESUMO
We review the adaptations of enzyme activity to different temperatures. Psychrophilic (cold-adapted) enzymes show significantly different activation parameters (lower activation enthalpies and entropies) from their mesophilic counterparts. Furthermore, there is increasing evidence that the temperature dependence of many enzyme-catalyzed reactions is more complex than is widely believed. Many enzymes show curvature in plots of activity versus temperature that is not accounted for by denaturation or unfolding. This is explained by macromolecular rate theory: A negative activation heat capacity for the rate-limiting chemical step leads directly to predictions of temperature optima; both entropy and enthalpy are temperature dependent. Fluctuations in the transition state ensemble are reduced compared to the ground state. We show how investigations combining experiment with molecular simulation are revealing fundamental details of enzyme thermoadaptation that are relevant for understanding aspects of enzyme evolution. Simulations can calculate relevant thermodynamic properties (such as activation enthalpies, entropies, and heat capacities) and reveal the molecular mechanisms underlying experimentally observed behavior.
Assuntos
Biocatálise , Enzimas/metabolismo , Temperatura , Entropia , Cinética , Modelos BiológicosRESUMO
PhoH2 proteins are found in a very diverse range of microorganisms that span bacteria and archaea. These proteins are composed of two domains: an N-terminal PIN-domain fused with a C-terminal PhoH domain. Collectively this fusion functions as an RNA helicase and ribonuclease. In other genomic contexts, PINdomains and PhoHdomains are separate but adjacent suggesting association to achieve similar function. Exclusively among the mycobacteria, PhoH2 proteins are encoded in the genome with an upstream gene, phoAT, which is thought to play the role of an antitoxin (in place of the traditional VapB antitoxin that lies upstream of the 47 other PINdomains in the mycobacterial genome). This review examines PhoH2 proteins as a whole and describes the bioinformatics, biochemical, structural, and biological properties of the two domains that make up PhoH2: PIN and PhoH. We review the transcriptional regulators of phoH2 from two mycobacterial species and speculate on the function of PhoH2 proteins in the context of a Type II toxin-antitoxin system which are thought to play a role in the stress response in bacteria.