Uncovering differential equations from data with hidden variables.

SINDy is a method for learning system of differential equations from data by solving a sparse linear regression optimization problem [Brunton, Proctor, and Kutz, Proc. Natl. Acad. Sci. USA 113, 3932 (2016)PNASA60027-842410.1073/pnas.1517384113]. In this article, we propose an extension of the SINDy method that learns systems of differential equations in cases where some of the variables are not observed. Our extension is based on regressing a higher order time derivative of a target variable onto a dictionary of functions that includes lower order time derivatives of the target variable. We evaluate our method by measuring the prediction accuracy of the learned dynamical systems on synthetic data and on a real data set of temperature time series provided by the Réseau de Transport d'Électricité. Our method provides high quality short-term forecasts and it is orders of magnitude faster than competing methods for learning differential equations with latent variables.

Hospitalized population diagnosed with Covid-19 in public health centers in the southeastern region of Greater Buenos Aires / [Población hospitalizada con diagnóstico de Covid-19 en los centros de salud públicos de la región sudeste del Gran Buenos Aires]

Introduction: The present work describes the clinical characteristics and interventions to minimize morbidity and mortality in hospitalized patients diagnosed with COVID-19. Methods: It is a prospective cohort investigation of patients who received a response from the Health Centers in the southeast region (RS) of the metropolitan area (AMBA) from April 8 to September 30, 2020. A Situation Room was used epidemiological with two monitoring and follow-up boards, one for bed management and the other for patient management. Results: During the analyzed period, 2,588 patients with confirmed COVID-19 diagnosis were admitted, 1,943 with suspected COVID-19 pathology, and 1,464 subjects with other pathologies. 55% of the patients were men and the mean age was 51 years. There were 82.8% patients with pre-existing diseases, hypertension and diabetes were the most frequent. 14% were hospitalized in the Intensive Care Unit. The mortality of the cohort was 15.05%, mortality was higher for men, with a mean age of 60 years, 92.65% had some pre-existing disease. Conclusion: Our cohort is younger than other published works. Older people, men, and people with comorbidities are at increased risk for COVID-19-related mortality. The public health system was able to respond to the demand without collapsing the hospital institutions.

Introducción: En el presente trabajo se describen las características clínicas y las intervenciones para minimizar la morbimortalidad en pacientes hospitalizados con diagnóstico de COVID-19. Métodos: Es una investigación de cohorte prospectiva de pacientes que recibieron respuesta de los Centros de Salud en la región sudeste (RS) del área metropolitana (AMBA) desde el 8 de abril hasta el 30 de septiembre de 2020. Se utilizó una Sala de Situación epidemiológica con dos tableros de monitoreo y seguimiento, uno de gestión de camas y otro de gestión de pacientes. Resultados: Durante el periodo analizado se internaron2.588pacientes con diagnóstico COVID-19 confirmados, 1.943 con sospecha de patología COVID-19, y 1.464sujetos con otras patologías. El 55% de los pacientes eran hombres y la edad media fue de 51 años. Hubo 82,8% pacientes con enfermedades preexistentes, hipertensión y diabetes fueron las más frecuentes. El 14% fue hospitalizado en la Unidad de Terapia Intensiva. La mortalidad de la cohorte fue del 15,05%, la mortalidad fue mayor para los hombres, con una edad media de 60 años, el 92,65% tenía alguna enfermedad preexistente. Conclusión: Nuestra cohorte es más joven que otros trabajos publicados. Las personas mayores, los hombres y las personas con comorbilidades tienen mayor riesgo de mortalidad relacionada con COVID-19. El sistema de salud público pudo responder a la demanda sin llegar a colapsar las instituciones hospitalarias.

Hemeproteins as Targets for Sulfide Species.

Significance: Sulfides are endogenous and ubiquitous signaling species that share the hemeproteins as biochemical targets with O2, nitric oxide, and carbon monoxide. The description of the binding mechanisms is mandatory to anticipate the biochemical relevance of the interaction. Recent Advances: The binding of sulfide to ferric hemeproteins has been described in more than 40 systems, including native proteins, mutants, and model systems. Mechanisms of sulfide binding to ferric hemeproteins have been examined by a combination of kinetic and computational experiments. The distal control of the association process, dissected into the migration of the ligand to the active site and the binding event, reveals that neutral hydrogen sulfide (H2S) reaches the active site and is the predominant binding ligand, while the HS- is excluded by the protein matrix. Experiments with model compounds, devoid of a protein scaffold, reveal that both H2S and HS- can bind the ferric heme if accessing the site. A critical role of the proximal ligand in the prevention of the metal-centered reduction has been experimentally assessed. For metmyoglobin and methemoglobin, the coordination of sulfide leads to noncanonical functions: sulfide storage and its oxidative detoxification have been evidenced under physiological and excess sulfide concentrations, respectively. Critical Issues: The bound species is suggested to predominate in the monoprotonated form, although spectroscopic evidence is pending. Future Directions: A description of the role of hemeproteins as biochemical targets for inorganic sulfide requires understanding the reactivity of bound sulfide, for example: the metal-centered reduction, the reaction with excess sulfide, oxidants, or other gasotransmitters, among other biomolecules.

Lessons learned about steered molecular dynamics simulations and free energy calculations.

The calculation of free energy profiles is central in understanding differential enzymatic activity, for instance, involving chemical reactions that require QM-MM tools, ligand migration, and conformational rearrangements that can be modeled using classical potentials. The use of steered molecular dynamics (sMD) together with the Jarzynski equality is a popular approach in calculating free energy profiles. Here, we first briefly review the application of the Jarzynski equality to sMD simulations, then revisit the so-called stiff-spring approximation and the consequent expectation of Gaussian work distributions and, finally, reiterate the practical utility of the second-order cumulant expansion, as it coincides with the parametric maximum-likelihood estimator in this scenario. We illustrate this procedure using simulations of CO, both in aqueous solution and in a carbon nanotube as a model system for biologically relevant nanoheterogeneous environments. We conclude the use of the second-order cumulant expansion permits the use of faster pulling velocities in sMD simulations, without introducing bias due to large dispersion in the non-equilibrium work distribution.

On the accurate estimation of free energies using the jarzynski equality.

The Jarzynski equality is one of the most widely celebrated and scrutinized nonequilibrium work theorems, relating free energy to the external work performed in nonequilibrium transitions. In practice, the required ensemble average of the Boltzmann weights of infinite nonequilibrium transitions is estimated as a finite sample average, resulting in the so-called Jarzynski estimator, ΔF^J . Alternatively, the second-order approximation of the Jarzynski equality, though seldom invoked, is exact for Gaussian distributions and gives rise to the Fluctuation-Dissipation estimator ΔF^FD . Here we derive the parametric maximum-likelihood estimator (MLE) of the free energy ΔF^ML considering unidirectional work distributions belonging to Gaussian or Gamma families, and compare this estimator to ΔF^J . We further consider bidirectional work distributions belonging to the same families, and compare the corresponding bidirectional ΔF^ML∗ to the Bennett acceptance ratio ( ΔF^BAR ) estimator. We show that, for Gaussian unidirectional work distributions, ΔF^FD is in fact the parametric MLE of the free energy, and as such, the most efficient estimator for this statistical family. We observe that ΔF^ML and ΔF^ML∗ perform better than ΔF^J and ΔF^BAR , for unidirectional and bidirectional distributions, respectively. These results illustrate that the characterization of the underlying work distribution permits an optimal use of the Jarzynski equality. © 2018 Wiley Periodicals, Inc.

Cold-Adaptation Signatures in the Ligand Rebinding Kinetics to the Truncated Hemoglobin of the Antarctic Bacterium Pseudoalteromonas haloplanktis TAC125.

Cold-adapted organisms have evolved proteins endowed with higher flexibility and lower stability in comparison to their thermophilic homologues, resulting in enhanced reaction rates at low temperatures. In this context, protein-bound water molecules were suggested to play a major role, and their weaker interactions at protein active sites have been associated with cold adaptation. In this work, we tested this hypothesis on truncated hemoglobins (a family of microbial heme-proteins of yet-unclear function) applying molecular dynamics simulations and ligand-rebinding kinetics on a protein from the Antarctic bacterium Pseudoalteromonas haloplanktis TAC125 in comparison with its thermophilic Thermobifida fusca homologue. The CO rebinding kinetics of the former highlight several geminate phases, with an unusually long-lived geminate intermediate. An articulated tunnel with at least two distinct docking sites was identified by analysis of molecular dynamics simulations and was suggested to be at the origin of the unusual geminate rebinding phase. Water molecules are present in the distal pocket, but their stabilization by TrpG8, TyrB10, and HisCD1 is much weaker than in thermophilic Thermobifida fusca truncated hemoglobin, resulting in a faster geminate rebinding. Our results support the hypothesis that weaker water-molecule interactions at the reaction site are associated with cold adaptation.

Mechanism of Sulfide Binding by Ferric Hemeproteins.

The reaction of hydrogen sulfide (H2S) with hemeproteins is a key physiological reaction; still, its mechanism and implications are not completely understood. In this work, we propose a combination of experimental and theoretical tools to shed light on the reaction in model system microperoxidase 11 (MP11-FeIII) and myoglobin (Mb-FeIII), from the estimation of the intrinsic binding constants of the species H2S and hydrosulfide (HS-), and the computational description of the overall binding process. Our results show that H2S and HS- are the main reactive species in Mb-FeIII and MP11-FeIII, respectively, and that the magnitude of their intrinsic binding constants are similar to most of the binding constants reported so far for hemeproteins systems and model compounds. However, while the binding of HS- to Mb-FeIII was negligible, the binding of H2S to MP11-FeIII was significant, providing a frame for a discriminated analysis of both species and revealing differential mechanistic aspects. A joint inspection of the kinetic data and the free energy profiles of the binding processes suggests that a dissociative mechanism with the release of a coordinated water molecule as rate limiting step is operative in the binding of H2S to Mb-FeIII and that the binding of HS- is prevented in the access to the protein matrix. For the MP11-FeIII case, where no access restrictions for the ligands are present, an associative component in the mechanism seems to be operative. Overall, the results suggest that if accessing the active site then both H2S and HS- are capable of binding a ferric heme moiety.

Access and Binding of H2S to Hemeproteins: The Case of HbI of Lucina pectinata.

Hydrogen sulfide (H2S) was recently discovered as a gasotransmitter, capable of coordinating to the heme iron of hemeproteins. H2S is unique for its ability to render varying concentrations of the nucleophilic conjugate bases (HS(-) or S(2-)), either as free or bound species with expected outcomes on its further reactivity. There is no direct evidence about which species (H2S, HS(-), or S(2-)) coordinates to the iron. We performed computer simulations to address the migration and binding processes of H2S species to the hemoglobin I of Lucina pectinata, which exhibits the highest affinity for the substrate measured to date. We found that H2S is the most favorable species in the migration from the bulk to the active site, through an internal pathway of the protein. After the coordination of H2S, an array of clustered water molecules modifies the active site environment, and assists in the subsequent deprotonation of the ligand, forming Fe(III)-SH(-). The feasibility of the second deprotonation of the coordinated ligand is also discussed.

A quantitative model for oxygen uptake and release in a family of hemeproteins.

MOTIVATION: Hemeproteins have many diverse functions that largely depend on the rate at which they uptake or release small ligands, like oxygen. These proteins have been extensively studied using either simulations or experiments, albeit only qualitatively and one or two proteins at a time. RESULTS: We present a physical-chemical model, which uses data obtained exclusively from computer simulations, to describe the uptake and release of oxygen in a family of hemeproteins, called truncated hemoglobins (trHbs). Through a rigorous statistical analysis we demonstrate that our model successfully recaptures all the reported experimental oxygen association and dissociation kinetic rate constants, thus allowing us to establish the key factors that determine the rates at which these hemeproteins uptake and release oxygen. We found that internal tunnels as well as the distal site water molecules control ligand uptake, whereas oxygen stabilization by distal site residues controls ligand release. Because these rates largely determine the functions of these hemeproteins, these approaches will also be important tools in characterizing the trHbs members with unknown functions. CONTACT: lboechi@ic.fcen.uba.ar SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Evolutionary and Functional Relationships in the Truncated Hemoglobin Family.

Predicting function from sequence is an important goal in current biological research, and although, broad functional assignment is possible when a protein is assigned to a family, predicting functional specificity with accuracy is not straightforward. If function is provided by key structural properties and the relevant properties can be computed using the sequence as the starting point, it should in principle be possible to predict function in detail. The truncated hemoglobin family presents an interesting benchmark study due to their ubiquity, sequence diversity in the context of a conserved fold and the number of characterized members. Their functions are tightly related to O2 affinity and reactivity, as determined by the association and dissociation rate constants, both of which can be predicted and analyzed using in-silico based tools. In the present work we have applied a strategy, which combines homology modeling with molecular based energy calculations, to predict and analyze function of all known truncated hemoglobins in an evolutionary context. Our results show that truncated hemoglobins present conserved family features, but that its structure is flexible enough to allow the switch from high to low affinity in a few evolutionary steps. Most proteins display moderate to high oxygen affinities and multiple ligand migration paths, which, besides some minor trends, show heterogeneous distributions throughout the phylogenetic tree, again suggesting fast functional adaptation. Our data not only deepens our comprehension of the structural basis governing ligand affinity, but they also highlight some interesting functional evolutionary trends.

The N-terminal pre-A region of Mycobacterium tuberculosis 2/2HbN promotes NO-dioxygenase activity.

UNLABELLED: A unique defense mechanisms by which Mycobacterium tuberculosis protects itself from nitrosative stress is based on the O2 -dependent NO-dioxygenase (NOD) activity of truncated hemoglobin 2/2HbN (Mt2/2HbN). The NOD activity largely depends on the efficiency of ligand migration to the heme cavity through a two-tunnel (long and short) system; recently, it was also correlated with the presence at the Mt2/2HbN N-terminus of a short pre-A region, not conserved in most 2/2HbNs, whose deletion results in a drastic reduction of NO scavenging. In the present study, we report the crystal structure of Mt2/2HbN-ΔpreA, lacking the pre-A region, at a resolution of 1.53 Å. We show that removal of the pre-A region results in long range effects on the protein C-terminus, promoting the assembly of a stable dimer, both in the crystals and in solution. In the Mt2/2HbN-ΔpreA dimer, access of heme ligands to the short tunnel is hindered. Molecular dynamics simulations show that the long tunnel branch is the only accessible pathway for O2 -ligand migration to/from the heme, and that the gating residue Phe(62)E15 partly restricts the diameter of the tunnel. Accordingly, kinetic measurements indicate that the kon value for peroxynitrite isomerization by Mt2/2HbN-ΔpreA-Fe(III) is four-fold lower relative to the full-length protein, and that NO scavenging by Mt2/2HbN-ΔpreA-Fe(II)-O2 is reduced by 35-fold. Therefore, we speculate that Mt2/2HbN evolved to host the pre-A region as a mechanism for preventing dimerization, thus reinforcing the survival of the microorganism against the reactive nitrosative stress in macrophages. DATABASE: Coordinates and structure factors have been deposited in the Protein Data Bank under accession number 5AB8.

Bridging Theory and Experiment to Address Structural Properties of Truncated Haemoglobins: Insights from Thermobifida fusca HbO.

In this chapter, we will discuss the paradigmatic case of Thermobifida fusca (Tf-trHb) HbO in its ferrous and ferric states and its behaviour towards a battery of possible ligands. This choice was dictated by the fact that it has been one of the most extensively studied truncated haemoglobins, both in terms of spectroscopic and molecular dynamics studies. Tf-trHb typifies the structural properties of group II trHbs, as the active site is characterized by a highly polar distal environment in which TrpG8, TyrCD1, and TyrB10 provide three potential H-bond donors in the distal cavity capable of stabilizing the incoming ligands. The role of these residues in key topological positions, and their interplay with the iron-bound ligands, has been addressed in studies carried out on the CO, F(-), OH(-), CN(-), and HS(-) adducts formed with the wild-type protein and a combinatorial set of mutants, in which the distal polar residues, TrpG8, TyrCD1, and TyrB10, have been singly, doubly, or triply replaced by a Phe residue. In this context, such a complete analysis provides an excellent benchmark for the investigation of the relationship between protein structure and function, allowing one to translate physicochemical properties of the active site into the observed functional behaviour. Tf-trHb will be compared with other members of the group II trHbs and, more generally, with members of the other trHb subgroups.

Structural flexibility of the heme cavity in the cold-adapted truncated hemoglobin from the Antarctic marine bacterium Pseudoalteromonas haloplanktis TAC125.

Truncated hemoglobins build one of the three branches of the globin protein superfamily. They display a characteristic two-on-two α-helical sandwich fold and are clustered into three groups (I, II and III) based on distinct structural features. Truncated hemoglobins are present in eubacteria, cyanobacteria, protozoa and plants. Here we present a structural, spectroscopic and molecular dynamics characterization of a group-II truncated hemoglobin, encoded by the PSHAa0030 gene from Pseudoalteromonas haloplanktis TAC125 (Ph-2/2HbO), a cold-adapted Antarctic marine bacterium hosting one flavohemoglobin and three distinct truncated hemoglobins. The Ph-2/2HbO aquo-met crystal structure (at 2.21 Å resolution) shows typical features of group-II truncated hemoglobins, namely the two-on-two α-helical sandwich fold, a helix Φ preceding the proximal helix F, and a heme distal-site hydrogen-bonded network that includes water molecules and several distal-site residues, including His(58)CD1. Analysis of Ph-2/2HbO by electron paramagnetic resonance, resonance Raman and electronic absorption spectra, under varied solution conditions, shows that Ph-2/2HbO can access diverse heme ligation states. Among these, detection of a low-spin heme hexa-coordinated species suggests that residue Tyr(42)B10 can undergo large conformational changes in order to act as the sixth heme-Fe ligand. Altogether, the results show that Ph-2/2HbO maintains the general structural features of group-II truncated hemoglobins but displays enhanced conformational flexibility in the proximity of the heme cavity, a property probably related to the functional challenges, such as low temperature, high O2 concentration and low kinetic energy of molecules, experienced by organisms living in the Antarctic environment.

Reactivity of inorganic sulfide species toward a heme protein model.

The reactivity of inorganic sulfide species toward heme peptides was explored under biorelevant conditions in order to unravel the molecular details of the reactivity of the endogenous hydrogen sulfide toward heme proteins. Unlike ferric porphyrinates, which are reduced by inorganic sulfide, some heme proteins can form stable Fe(III)-sulfide adducts. To isolate the protein factors ruling the redox chemistry, we used as a system model, the undecapeptide microperoxidase (MP11), a heme peptide derived from cytochrome c proteolysis that retains the proximal histidine bound to the Fe(III) atom. Upon addition of gaseous hydrogen sulfide (H2S) at pH 6.8, the UV-vis spectra of MP11 closely resembled those of the low-spin ferric hydroxo complex (only attained at an alkaline pH) and cysteine or alkylthiol derivatives, suggesting that the Fe(III) reduction was prevented. The low-frequency region of the resonance Raman spectrum revealed the presence of an Fe(III)-S band at 366 cm(-1) and the general features of a low-spin hexacoordinated heme. Anhydrous sodium sulfide (Na2S) was the source of sulfide of choice for the kinetic evaluation of the process. Theoretical calculations showed no distal stabilization mechanisms for bound sulfide species in MP11, highlighting a key role of the proximal histidine for the stabilization of the Fe(III)-S adducts of heme compounds devoid of distal counterparts, which is significant with regard to the biochemical reactivity of endogenous hydrogen sulfide.

Ligand uptake in Mycobacterium tuberculosis truncated hemoglobins is controlled by both internal tunnels and active site water molecules.

Mycobacterium tuberculosis, the causative agent of human tuberculosis, has two proteins belonging to the truncated hemoglobin (trHb) family. Mt-trHbN presents well-defined internal hydrophobic tunnels that allow O 2 and •NO to migrate easily from the solvent to the active site, whereas Mt-trHbO possesses tunnels interrupted by a few bulky residues, particularly a tryptophan at position G8. Differential ligand migration rates allow Mt-trHbN to detoxify •NO, a crucial step for pathogen survival once under attack by the immune system, much more efficiently than Mt-trHbO. In order to investigate the differences between these proteins, we performed experimental kinetic measurements, •NO decomposition, as well as molecular dynamics simulations of the wild type Mt-trHbN and two mutants, VG8F and VG8W. These mutations affect both the tunnels accessibility as well as the affinity of distal site water molecules, thus modifying the ligand access to the iron. We found that a single mutation allows Mt-trHbN to acquire ligand migration rates comparable to those observed for Mt-trHbO, confirming that ligand migration is regulated by the internal tunnel architecture as well as by water molecules stabilized in the active site.

Trypsinogen activation as observed in accelerated molecular dynamics simulations.

Serine proteases are involved in many fundamental physiological processes, and control of their activity mainly results from the fact that they are synthetized in an inactive form that becomes active upon cleavage. Three decades ago Martin Karplus's group performed the first molecular dynamics simulations of trypsin, the most studied member of the serine protease family, to address the transition from the zymogen to its active form. Based on the computational power available at the time, only high frequency fluctuations, but not the transition steps, could be observed. By performing accelerated molecular dynamics (aMD) simulations, an interesting approach that increases the configurational sampling of atomistic simulations, we were able to observe the N-terminal tail insertion, a crucial step of the transition mechanism. Our results also support the hypothesis that the hydrophobic effect is the main force guiding the insertion step, although substantial enthalpic contributions are important in the activation mechanism. As the N-terminal tail insertion is a conserved step in the activation of serine proteases, these results afford new perspective on the underlying thermodynamics of the transition from the zymogen to the active enzyme.

Molecular basis of thermal stability in truncated (2/2) hemoglobins.

BACKGROUND: Understanding the molecular mechanism through which proteins are functional at extreme high and low temperatures is one of the key issues in structural biology. To investigate this phenomenon, we have focused on two instructive truncated hemoglobins from Thermobifida fusca (Tf-trHbO) and Mycobacterium tuberculosis (Mt-trHbO); although the two proteins are structurally nearly identical, only the former is stable at high temperatures. METHODS: We used molecular dynamics simulations at different temperatures as well as thermal melting profile measurements of both wild type proteins and two mutants designed to interchange the amino acid residue, either Pro or Gly, at E3 position. RESULTS: The results show that the presence of a Pro at the E3 position is able to increase (by 8°) or decrease (by 4°) the melting temperature of Mt-trHbO and Tf-trHbO, respectively. We observed that the ProE3 alters the structure of the CD loop, making it more flexible. CONCLUSIONS: This gain in flexibility allows the protein to concentrate its fluctuations in this single loop and avoid unfolding. The alternate conformations of the CD loop also favor the formation of more salt-bridge interactions, together augmenting the protein's thermostability. GENERAL SIGNIFICANCE: These results indicate a clear structural and dynamical role of a key residue for thermal stability in truncated hemoglobins.

Ligand uptake modulation by internal water molecules and hydrophobic cavities in hemoglobins.

Internal water molecules play an active role in ligand uptake regulation, since displacement of retained water molecules from protein surfaces or cavities by incoming ligands can promote favorable or disfavorable effects over the global binding process. Detection of these water molecules by X-ray crystallography is difficult given their positional disorder and low occupancy. In this work, we employ a combination of molecular dynamics simulations and ligand rebinding over a broad time range to shed light into the role of water molecules in ligand migration and binding. Computational studies on the unliganded structure of the thermostable truncated hemoglobin from Thermobifida fusca (Tf-trHbO) show that a water molecule is in the vicinity of the iron heme, stabilized by WG8 with the assistance of YCD1, exerting a steric hindrance for binding of an exogenous ligand. Mutation of WG8 to F results in a significantly lower stabilization of this water molecule and in subtle dynamical structural changes that favor ligand binding, as observed experimentally. Water is absent from the fully hydrophobic distal cavity of the triple mutant YB10F-YCD1F-WG8F (3F), due to the lack of residues capable of stabilizing it nearby the heme. In agreement with these effects on the barriers for ligand rebinding, over 97% of the photodissociated ligands are rebound within a few nanoseconds in the 3F mutant case. Our results demonstrate the specific involvement of water molecules in shaping the energetic barriers for ligand migration and binding.

Structure, mechanism, and dynamics of UDP-galactopyranose mutase.

The flavoenzyme UDP-galactopyranose mutase (UGM) is a key enzyme in galactofuranose biosynthesis. The enzyme catalyzes the 6-to-5 ring contraction of UDP-galactopyranose to UDP-galactofuranose. Galactofuranose is absent in humans yet is an essential component of bacterial and fungal cell walls and a cell surface virulence factor in protozoan parasites. Thus, inhibition of galactofuranose biosynthesis is a valid strategy for developing new antimicrobials. UGM is an excellent target in this effort because the product of the UGM reaction represents the first appearance of galactofuranose in the biosynthetic pathway. The UGM reaction is redox neutral, which is atypical for flavoenzymes, motivating intense examination of the chemical mechanism and structural features that tune the flavin for its unique role in catalysis. These studies show that the flavin functions as nucleophile, forming a flavin-sugar adduct that facilitates galactose-ring opening and contraction. The 3-dimensional fold is novel and conserved among all UGMs, however the larger eukaryotic enzymes have additional secondary structure elements that lead to significant differences in quaternary structure, substrate conformation, and conformational flexibility. Here we present a comprehensive review of UGM three-dimensional structure, provide an update on recent developments in understanding the mechanism of the enzyme, and summarize computational studies of active site flexibility.

Substrate-dependent dynamics of UDP-galactopyranose mutase: Implications for drug design.

Trypanosoma cruzi is the causative agent of Chagas disease, a neglected tropical disease that represents one of the major health challenges of the Latin American countries. Successful efforts were made during the last few decades to control the transmission of this disease, but there is still no treatment for the 10 million adults in the chronic phase of the disease. In T. cruzi, as well as in other pathogens, the flavoenzyme UDP-galactopyranose mutase (UGM) catalyzes the conversion of UDP-galactopyranose to UDP-galactofuranose, a precursor of the cell surface ß-galactofuranose that is involved in the virulence of the pathogen. The fact that UGM is not present in humans makes inhibition of this enzyme a good approach in the design of new Chagas therapeutics. By performing a series of computer simulations of T. cruzi UGM in the presence or absence of an active site ligand, we address the molecular details of the mechanism that controls the uptake and retention of the substrate. The simulations suggest a modular mechanism in which each moiety of the substrate controls the flexibility of a different protein loop. Furthermore, the calculations indicate that interactions with the substrate diphosphate moiety are especially important for stabilizing the closed active site. This hypothesis is supported with kinetics measurements of site-directed mutants of T. cruzi UGM. Our results extend our knowledge of UGM dynamics and offer new alternatives for the prospective design of drugs.