Understanding Brain-Skeletal Muscle Crosstalk Impacting Metabolism and Movement.

Metabolism and movement, among the critical determinants in the survival and success of an organism, are tightly regulated by the brain and skeletal muscle. At the cellular level, mitochondria -that powers life, and myosin - the molecular motor of the cell, have both evolved to serve this purpose. Although independently, the skeletal muscle and brain have been intensively investigated for over a century, their coordinated involvement in metabolism and movement remains poorly understood. Therefore, a fundamental understanding of the coordinated involvement of the brain and skeletal muscle in metabolism and movement holds great promise in providing a window to a wide range of life processes and in the development of tools and approaches in disease detection and therapy. Recent developments in new tools, technologies and approaches, and advances in computing power and machine learning, provides for the first time the opportunity to establish a new field of study, the 'Science and Engineering of Metabolism and Movement'. This new field of study could provide substantial new insights and breakthrough into how metabolism and movement is governed at the systems level in an organism. The design and approach to accomplish this objective is briefly discussed in this article.

On the Origin of Hemoglobin Cooperativity under Non-equilibrium Conditions.

Abnormal hemoglobins can have major consequences for tissue delivery of oxygen. Correct diagnosis of hemoglobinopathies with altered oxygen affinity requires a determination of hemoglobin oxygen dissociation curve, which relates the hemoglobin oxygen saturation to the partial pressure of oxygen in the blood. Determination of the oxygen dissociation curve of human hemoglobin is typically carried out under conditions in which hemoglobin is in equilibrium with O2 at each partial pressure. However, in the human body due to the fast transit of red blood cells through tissues hemoglobin oxygen exchanges occur under non-equilibrium conditions. We describe the determination of non-equilibrium oxygen dissociation curve and show that under these conditions the true nature of hemoglobin cooperativity is revealed as emerging solely from the consecutive binding of oxygen to each one of the four subunits of hemoglobin until the entire tetramer is saturated. We call this form of cooperativity the sequential cooperativity of hemoglobin and define the simplest model that includes it as the minimalist model of hemoglobin. A single instantiation of this model accounts for ~70% of hemoglobin cooperativity under non-equilibrium conditions. The total cooperativity of hemoglobin can be viewed more correctly as the summation of two instantiations of the minimalist model (each one corresponding to a tetramer of low and high affinity for O2, respectively) in equilibrium with each other, as in the Monod-Wyman-Changeux model of hemoglobin. In addition to offering new insights on the nature of hemoglobin reaction with oxygen, the methodology described here for the determination of hemoglobin non-equilibrium oxygen dissociation curve provides a simple, fast, low-cost alternative to complex spectrophotometric methods, which is expected to be particularly valuable in regions where hemoglobinopathies are a significant public health problem, but where highly specialized laboratories capable of determining a traditional oxygen dissociation curve are not easily accessible.

Nanoscale imaging using differential expansion microscopy.

Expensive and time-consuming approaches of immunoelectron microscopy of biopsy tissues continues to serve as the gold-standard for diagnostic pathology. The recent development of the new approach of expansion microscopy (ExM) capable of fourfold lateral expansion of biological specimens for their morphological examination at approximately 70 nm lateral resolution using ordinary diffraction limited optical microscopy, is a major advancement in cellular imaging. Here we report (1) an optimized fixation protocol for retention of cellular morphology while obtaining optimal expansion, (2) an ExM procedure for up to eightfold lateral and over 500-fold volumetric expansion, (3) demonstrate that ExM is anisotropic or differential between tissues, cellular organelles and domains within organelles themselves, and (4) apply image analysis and machine learning (ML) approaches to precisely assess differentially expanded cellular structures. We refer to this enhanced ExM approach combined with ML as differential expansion microscopy (DiExM), applicable to profiling biological specimens at the nanometer scale. DiExM holds great promise for the precise, rapid and inexpensive diagnosis of disease from pathological specimen slides.

Human skeletal muscle cell atlas: Unraveling cellular secrets utilizing 'muscle-on-a-chip', differential expansion microscopy, mass spectrometry, nanothermometry and machine learning.

The 'Human Cell Atlas' project has been launched to obtain a comprehensive understanding of all cell types, the fundamental living units that constitute the human body. This is a global partnership and effort involving experts from many disciplines, from computer science, engineering to medicine, and is supported by several private and public organizations, among them, the Chan Zuckerberg Foundation, the National Institutes of Health, and Google, that will greatly benefit humanity. Nearly 37 trillion cells of various shapes, sizes, and composition, are precisely organized to constitute the human body. Humans, like all other living organisms, are dynamic, and therefore a comprehensive understanding of different cells in their various dynamic states is required to provide a reference map for the early diagnosis and various preventive approach to disease, and in the development of precision therapeutics. Skeletal muscles being the most abundant tissue and the largest locomotor and metabolic organ in the human body, requires a global understanding of its structure, composition, and function. The objective of creating a 'Human Skeletal Muscle Cell Atlas', necessitates therefore a comprehensive understanding of the emergent properties of skeletal muscle cell growth, development, structure, function and chemistry, under conditions of activity and inactivity. To achieve this objective would require a very precise yet rapid and cost-effective approach of combined multimodal imaging, including our new and novel 'Differential Expansion Microscopy', our 'Nanoscale Thermometry', combined with 'Mass Spectrometry', 'Motor Protein Motility Assay' and 'Machine Learning' tools.

High-throughput RNA sequencing reveals structural differences of orthologous brain-expressed genes between western lowland gorillas and humans.

The human brain and human cognitive abilities are strikingly different from those of other great apes despite relatively modest genome sequence divergence. However, little is presently known about the interspecies divergence in gene structure and transcription that might contribute to these phenotypic differences. To date, most comparative studies of gene structure in the brain have examined humans, chimpanzees, and macaque monkeys. To add to this body of knowledge, we analyze here the brain transcriptome of the western lowland gorilla (Gorilla gorilla gorilla), an African great ape species that is phylogenetically closely related to humans, but with a brain that is approximately one-third the size. Manual transcriptome curation from a sample of the planum temporale region of the neocortex revealed 12 protein-coding genes and one noncoding-RNA gene with exons in the gorilla unmatched by public transcriptome data from the orthologous human loci. These interspecies gene structure differences accounted for a total of 134 amino acids in proteins found in the gorilla that were absent from protein products of the orthologous human genes. Proteins varying in structure between human and gorilla were involved in immunity and energy metabolism, suggesting their relevance to phenotypic differences. This gorilla neocortical transcriptome comprises an empirical, not homology- or prediction-driven, resource for orthologous gene comparisons between human and gorilla. These findings provide a unique repository of the sequences and structures of thousands of genes transcribed in the gorilla brain, pointing to candidate genes that may contribute to the traits distinguishing humans from other closely related great apes.

Nodal versus Total Axonal Strain and the Role of Cholesterol in Traumatic Brain Injury.

Traumatic brain injury (TBI) is a health threat that affects every year millions of people involved in motor vehicle and sporting accidents, and thousands of soldiers in battlefields. Diffuse axonal injury (DAI) is one of the most frequent types of TBI leading to death. In DAI, the initial traumatic event is followed by a cascade of biochemical changes that take time to develop in full, so that symptoms may not become apparent until days or weeks after the original injury. Hence, DAI is a dynamic process, and the opportunity exists to prevent its progression provided the initial trauma can be predicted at the molecular level. Here, we present preliminary evidence from micro-finite element (FE) simulations that the mechanical response of central nervous system myelinated fibers is dependent on the axonal diameter, the ratio between axon diameter and fiber diameter (g-ratio), the microtubules density, and the cholesterol concentration in the axolemma and myelin. A key outcome of the simulations is that there is a significant difference between the overall level of strain in a given axonal segment and the level of local strain in the Ranvier nodes contained in that segment, with the nodal strain being much larger than the total strain. We suggest that the acquisition of this geometric and biochemical information by means of already available high resolution magnetic resonance imaging techniques, and its incorporation in current FE models of the brain will enhance the models capacity to predict the site and magnitude of primary axonal damage upon TBI.

Multidimensional mutual information methods for the analysis of covariation in multiple sequence alignments.

BACKGROUND: Several methods are available for the detection of covarying positions from a multiple sequence alignment (MSA). If the MSA contains a large number of sequences, information about the proximities between residues derived from covariation maps can be sufficient to predict a protein fold. However, in many cases the structure is already known, and information on the covarying positions can be valuable to understand the protein mechanism and dynamic properties. RESULTS: In this study we have sought to determine whether a multivariate (multidimensional) extension of traditional mutual information (MI) can be an additional tool to study covariation. The performance of two multidimensional MI (mdMI) methods, designed to remove the effect of ternary/quaternary interdependencies, was tested with a set of 9 MSAs each containing <400 sequences, and was shown to be comparable to that of the newest methods based on maximum entropy/pseudolikelyhood statistical models of protein sequences. However, while all the methods tested detected a similar number of covarying pairs among the residues separated by < 8 Å in the reference X-ray structures, there was on average less than 65% overlap between the top scoring pairs detected by methods that are based on different principles. CONCLUSIONS: Given the large variety of structure and evolutionary history of different proteins it is possible that a single best method to detect covariation in all proteins does not exist, and that for each protein family the best information can be derived by merging/comparing results obtained with different methods. This approach may be particularly valuable in those cases in which the size of the MSA is small or the quality of the alignment is low, leading to significant differences in the pairs detected by different methods.

Biapenem inactivation by B2 metallo ß-lactamases: energy landscape of the hydrolysis reaction.

BACKGROUND: A general mechanism has been proposed for metallo ß-lactamases (MßLs), in which deprotonation of a water molecule near the Zn ion(s) results in the formation of a hydroxide ion that attacks the carbonyl oxygen of the ß-lactam ring. However, because of the absence of X-ray structures that show the exact position of the antibiotic in the reactant state (RS) it has been difficult to obtain a definitive validation of this mechanism. METHODOLOGY/PRINCIPAL FINDINGS: We have employed a strategy to identify the RS, which does not rely on substrate docking and/or molecular dynamics. Starting from the X-ray structure of the enzyme:product complex (the product state, PS), a QM/MM scan was used to drive the reaction uphill from product back to reactant. Since in this process also the enzyme changes from PS to RS, we actually generate the enzyme:substrate complex from product and avoid the uncertainties associated with models of the reactant state. We used this strategy to study the reaction of biapenem hydrolysis by B2 MßL CphA. QM/MM simulations were carried out under 14 different ionization states of the active site, in order to generate potential energy surfaces (PESs) corresponding to a variety of possible reaction paths. CONCLUSIONS/SIGNIFICANCE: The calculations support a model for biapenem hydrolysis by CphA, in which the nucleophile that attacks the ß-lactam ring is not the water molecule located in proximity of the active site Zn, but a second water molecule, hydrogen bonded to the first one, which is used up in the reaction, and thus is not visible in the X-ray structure of the enzyme:product complex.

Accurate simulation and detection of coevolution signals in multiple sequence alignments.

BACKGROUND: While the conserved positions of a multiple sequence alignment (MSA) are clearly of interest, non-conserved positions can also be important because, for example, destabilizing effects at one position can be compensated by stabilizing effects at another position. Different methods have been developed to recognize the evolutionary relationship between amino acid sites, and to disentangle functional/structural dependencies from historical/phylogenetic ones. METHODOLOGY/PRINCIPAL FINDINGS: We have used two complementary approaches to test the efficacy of these methods. In the first approach, we have used a new program, MSAvolve, for the in silico evolution of MSAs, which records a detailed history of all covarying positions, and builds a global coevolution matrix as the accumulated sum of individual matrices for the positions forced to co-vary, the recombinant coevolution, and the stochastic coevolution. We have simulated over 1600 MSAs for 8 protein families, which reflect sequences of different sizes and proteins with widely different functions. The calculated coevolution matrices were compared with the coevolution matrices obtained for the same evolved MSAs with different coevolution detection methods. In a second approach we have evaluated the capacity of the different methods to predict close contacts in the representative X-ray structures of an additional 150 protein families using only experimental MSAs. CONCLUSIONS/SIGNIFICANCE: Methods based on the identification of global correlations between pairs were found to be generally superior to methods based only on local correlations in their capacity to identify coevolving residues using either simulated or experimental MSAs. However, the significant variability in the performance of different methods with different proteins suggests that the simulation of MSAs that replicate the statistical properties of the experimental MSA can be a valuable tool to identify the coevolution detection method that is most effective in each case.

Biapenem inactivation by B2 metallo ß-lactamases: energy landscape of the post-hydrolysis reactions.

BACKGROUND: The first line of defense by bacteria against ß-lactam antibiotics is the expression of ß-lactamases, which cleave the amide bond of the ß-lactam ring. In the reaction of biapenem inactivation by B2 metallo ß-lactamases (MßLs), after the ß-lactam ring is opened, the carboxyl group generated by the hydrolytic process and the hydroxyethyl group (common to all carbapenems) rotate around the C5-C6 bond, assuming a new position that allows a proton transfer from the hydroxyethyl group to C2, and a nucleophilic attack on C3 by the oxygen atom of the same side-chain. This process leads to the formation of a bicyclic compound, as originally observed in the X-ray structure of the metallo ß-lactamase CphA in complex with product. METHODOLOGY/PRINCIPAL FINDINGS: QM/MM and metadynamics simulations of the post-hydrolysis steps in solution and in the enzyme reveal that while the rotation of the hydroxyethyl group can occur in solution or in the enzyme active site, formation of the bicyclic compound occurs primarily in solution, after which the final product binds back to the enzyme. The calculations also suggest that the rotation and cyclization steps can occur at a rate comparable to that observed experimentally for the enzymatic inactivation of biapenem only if the hydrolysis reaction leaves the N4 nitrogen of the ß-lactam ring unprotonated. CONCLUSIONS/SIGNIFICANCE: The calculations support the existence of a common mechanism (in which ionized N4 is the leaving group) for carbapenems hydrolysis in all MßLs, and suggest a possible revision of mechanisms for B2 MßLs in which the cleavage of the ß-lactam ring is associated with or immediately followed by protonation of N4. The study also indicates that the bicyclic derivative of biapenem has significant affinity for B2 MßLs, and that it may be possible to obtain clinically effective inhibitors of these enzymes by modification of this lead compound.

Protein phosphorylation and prevention of cytochrome oxidase inhibition by ATP: coupled mechanisms of energy metabolism regulation.

Rapid regulation of oxidative phosphorylation is crucial for mitochondrial adaptation to swift changes in fuels availability and energy demands. An intramitochondrial signaling pathway regulates cytochrome oxidase (COX), the terminal enzyme of the respiratory chain, through reversible phosphorylation. We find that PKA-mediated phosphorylation of a COX subunit dictates mammalian mitochondrial energy fluxes and identify the specific residue (S58) of COX subunit IV-1 (COXIV-1) that is involved in this mechanism of metabolic regulation. Using protein mutagenesis, molecular dynamics simulations, and induced fit docking, we show that mitochondrial energy metabolism regulation by phosphorylation of COXIV-1 is coupled with prevention of COX allosteric inhibition by ATP. This regulatory mechanism is essential for efficient oxidative metabolism and cell survival. We propose that S58 COXIV-1 phosphorylation has evolved as a metabolic switch that allows mammalian mitochondria to rapidly toggle between energy utilization and energy storage.

The contribution of coevolving residues to the stability of KDO8P synthase.

BACKGROUND: The evolutionary tree of 3-deoxy-D-manno-octulosonate 8-phosphate (KDO8P) synthase (KDO8PS), a bacterial enzyme that catalyzes a key step in the biosynthesis of bacterial endotoxin, is evenly divided between metal and non-metal forms, both having similar structures, but diverging in various degrees in amino acid sequence. Mutagenesis, crystallographic and computational studies have established that only a few residues determine whether or not KDO8PS requires a metal for function. The remaining divergence in the amino acid sequence of KDO8PSs is apparently unrelated to the underlying catalytic mechanism. METHODOLOGY/PRINCIPAL FINDINGS: The multiple alignment of all known KDO8PS sequences reveals that several residue pairs coevolved, an indication of their possible linkage to a structural constraint. In this study we investigated by computational means the contribution of coevolving residues to the stability of KDO8PS. We found that about 1/4 of all strongly coevolving pairs probably originated from cycles of mutation (decreasing stability) and suppression (restoring it), while the remaining pairs are best explained by a succession of neutral or nearly neutral covarions. CONCLUSIONS/SIGNIFICANCE: Both sequence conservation and coevolution are involved in the preservation of the core structure of KDO8PS, but the contribution of coevolving residues is, in proportion, smaller. This is because small stability gains or losses associated with selection of certain residues in some regions of the stability landscape of KDO8PS are easily offset by a large number of possible changes in other regions. While this effect increases the tolerance of KDO8PS to deleterious mutations, it also decreases the probability that specific pairs of residues could have a strong contribution to the thermodynamic stability of the protein.

Common basis for the mechanism of metallo and non-metallo KDO8P synthases.

The three-dimensional structures of metal and non-metal enzymes that catalyze the same reaction are often quite different, a clear indication of convergent evolution. However, there are interesting cases in which the same scaffold supports both a metal and a non-metal catalyzed reaction. One of these is 3-deoxy-D-manno-octulosonate 8-phosphate (KDO8P) synthase (KDO8PS), a bacterial enzyme that catalyzes the synthesis of KDO8P and inorganic phosphate (P(i)) from phosphoenolpyruvate (PEP), arabinose 5-phosphate (A5P), and water. This reaction is one of the key steps in the biosynthesis of bacterial endotoxins. The evolutionary tree of KDO8PS is evenly divided between metal and non-metal forms, both having essentially identical structures. Mutagenesis and crystallographic studies suggest that one or two residues at most determine whether or not KDO8PS requires a metal for function, a clear example of "minimalist evolution". Quantum mechanical/molecular mechanical (QM/MM) simulations of both the enzymatic and non-enzymatic synthesis of KDO8P have revealed the mechanism underlying the switch between metal and non-metal dependent catalysis. The principle emerging from these studies is that this conversion is possible in KDO8PS because the metal is not involved in an activation process, but primarily contributes to orienting properly the reactants to lower the activation energy, an action easily mimicked by amino acid side-chains.

Defining the pathogenesis of the human Atp12p W94R mutation using a Saccharomyces cerevisiae yeast model.

Studies in yeast have shown that a deficiency in Atp12p prevents assembly of the extrinsic domain (F(1)) of complex V and renders cells unable to make ATP through oxidative phosphorylation. De Meirleir et al. (De Meirleir, L., Seneca, S., Lissens, W., De Clercq, I., Eyskens, F., Gerlo, E., Smet, J., and Van Coster, R. (2004) J. Med. Genet. 41, 120-124) have reported that a homozygous missense mutation in the gene for human Atp12p (HuAtp12p), which replaces Trp-94 with Arg, was linked to the death of a 14-month-old patient. We have investigated the impact of the pathogenic W94R mutation on Atp12p structure/function. Plasmid-borne wild type human Atp12p rescues the respiratory defect of a yeast ATP12 deletion mutant (Deltaatp12). The W94R mutation alters the protein at the most highly conserved position in the Pfam sequence and renders HuAtp12p insoluble in the background of Deltaatp12. In contrast, the yeast protein harboring the corresponding mutation, ScAtp12p(W103R), is soluble in the background of Deltaatp12 but not in the background of Deltaatp12Deltafmc1, a strain that also lacks Fmc1p. Fmc1p is a yeast mitochondrial protein not found in higher eukaryotes. Tryptophan 94 (human) or 103 (yeast) is located in a positively charged region of Atp12p, and hence its mutation to arginine does not alter significantly the electrostatic properties of the protein. Instead, we provide evidence that the primary effect of the substitution is on the dynamic properties of Atp12p.

The energy landscape of 3-deoxy-D-manno-octulosonate 8-phosphate synthase.

3-Deoxy-d-manno-octulosonate 8-phosphate (KDO8P) synthase catalyzes the condensation of arabinose 5-phosphate (A5P) and phosphoenolpyruvate (PEP) to form KDO8P, a key precursor in the biosynthesis of the endotoxin of Gram-negative bacteria. Earlier studies have established that the condensation occurs with a syn addition of water to the si side of C2(PEP) and of C3(PEP) to the re side of C1(A5P). Two stepwise mechanisms have been proposed for this reaction. One involves a transient carbanion intermediate, formed by attack of water or a hydroxide ion on C2(PEP). The other involves a transient oxocarbenium zwitterionic intermediate, formed by direct attack of C3(PEP) onto C1(A5P), followed by reaction of water at C2. In both cases, the transient intermediates are expected to converge to a more stable tetrahedral intermediate, which decays into KDO8P and inorganic phosphate. In this study we calculated the potential energy surfaces (PESs) associated with all possible reaction paths in the active site of KDO8PS: the path involving a syn addition of water to the si side of C2(PEP) and of C3(PEP) to the re side of C1(A5P), with the PEP phosphate group deprotonated, has the lowest energy barrier ( approximately 14 kcal/mol) and is strongly exoergonic (reaction energy of -38 kcal/mol). Consistent with the experimental observations, other potential reaction paths, like an anti addition of water to the re side of C2(PEP) or addition of C3(PEP) to the si side of C1(A5P), are associated with much higher barriers. An important new finding of this study is that the lowest energy reaction path does not correspond to either one of the pure stepwise mechanisms proposed formerly but can be described instead as a partially concerted reaction between PEP, A5P, and water. The success in using PESs to reproduce established features of the reaction and to discriminate between different mechanisms suggests that this approach may be of general utility in the study of other enzymatic reactions.

Chaperones of F1-ATPase.

Mitochondrial F(1)-ATPase contains a hexamer of alternating alpha and beta subunits. The assembly of this structure requires two specialized chaperones, Atp11p and Atp12p, that bind transiently to beta and alpha. In the absence of Atp11p and Atp12p, the hexamer is not formed, and alpha and beta precipitate as large insoluble aggregates. An early model for the mechanism of chaperone-mediated F(1) assembly (Wang, Z. G., Sheluho, D., Gatti, D. L., and Ackerman, S. H. (2000) EMBO J. 19, 1486-1493) hypothesized that the chaperones themselves look very much like the alpha and beta subunits, and proposed an exchange of Atp11p for alpha and of Atp12p for beta; the driving force for the exchange was expected to be a higher affinity of alpha and beta for each other than for the respective chaperone partners. One important feature of this model was the prediction that as long as Atp11p is bound to beta and Atp12p is bound to alpha, the two F(1) subunits cannot interact at either the catalytic site or the noncatalytic site interface. Here we present the structures of Atp11p from Candida glabrata and Atp12p from Paracoccus denitrificans, and we show that some features of the Wang model are correct, namely that binding of the chaperones to alpha and beta prevents further interactions between these F(1) subunits. However, Atp11p and Atp12p do not resemble alpha or beta, and it is instead the F(1) gamma subunit that initiates the release of the chaperones from alpha and beta and their further assembly into the mature complex.

Electronic structure of the metal center in the Cd(2+), Zn(2+), and Cu(2+) substituted forms of KDO8P synthase: implications for catalysis.

Aquifex aeolicus 3-deoxy-d-manno-octulosonate 8-phosphate synthase (KDO8PS) is active with a variety of different divalent metal ions bound in the active site. The Cd(2+), Zn(2+), and Cu(2+) substituted enzymes display similar values of k(cat) and similar dependence of K(m)(PEP) and K(m)(A5P) on both substrate and product concentrations. However, the flux-control coefficients for some of the catalytically relevant reaction steps are different in the presence of Zn(2+) or Cu(2+), suggesting that the type of metal bound in the active site affects the behavior of the enzyme in vivo. The type of metal also affects the rate of product release in the crystal environment. For example, the crystal structure of the Cu(2+) enzyme incubated with phosphoenolpyruvate (PEP) and arabinose 5-phosphate (A5P) shows the formed product, 3-deoxy-d-manno-octulosonate 8-phosphate (KDO8P), still bound in the active site in its linear conformation. This observation completes our structural studies of the condensation reaction, which altogether have provided high-resolution structures for the reactants, the intermediate, and the product bound forms of KDO8PS. The crystal structures of the Cd(2+), Zn(2+), and Cu(2+) substituted enzymes show four residues (Cys-11, His-185, Glu-222, and Asp-233) and a water molecule as possible metal ligands. Combined quantum mechanics/molecular mechanics (QM/MM) geometry optimizations reveal that the metal centers have a delocalized electronic structure, and that their true geometry is square pyramidal for Cd(2+) and Zn(2+) and distorted octahedral or distorted tetrahedral for Cu(2+). These geometries are different from those obtained by QM optimization in the gas phase (tetrahedral for Cd(2+) and Zn(2+), distorted tetrahedral for Cu(2+)) and may represent conformations of the metal center that minimize the reorganization energy between the substrate-bound and product-bound states. The QM/MM calculations also show that when only PEP is bound to the enzyme the electronic structure of the metal center is optimized to prevent a wasteful reaction of PEP with water.

Structural and mechanistic changes along an engineered path from metallo to nonmetallo 3-deoxy-D-manno-octulosonate 8-phosphate synthases.

There are two classes of KDO8P synthases characterized respectively by the presence or absence of a metal in the active site. The nonmetallo KDO8PS from Escherichia coli and the metallo KDO8PS from Aquifex aeolicus are the best characterized members of each class. All amino acid residues that make important contacts with the substrates are conserved in both enzymes with the exception of Pro-10, Cys-11, Ser-235, and Gln-237 of the A. aeolicus enzyme, which correspond respectively to Met-25, Asn-26, Pro-252, and Ala-254 in the E. coli enzyme. Interconversion between the two forms of KDO8P synthases can be achieved by substituting the metal-coordinating cysteine of metallo synthases with the corresponding asparagine of nonmetallo synthases, and vice versa. In this report we describe the structural changes elicited by the C11N mutation and by three combinations of mutations (P10M/C11N, C11N/S235P/Q237A, and P10M/C11N/S235P/Q237A) situated along possible evolutionary paths connecting the A. aeolicus and the E. coli enzyme. All four mutants are not capable of binding metal and lack the structural asymmetry among subunits with regard to substrate binding and conformation of the L7 loop, which is typical of A. aeolicus wild-type KDO8PS but is absent in the E. coli enzyme. Despite the lack of the active site metal, the mutant enzymes display levels of activity ranging from 46% to 24% of the wild type. With the sole exception of the quadruple mutant, metal loss does not affect the thermal stability of KDO8PS. The free energy of unfolding in water is also either unchanged or even increased in the mutant enzymes, suggesting that the primary role of the active site metal in A. aeolicus KDO8PS is not to increase the enzyme stability. In all four mutants A5P binding displaces a water molecule located on the si side of PEP. In particular, in the double and triple mutant, A5P binds with the aldehyde carbonyl in hydrogen bond distance of Asn-11, while in the wild type this functional group points away from Cys-11. This alternative conformation of A5P is likely to have functional significance as it resembles the conformation of the acyclic reaction intermediate, which is observed here for the first time in some of the active sites of the triple mutant. The direct visualization of this intermediate by X-ray crystallography confirms earlier mechanistic models of KDO8P synthesis. In particular, the configuration of the C2 chiral center of the intermediate supports a model of the reaction in nonmetallo KDO8PS, in which water attacks an oxocarbenium ion or PEP from the si side of C2. Several explanations are offered to reconcile this observation with the fact that no water molecule is observed at this position in the mutant enzymes in the presence of both PEP and A5P. Significant differences were observed between the wild-type and the mutant enzymes in the Km values for PEP and A5P and in the Kd values for inorganic phosphate and R5P. These differences may reflect an evolutionary adaptation of metallo and nonmetallo KDO8PS's to the cellular concentrations of these metabolites in their respective hosts.

The catalytic and conformational cycle of Aquifex aeolicus KDO8P synthase: role of the L7 loop.

KDO8P synthase catalyzes the condensation of arabinose 5-phosphate (A5P) and phosphoenolpyruvate (PEP) to form the 8-carbon sugar KDO8P and inorganic phosphate (P(i)). The X-ray structure of the wild-type enzyme shows that when both PEP and A5P bind, the active site becomes isolated from the environment due to a conformational change of the L7 loop. The structures of the R106G mutant, without substrates, and with PEP and PEP plus A5P bound, were determined and reveal that in R106G closure of the L7 loop is impaired. The structural perturbations originating from the loss of the Arg(106) side chain point to a role of the L2 loop in stabilizing the closed conformation of the L7 loop. Despite the increased exposure of the R106G active site, no abnormal reaction of PEP with water was observed, ruling out the hypothesis that the primary function of the L7 loop is to shield the active site from bulk solvent during the condensation reaction. However, the R106G enzyme displays several kinetic abnormalities on both the substrate side (smaller K(m)(PEP), larger K(i)(A5P) and K(m)(A5P)) and the product side (smaller K(i)(Pi) and K(i)(KDO8P)) of the reaction. As a consequence, the mutant enzyme is less severely inhibited by A5P and more severely inhibited by P(i) and KDO8P. Simulations of the flux of KDO8P synthesis under metabolic steady-state conditions (constant concentration of reactants and products over time) suggest that in vivo R106G is expected to perform optimally in a narrower range of substrate and product concentrations than the wild-type enzyme.

Removal of a methyl group causes global changes in p-hydroxybenzoate hydroxylase.

p-Hydroxybenzoate hydroxylase (PHBH) is a homodimeric flavoprotein monooxygenase that catalyzes the hydroxylation of p-hydroxybenzoate to form 3,4-dihydroxybenzoate. Controlled catalysis is achieved by movement of the flavin and protein between three conformations, in, out, and open [Entsch, B., et al. (2005) Arch. Biochem. Biophys. 433, 297-311]. The open conformation is important for substrate binding and product release, the in conformation for reaction with oxygen and hydroxylation, and the out conformation for the reduction of FAD by NADPH. The open conformation is similar to the structure of Arg220Gln-PHBH in which the backbone peptide loop of residues 43-46, located on the si side of the flavin, is rotated. In this paper, we examine the structure and properties of the Ala45Gly-PHBH mutant enzyme. The crystal structure of the Ala45Gly enzyme is an asymmetric dimer, with one monomer similar (but not identical) to wild-type PHBH, while the other monomer has His72 flipped into solvent and replaced with Glu73 as one of several changes in the structure. The two structures correlate with evidence from kinetic studies for two forms of Ala45Gly-PHBH. One form of the enzyme dominates turnover and hydroxylates, while the other contributes little to turnover and fails to hydroxylate. Ala45Gly-PHBH favors the in conformation over alternative conformations. The effect of this mutation on the structure and function of PHBH illustrates the importance of the si side loop in the conformational state of PHBH and, consequently, the function of the enzyme. This work demonstrates some general principles of how enzymes use conformational movements to allow both access and egress of substrates and product, while restricting access to the solvent at a critical stage in catalysis.