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PubMed1992

Characterization and regulation of the 58,000-dalton cellular inhibitor of the interferon-induced, dsRNA-activated protein kinase.

The P68 protein kinase is a serine/threonine kinase induced by interferon treatment and activated by double-stranded RNAs (dsRNAs). Once activated, the kinase phosphorylates its natural substrate, the alpha subunit of eukaryotic initiation factor 2 (eIF-2) leading to potential limitations in functional eIF-2 and decreases in protein synthesis initiation. We have recently purified from influenza virus-infected cells a P68 kinase inhibitor, found to be a 58-kDa cellular protein. We have now investigated the mechanisms by which the 58-kDa inhibitor regulates P68 kinase activity and how the inhibitor itself is controlled. The 58-kDa inhibitor did not function by degrading or sequestering the dsRNA activator of P68 but could repress phosphorylation of eIF-2 alpha by an already activated protein kinase. Utilizing antibody prepared against a 58-kDa-specific peptide, we showed that the 58-kDa proteins from infected and uninfected cells were present in equivalent amounts. Although kinase inhibitory activity could not be detected in crude uninfected cell extracts, ammonium sulfate treatment unmasked this activity and allowed purification of the cellular inhibitor with identical chromatographic properties as that from influenza virus-infected cells. Finally, we have identified and partially purified a specific inhibitor of the 58-kDa protein which we refer to as an "anti-inhibitor." Based on these data, we present a model depicting the complex regulation of the interferon-induced protein kinase in eukaryotic cells.

Amino Acid Sequence↗

PubMed1997

Identification of synaptic connections in neural ensembles by graphical models.

A method for the identification of direct synaptic connections in a larger neural net is presented. It is based on a conditional correlation graph for multivariate point processes. The connections are identified via the partial spectral coherence of two neurons, given all others. It is shown how these coherences can be calculated by inversion of the spectral density matrix. In simulations with GENESIS, we discuss the relevance of the method for identifying different neural ensembles including an excitatory feedback loop and networks with lateral inhibitions.

Computer Graphics↗

PubMed1997

Structure of the collagen-binding domain from a Staphylococcus aureus adhesin.

The crystal structure of the recombinant 19,000 M(r) binding domain from the Staphylococcus aureus collagen adhesin has been determined at 2 A resolution. The domain fold is a jelly-roll, composed of two antiparallel beta-sheets and two short alpha-helices. Triple-helical collagen model probes were used in a systematic docking search to identify the collagen-binding site. A groove on beta-sheet I exhibited the best surface complementarity to the collagen probes. This site partially overlaps with the peptide sequence previously shown to be critical for collagen binding. Recombinant proteins containing single amino acid mutations designed to disrupt the surface of the putative binding site exhibited significantly lower affinities for collagen. Here we present a structural perspective for the mode of collagen binding by a bacterial surface protein.

Adhesins, Bacterial↗

PubMed1995

Quantitative changes of the two major AgNOR proteins, nucleolin and protein B23, related to stimulation of rDNA transcription.

The relationship between the amount of the two major AgNOR proteins, nucleolin and protein B23, and the rate of ribosomal RNA (rRNA) synthesis was studied in cortisol-treated and regenerating rat hepatocytes after partial hepatectomy. In both experimental models the synthesis of rRNA was greatly stimulated, but only in regenerating hepatocytes was the increased synthesis associated with cells entering the mitotic cycle. Nucleolin and protein B23 were identified on SDS-polyacrylamide gels of nuclear proteins transferred to nitrocellulose and detected (1) by immunoreaction with specific monoclonal antibodies revealed by second antibodies coupled to peroxidase followed by chemiluminescence or (2) by the silver-staining procedure for AgNOR proteins. Nucleolin and protein B23 were then quantified by computerized densitometric analysis of the immunolabeling signals or the silver-stained bands at 105 kDa (nucleolin) and 39 kDa (protein B23). The synthesis of rRNA was measured by evaluating the amount of radioactivity incorporated into pre-rRNA after [3H]orotic acid injection. Densitometric analysis of silver-stained bands and immunolabeling signals showed no change in nucleolin and protein B23 amounts in cortisol-stimulated hepatocytes, whereas a moderate increase was found in regenerating hepatocytes at 12 h after partial hepatectomy. In both cortisol-stimulated and regenerating hepatocytes the synthesis of rRNA was highly increased (2.6-fold and 4.3-fold above the control level, respectively). To ascertain the relationship between quantitative changes in nucleolin and protein B23 and stimulation of rRNA transcriptional activity in regenerating hepatocytes, the quantitative distribution of these proteins was also investigated in the early times of regeneration using silver-stained nitrocellulose-transblotted nuclear proteins. The quantity of protein B23 was unchanged until 12 h after partial hepatectomy, whereas nucleolin appeared to be slightly increased at 9 h (1.15-fold above the control value) after partial hepatectomy. On the other hand, at just 6 h after partial hepatectomy, a significant increase of rRNA synthesis occurred in regenerating rat hepatocytes (1.8-fold above the control value). These data demonstrated that stimulation of rRNA transcriptional activity occurring in rat hepatocytes after cortisol treatment and in the early times after partial hepatectomy was not associated with quantitative changes in the amounts of nucleolin and protein B23.

Animals↗

PubMed1982

Structure-antitumor activity relationships of 9-anilinoacridines using pattern recognition.

A pattern-recognition analysis using the ADAPT system was performed on a set of 9-anilinoacridine antitumor agents, to determine whether computer-generated descriptors could be used to separate active from inactive compounds. A training set of 213 compounds was chosen by random computer selection from a list of 776 structures. Maximal increase in life span at the LD10 dosage, a response which is difficult to model using traditional Hansch analysis, was used as the measure of biological activity. A set of 18 molecular descriptors, including fragment, substructure environment, and physicochemical property descriptors (molar refraction, partial electronic charge) was identified which could correctly classify 94% of the compounds in the training set (97% of active and 85% of inactive compounds). Eight of the inactive compounds that were misclassified contained amino substituents, suggesting a role for ionization. The weight vector that was obtained from the training set was applied to a prediction set of 50 compounds that were not included in the original analysis and to a set of 69 structures drawn from the recent literature. The prediction set results, ranging from 73 to 86% correct, were lower than those of the training set, but they clearly indicate that pattern-recognition techniques can be useful in the screening of proposed or already existing agents and especially useful for the identification of active compounds.

Aminoacridines↗

PubMed2004

Filling gaps in a metabolic network using expression information.

MOTIVATION: The metabolic models of both newly sequenced and well-studied organisms contain reactions for which the enzymes have not been identified yet. We present a computational approach for identifying genes encoding such missing metabolic enzymes in a partially reconstructed metabolic network. RESULTS: The metabolic expression placement (MEP) method relies on the coexpression properties of the metabolic network and is complementary to the sequence homology and genome context methods that are currently being used to identify missing metabolic genes. The MEP algorithm predicts over 20% of all known Saccharomyces cerevisiae metabolic enzyme-encoding genes within the top 50 out of 5594 candidates for their enzymatic function, and 70% of metabolic genes whose expression level has been significantly perturbed across the conditions of the expression dataset used. AVAILABILITY: Freely available (in Supplementary information).

Algorithms↗

PubMed2002

Analyses of genotypic diversity among North, South, and Central American isolates of sugarcane yellow leaf virus: evidence for Colombian origins and for intraspecific spatial phylogenetic variation.

We have analyzed the genotypic diversity of sugarcane yellow leaf virus (SCYLV) collected from North, South, and Central America by fingerprinting assays and selective cDNA cloning and sequencing. One group of isolates from Colombia, designated the C-population, has been identified as residing at the root node between a separable superpopulation structure of SCYLV and other members of the family Luteoviridae, indicating that the progenitor viruses of the North, South, and Central American isolates of the SCYLV superpopulation most likely arose from a C-population structure. From a model of intrafamilial evolution (F. Moonan et al., Virology 269:156-171, 2000), a prediction could be made that within the SCYLV species, the capacity of genomic sequence divergence would range from lowest in the capsid protein open reading frame 3 (ORF 3) to highest in a region spanning across the carboxy-terminal end of the RNA-dependent RNA polymerase ORF. We have demonstrated the validity and applicability of this intrafamilial model for the prediction of intraspecies SCYLV diversity. Analysis of spatial phylogenetic variation (SPV) within the SCYLV isolates could not be assessed by application of a "partial likelihoods assessed through optimization" (PLATO)-derived intraspecies model alone. However, application of a PLATO-derived intrafamilial model with the intraspecies-derived model allowed distinction of three forms of SPV. Two of the SPV forms identified correspond to the extremes in a continuum of sequence evolution displayed in a SCYLV superpopulation structure, and the third form was diagnostic of a C-population structure. The application of these types of models has value in terms of predicting the types of SCYLV intraspecies diversity that may exist worldwide, and in general, may be useful in application for more informed design of transgenes for use in the elicitation of homology-dependent virus resistance mechanisms in transgenic plants.

Central America↗

PubMed1996

Sensitivity of high-speed "perfusion-sensitive" magnetic resonance imaging to mild cerebral ischemia.

This study assessed the sensitivity of contrast-enhanced dynamic echo-planar imaging to subtotal stenosis of the middle cerebral artery as a model of mildly compromised cerebral blood supply. Dynamic data was analyzed in terms of the relative cerebral blood volume (rCBV) and bolus peak arrival time (BPAT), and the prognostic utility of these parameters was compared with measurements of the regional apparent diffusion coefficient of water (ADC) with the goal of identifying tissue at risk of future infarct. Dynamic echo-planar MRI in conjunction with bolus administration of a magnetic susceptibility contrast agent was used in a cat model of acute, unilateral cerebral ischemia, induced by partial occlusion (stenosis) of the right middle cerebral artery. The contrast agent transit was analyzed in terms of the regional time of arrival of the peak bolus-induced signal loss as well as the time integral of agent concentration. Pixel-by-pixel maps of cerebrovascular parameters (rCBV, BPAT) were constructed along with spatial maps of the ADC, derived from diffusion-weighted MR images at the same anatomical level. Arterial stenosis was maintained for a 6 h period, after which histological determination of tissue viability was obtained. Maps of BPAT showed sensitivity to mild flow perturbations not detectable from cerebral blood volume estimations from the same bolus injection or from determinations of the apparent diffusion coefficient of water. Of nine animals subjected to subtotal stenosis, BPAT identified compromised tissue in all nine after 1 h of stenosis. No animals had differences in rCBV or ADC at this point. Stenosis was maintained for 6 h in 7 of the cats. After 6 h, two cats had developed identifiable injury on ADC and rCBV maps. Of the remaining five, where rCBV and ADC appeared normal even after 6 h, three exhibited abnormal histological staining, whereas two indeed appeared normal. In the other two cats where initial subtotal stenosis was later made total, the anatomical region identified as "compromised" during stenosis, by the appearance of delayed bolus peak arrival, matched the area of subsequent infarct after total occlusion of the same vessel. Echo planar imaging in conjunction with bolus administration of a magnetic susceptibility contrast agent appears sensitive to mild perturbations to blood supply. These perturbations may not be resolved on synthesized maps of relative cerebral blood volume or apparent diffusion coefficient. Although "compromised" blood supply does not necessarily lead to infarct (over the 6-h course of this study), it may represent tissue particularly at risk of infarct in the event of further insult.

Animals↗

PubMed1988

Explaining the declining rate of physician utilization among the oldest-old.

This article reports the results of a regression-based cohort analysis of physician utilization rates among eight 4-year cohorts of elderly Americans over an 8-year period. Data on 99,445 noninstitutionalized individuals aged 56-95 were taken from the pooled 1972-73, 1976-77, and 1980-81 Health Interview Surveys. The parameters of the behavioral model of health services utilization were then estimated separately for each cohort within each pooled survey. The partial unstandardized regression coefficients obtained were used to construct standard cohort tables, which were then visually and statistically compared to identify any changes due to aging, period, or cohort effects. The findings suggest that the declining rate of physician utilization among the oldest-old results from an accelerated decreased response to health-related limitations in activity. Six plausible explanations for this changing response are considered.

Aged↗

PubMed2006

Fast H-vacancy dynamics during alanate decomposition by anelastic spectroscopy. proposition of a model for Ti-enhanced hydrogen transport.

A systematic study of the dehydrogenation process of undoped and of catalyzed NaAlH4 by means of anelastic spectroscopy is presented. Evidence is reported of the formation of a highly mobile species during decomposition, which has been identified in off-stoichiometric AlH6-x units, giving rise to fast H vacancy local dynamics. The formation of such stoichiometry defects starts at temperatures much lower in Ti doped than in undoped samples, and concomitantly with the decomposition reaction. The catalyst atoms decrease the energy barrier to be overcome by H to break the bond, thus enhancing the kinetics of the chemical reactions and decreasing the temperature at which the dehydrogenation processes take place. The experimental data show that not all the hydrogen released by the formula units during the evolution of decomposition evolves out of the sample, but part of it remains in the lattice and migrates on a long-range scale within the sample. We identify, in this H mobilized population, the species which induces the fast tetragonal to monoclinic phase transformation accompanying decomposition. A partial spontaneous thermally activated regression of decomposition has also been observed by aging experiments. A model is proposed which accounts for the action of the Ti catalyst and for the atomistic mechanism of decomposition.

Journal Article↗

PubMed2026

Machine Learning and Metabolomics to Characterize Warburg-Like Metabolic Subtypes in Human Retinal Endothelial Cells Exposed to Risk Factors Associated With Proliferative Diabetic Retinopathy.

PURPOSE: High glucose (HG), hypoxia (Hyp), and their combination are major risk factors for proliferative diabetic retinopathy (PDR). Although these conditions induce features of the Warburg-like metabolic reprogramming in human retinal endothelial cells (HRECs), it remains unclear whether they produce distinct metabolic and angiogenic subtypes. This study aimed to characterize the Warburg-like-associated metabolic heterogeneity induced by these PDR-related risk factors and evaluate the ability of supervised machine-learning models to distinguish these subtypes. METHODS: HRECs were cultured under normoglycemic, HG, Hyp (2% O2), and combined HG-Hyp conditions. Untargeted LC-MS/MS metabolomics quantified metabolites spanning carbohydrates, amino acids, nucleotides, and lipids. Principal component analysis (PCA) assessed overall metabolic variation, and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis identified metabolic pathways associated with angiogenesis. In vitro angiogenesis assays measured endothelial tube formation and branching. Nine supervised classifiers (decision tree, logistic regression, naïve Bayes, random forest, K-Nearest Neighbors, neural network, gradient boosting, AdaBoost, and Support Vector Machine) were trained on the highest-ranked metabolites selected by the Information Gain Ratio feature-ranking approach. Model performance was evaluated using 10-fold cross-validation, leave-one-out cross-validation (LOOCV), permutation testing, and a classifier stability analysis under biologically meaningful distributional shift using an independent chemically induced hypoxia model (CoCl2). RESULTS: PCA revealed partial separation of metabolic profiles across conditions, indicating different Warburg-like metabolic subtypes. The combined HG-Hyp condition exhibited enhanced angiogenic potential relative to either HG or Hyp alone. KEGG pathway enrichment analysis identified fatty acid biosynthesis and elongation among the most significantly enriched pathways in HRECs under combined HG-Hyp conditions, alongside amino sugar and nucleotide sugar metabolism, glycerophospholipid metabolism, the pentose phosphate pathway, and glycolysis/gluconeogenesis. Supervised machine-learning classifiers distinguished these metabolic subtypes, with AdaBoost and gradient Boosting showing the most balanced, reproducible performance across 10-fold cross-validation, LOOCV, and permutation testing, and remaining the most reliable classifiers under domain-shift testing (area under the curve = 0.88, P = 0.0061). CONCLUSIONS: In this exploratory analysis, HG, Hyp, and their combination drive metabolically and functionally distinct subtypes of Warburg-like metabolic reprogramming in HRECs, with HG-Hyp in combination producing a highly angiogenic phenotype. Boosting-based ensemble classifiers provide a promising framework for detecting these subtypes even under domain-shift conditions, warranting validation in larger independent datasets. TRANSLATIONAL RELEVANCE: Integrating metabolomics with machine-learning classification offers a strategy to identify Warburg-like metabolic subtypes in retinal endothelial cells, providing insights into angiogenic mechanisms and guiding the development of targeted diagnostics or therapeutics for PDR.

Humans↗

PubMed2006

Substitution of critical isoleucines in the KH domains of Drosophila fragile X protein results in partial loss-of-function phenotypes.

Fragile X mental retardation proteins (FMRP) are RNA-binding proteins that interact with a subset of cellular RNAs. Several RNA-binding domains have been identified in FMRP, but the contribution of these individual domains to FMRP function in an animal model is not well understood. In this study, we have generated flies with point mutations in the KH domains of the Drosophila melanogaster fragile X gene (dfmr1) in the context of a genomic rescue fragment. The substitutions of conserved isoleucine residues within the KH domains with asparagine are thought to impair binding of RNA substrates and perhaps the ability of FMRP to assemble into mRNP complexes. The mutants were analyzed for defects in development and behavior that are associated with deletion null alleles of dfmr1. We find that these KH domain mutations result in partial loss of function or no significant loss of function for the phenotypes assayed. The phenotypes resulting from these KH domain mutants imply that the capacities of the mutant proteins to bind RNA and form functional mRNP complexes are not wholly disrupted and are consistent with biochemical models suggesting that RNA-binding domains of FMRP can function independently.

Analysis of Variance↗

PubMed2003

Development of a Listeria monocytogenes EGDe partial proteome reference map and comparison with the protein profiles of food isolates.

A partially annotated proteome reference map of the food pathogen Listeria monocytogenes was developed for exponentially growing cells under standardized, optimal conditions by using the sequenced strain EGDe (serotype 1/2a) as a model organism. The map was developed by using a reproducible total protein extraction and two-dimensional (2-D) polyacrylamide gel electrophoresis analysis procedure, and it contained 33 identified proteins representing the four main protein functional classes. In order to facilitate analysis of membrane proteins, a protein compartmentalization procedure was assessed. The method used provided partial fractionation of membrane and cytosolic proteins. The total protein 2-D profiles of three serotype 1/2a strains and one serotype 1/2b strain isolated from food were compared to the L. monocytogenes EGDe proteome. An average of 13% of the major protein spots in the food strain proteomes were not matched in the strain EGDe proteome. The variation was greater for the less intense spots, and on average 28% of these spots were not matched. Two of the proteins identified in L. monocytogenes EGDe were missing in one or more of the food isolates. These two proteins were proteins involved in the main glycolytic pathway and in metabolism of coenzymes and prosthetic groups. The two corresponding genes were found by PCR amplification to be present in the four food isolates. Our results show that the L. monocytogenes EGDe reference map is a valuable starting point for analyses of strains having various origins and could be useful for analyzing the proteomes of different isolates of this pathogen.

Animals↗

PubMed2024

Loss-of-function in RBBP5 results in a syndromic neurodevelopmental disorder associated with microcephaly.

PURPOSE: Epigenetic dysregulation has been associated with many inherited disorders. RBBP5 (HGNC:9888) encodes a core member of the protein complex that methylates histone 3 lysine-4 and has not been implicated in human disease. METHODS: We identify 5 unrelated individuals with de novo heterozygous variants in RBBP5. Three nonsense/frameshift and 2 missense variants were identified in probands with neurodevelopmental symptoms, including global developmental delay, intellectual disability, microcephaly, and short stature. Here, we investigate the pathogenicity of the variants through protein structural analysis and transgenic Drosophila models. RESULTS: Both missense p.(T232I) and p.(E296D) variants affect evolutionarily conserved amino acids located at the interface between RBBP5 and the nucleosome. In Drosophila, overexpression analysis identifies partial loss-of-function mechanisms when the variants are expressed using the fly Rbbp5 or human RBBP5 cDNA. Loss of Rbbp5 leads to a reduction in brain size. The human reference or variant transgenes fail to rescue this loss and expression of either missense variant in an Rbbp5 null background results in a less severe microcephaly phenotype than the human reference, indicating both missense variants are partial loss-of-function alleles. CONCLUSION: Haploinsufficiency of RBBP5 observed through de novo null and hypomorphic loss-of-function variants is associated with a syndromic neurodevelopmental disorder.

Humans↗

PubMed2006

Divergent roles of superoxide and nitric oxide in reduced-size liver ischemia and reperfusion injury: Implications for partial liver transplantation.

Hepatic resection with concomitant periods of ischemia and reperfusion (I/R) are required to perform partial liver transplantation procedures such as split liver or living donor transplantation. Although great progress has been made using these types of surgeries, there remains substantial risk to both donors and recipients, with a significant number of patients developing liver injury and failure during the course these operations. Therefore, there is need to investigate the different mechanisms responsible for the tissue injury induced by ischemia and reperfusion of a reduced-size liver (RSL+I/R) with the ultimate objective of developing new therapeutic agents that may limit hepatocellular damage induced during partial liver transplantation. This review summarizes recent studies that have been performed in a mouse model of RSL+I/R. In addition, we present data demonstrating how the pathophysiological mechanisms identified in this model compare to those observed in a rat model of RSL transplantation.

Journal Article↗

PubMed1996

Multivariable predictive models for adverse outcome of invasive meningococcal disease in children.

For prediction of adverse outcome (AO, defined as death or limb amputation) of invasive meningococcal disease (IMD) in children, two multivariable models were derived and validated by reviewing the data in the medical records of patients with IMD, who ranged from birth to 19 years of age, at three pediatric referral hospitals between 1985 and 1990 (derivation set, n = 153, 19 AO) and between 1991 and 1994 (validation set, n = 92, 11 AO). Variables in the derivation set significantly associated with AO (p < 0.05) were entered into a logistic regression analysis. Because coagulation studies (prothrombin time, partial thromboplastin time, and serum fibrinogen concentration) were available for only 50% of patients, two analyses were performed, either excluding (model 1) or including (model 2) coagulation studies. These analyses identified an absolute neutrophil count less than 3000/mm3, poor perfusion, and a platelet count less than 150,000/mm3 (model 1), and a serum fibrinogen concentration less than 2.5 gm/L (250 mg/dl) and an absolute neutrophil count less than 3000/mm3 (model 2), as independent predictors of AO (p < 0.05). When the models were tested on the validation set, the presence of at least two of the three predictors in model 1 had a sensitivity of 82% and a specificity of 97% in predicting AO; the presence of both predictors in model 2 had a sensitivity of 89% and a specificity of 97%. These models can reliably identify patients with IMD at high risk of AO for whom consideration of novel therapies is justified.

Adolescent↗

PubMed1998

Photoprotection: sunscreens and the immunomodulatory effects of UV irradiation.

UV-B irradiation (UVR) of the host, in both humans and animal models, induces dose-related acute and chronic changes in skin which include erythema and photoageing, and induction of cancer. It can also induce modulation of immune responses of the host to antigens presented following irradiation. Commercially-available, broad-spectrum, high SPF (15, 15 + ) sunscreens protect against most effects of UV irradiation. An exception is the effects of UVR on immune responsiveness, with varying degrees of protection having been reported. We examined a system of UV-induced systemic suppression of contact hypersensitivity (CHS) responses in BALB/c mice. A range of commercially-available, broad spectrum, high SPF (15 + ) sunscreens demonstrated at best partial protection against systemic immunosuppression, yet were able to protect against two hallmarks of acute UVR-induced damage: skin oedema and keratinocyte proliferation. Two major models have been identified for the induction of immunosuppression following UVR, one identifying trans-urocanic acid (trans-UCA; deaminated histidine, located in the stratum corneum) as the critical photoreceptor, the other featuring DNA. UVR of trans-UCA produces cis-UCA, which itself is immunomodulatory. There was some abrogation of trans to cis isomerisation of urocanic acid in UV-irradiated, sunscreen-protected mice. However, the majority of the immunomodulation seen in these mice was abrogated by pretreatment with a monoclonal antibody to cis-urocanic acid. It is possible to induce formation of cis-urocanic acid in BALB/c skin in the absence of immunosuppression, using lower doses of UV radiation, indicating that formation of cis-urocanic acid in the stratum corneum is not necessarily sufficient to induce immunosuppression in the UV-irradiated host. The mechanisms of induction of the immunomodulated state in the UV-irradiated host are potentially diverse and the subject of ongoing debate. Our studies maintain a role for cis-UCA, and form the basis for further studies on its involvement in immunomodulation by UVR in sunscreen-protected hosts.

Animals↗

PubMed2001

Probing catalysis by Escherichia coli dTDP-glucose-4,6-dehydratase: identification and preliminary characterization of functional amino acid residues at the active site.

A model of the Escherichia coli dTDP-glucose-4,6-dehydratase (4,6-dehydratase) active site has been generated by combining amino acid sequence alignment information with the 3-dimensional structure of UDP-galactose-4-epimerase. The active site configuration is consistent with the partially refined 3-dimensional structure of 4,6-dehydratase, which lacks substrate-nucleotide but contains NAD(+) (PDB file ). From the model, two groups of active site residues were identified. The first group consists of Asp135(DEH), Glu136(DEH), Glu198(DEH), Lys199(DEH), and Tyr301(DEH). These residues are near the substrate-pyranose binding pocket in the model, they are completely conserved in 4,6-dehydratase, and they differ from the corresponding equally well-conserved residues in 4-epimerase. The second group of residues is Cys187(DEH), Asn190(DEH), and His232(DEH), which form a motif on the re face of the cofactor nicotinamide binding pocket that resembles the catalytic triad of cysteine-proteases. The importance of both groups of residues was tested by mutagenesis and steady-state kinetic analysis. In all but one case, a decrease in catalytic efficiency of approximately 2 orders of magnitude below wild-type activity was observed. Mutagenesis of each of these residues, with the exception of Cys187(DEH), which showed near-wild-type activity, clearly has important negative consequences for catalysis. The allocation of specific functions to these residues and the absolute magnitude of these effects are obscured by the complex chemistry in this multistep mechanism. Tools will be needed to characterize each chemical step individually in order to assign loss of catalytic efficiency to specific residue functions. To this end, the effects of each of these variants on the initial dehydrogenation step were evaluated using a the substrate analogue dTDP-xylose. Additional steady-state techniques were employed in an attempt to further limit the assignment of rate limitation. The results are discussed within the context of the 4,6-dehydratase active site model and chemical mechanism.

Amino Acid Substitution↗