Debra Dunaway-Mariano

University of New Mexico, Albuquerque, New Mexico, United States

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Publications (200)881.22 Total impact

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  • [Show abstract] [Hide abstract]
    ABSTRACT: In the current study, 1,2-diarylpropan-1,3-diols, containing varying numbers of methoxy substituents that mimic β-1 type units in lignins, were prepared and subjected to photochemical and enzymatic SET oxidative C-C bond cleavage reactions to explore how product distributions and reactivity profiles depend on the numbers and positions of arene ring methoxy-substituents. For this purpose, product distributions of SET-promoted photochemical reactions of the β-1 model compounds and the characteristics of lignin peroxidase catalyzed bond cleavage reactions of these substances were explored. The results show that both the photochemical and enzymatic reactions, which are known to occur by initial SET to form arylpropanoid cation radicals, generate predominantly aldehydes and β-hydroxyketones through cation radical C1-C2 bond cleavage pathways. In addition, analysis of the relative quantum efficiencies of the SET photochemical processes shows that they do not depend greatly on the numbers and positions of arene ring methoxy substituents of the β-1 model compounds.
    Tetrahedron 04/2015; 71(24). DOI:10.1016/j.tet.2015.04.077 · 2.82 Impact Factor
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    ABSTRACT: Large-scale activity profiling of enzyme superfamilies provides information about cellular functions as well as the intrinsic binding capabilities of conserved folds. Herein, the functional space of the ubiquitous haloalkanoate dehalogenase superfamily (HADSF) was revealed by screening a customized substrate library against >200 enzymes from representative prokaryotic species, enabling inferred annotation of ∼35% of the HADSF. An extremely high level of sub-strate ambiguity was revealed, with the majority of HADSF enzymes using more than five substrates. Substrate profiling allowed assignment of function to previously unannotated enzymes with known structure, uncovered potential new pathways, and identified iso-functional orthologs from evolutionarily distant taxonomic groups. Intriguingly, the HADSF subfamily having the least structural elaboration of the Rossmann fold catalytic domain was the most specific, consistent with the concept that domain insertions drive the evolution of new functions and that the broad specificity observed in HADSF may be a relic of this process. evolution | specificity | phosphatase | substrate screen | promiscuity S ince the first genomes were sequenced, there has been an exponential increase in the number of protein sequences deposited into databases worldwide. At the time of this writing the UniProtKB/TrEMBL database contains over 32 million protein sequences. Although this increase in sequence data has dramatically enhanced our understanding of the genomic organization of organisms, as the number of protein sequences grows, the proportion of firm functional assignments diminishes. Traditionally, methods of functional annotation involve comparing sequence identity between experimentally characterized proteins and newly sequenced ones, typically via BLAST (1). In cases where significant sequence similarity cannot be ascertained, proteins are annotated as " hypothetical " or " putative. " Moreover, the decrease in sequence identity leads to an increased uncertainty in functional assignment, especially as the phylogenetic distance between organisms grows, limiting iso-functional ortholog discovery. As the number of newly sequenced genomes grows larger, more protein sequences are likely to be misannotated, oftentimes resulting in the propagation of incorrect functional annotation across newly identified sequences. To tackle the problem of un-annotated or misannotated proteins, newer methods for computational assignment have been created with varying degrees of success (2). Although these methods outperform historical methods, continued improvement is necessary to ensure accurate annotation of function (2). A greater swath of functional space can be covered by screening substrates in a high-throughput manner on multiple enzymes from a family (3, 4). Family-wide substrate profiling offers a data-rich resource. The use of sparse screening of sequence space and a diversified library permits the determination of substrate specificity profiles to provide a family-wide view of the range of substrates and insight into the structure of the prototypical substrate. Where structures are available, correlation between substrate range and structural determinants of specificity can be achieved. In addition, the approach has utility in genomic annotation (inferred function), iso-functional ortho-log assignment, and the assignment of in vitro substrate profiles to orphaned PDB entries (enzymes with structure but no function , or SNFs). Here we report the application of in vitro high-throughput functional screening of metabolites and related compounds at the superfamily level. We use as an example prokaryotic members of the haloalkanoic acid dehalogenase superfamily (HADSF), a diverse superfamily of enzymes (5) that catalyze a wide range of reactions involving the formation of a covalent intermediate with an active-site aspartate. Reactions catalyzed by this superfamily include dehalogenation (6) as well as Mg
    Proceedings of the National Academy of Sciences 03/2015; 112(16). DOI:10.1073/pnas.1423570112 · 9.81 Impact Factor
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    ABSTRACT: Large-scale activity profiling of enzyme superfamilies provides information about cellular functions as well as the intrinsic binding capabilities of conserved folds. Herein, the functional space of the ubiquitous haloalkanoate dehalogenase superfamily (HADSF) was revealed by screening a customized substrate library against >200 enzymes from representative prokaryotic species, enabling inferred annotation of ∼35% of the HADSF. An extremely high level of sub-strate ambiguity was revealed, with the majority of HADSF enzymes using more than five substrates. Substrate profiling allowed assignment of function to previously unannotated enzymes with known structure, uncovered potential new pathways, and identified iso-functional orthologs from evolutionarily distant taxonomic groups. Intriguingly, the HADSF subfamily having the least structural elaboration of the Rossmann fold catalytic domain was the most specific, consistent with the concept that domain insertions drive the evolution of new functions and that the broad specificity observed in HADSF may be a relic of this process. evolution | specificity | phosphatase | substrate screen | promiscuity S ince the first genomes were sequenced, there has been an exponential increase in the number of protein sequences deposited into databases worldwide. At the time of this writing the UniProtKB/TrEMBL database contains over 32 million protein sequences. Although this increase in sequence data has dramatically enhanced our understanding of the genomic organization of organisms, as the number of protein sequences grows, the proportion of firm functional assignments diminishes. Traditionally, methods of functional annotation involve comparing sequence identity between experimentally characterized proteins and newly sequenced ones, typically via BLAST (1). In cases where significant sequence similarity cannot be ascertained, proteins are annotated as " hypothetical " or " putative. " Moreover, the decrease in sequence identity leads to an increased uncertainty in functional assignment, especially as the phylogenetic distance between organisms grows, limiting iso-functional ortholog discovery. As the number of newly sequenced genomes grows larger, more protein sequences are likely to be misannotated, oftentimes resulting in the propagation of incorrect functional annotation across newly identified sequences. To tackle the problem of un-annotated or misannotated proteins, newer methods for computational assignment have been created with varying degrees of success (2). Although these methods outperform historical methods, continued improvement is necessary to ensure accurate annotation of function (2). A greater swath of functional space can be covered by screening substrates in a high-throughput manner on multiple enzymes from a family (3, 4). Family-wide substrate profiling offers a data-rich resource. The use of sparse screening of sequence space and a diversified library permits the determination of substrate specificity profiles to provide a family-wide view of the range of substrates and insight into the structure of the prototypical substrate. Where structures are available, correlation between substrate range and structural determinants of specificity can be achieved. In addition, the approach has utility in genomic annotation (inferred function), iso-functional ortho-log assignment, and the assignment of in vitro substrate profiles to orphaned PDB entries (enzymes with structure but no function , or SNFs). Here we report the application of in vitro high-throughput functional screening of metabolites and related compounds at the superfamily level. We use as an example prokaryotic members of the haloalkanoic acid dehalogenase superfamily (HADSF), a diverse superfamily of enzymes (5) that catalyze a wide range of reactions involving the formation of a covalent intermediate with an active-site aspartate. Reactions catalyzed by this superfamily include dehalogenation (6) as well as Mg
    Proceedings of the National Academy of Sciences 03/2015; · 9.81 Impact Factor
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    ABSTRACT: Catalytic promiscuity and substrate ambiguity are keys to evolvability, which in turn is pivotal to the successful acquisition of novel biological functions. Action on multiple substrates (substrate ambiguity) can be harnessed for performance of functions in the cell that supersede catalysis of a single metabolite. These functions include proofreading, scavenging of nutrients, removal of anti-metabolites, balancing of metabolite pools, and establishing system redundancy. In this review we present examples of enzymes that perform these cellular roles by leveraging substrate ambiguity and then present the structural features that support both specificity and ambiguity. We focus on the phosphatases of the haloalkanoate dehalogenase superfamily and the thioesterases of the hotdog-fold superfamily.
    Journal of Biological Chemistry 09/2014; 289(44). DOI:10.1074/jbc.R114.572990 · 4.57 Impact Factor
  • Chetanya Pandya, Debra Dunaway-Mariano, Yu Xia, Karen N Allen
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    ABSTRACT: In multi-domain proteins, the domains typically run end-to-end, i.e. one domain follows the C-terminus of another domain. However, approximately 10% of multi-domain proteins are formed by insertion of one domain sequence into that of another domain. Detecting such insertions within protein sequences is a fundamental challenge in structural biology. The haloacid dehalogenase superfamily (HADSF) serves as a challenging model system wherein a variable cap domain (˜5-200 residues in length) accessorizes the ubiquitous Rossmann-fold core domain, with variations in insertion site and topology corresponding to different classes of cap-types. Herein, we describe a comprehensive computational strategy, CapPredictor, for determining large, variable domain insertions in protein sequences. Using a novel sequence alignment algorithm in conjunction with a structure-guided sequence profile from 154 core-domain-only structures, more than 40,000 HADSF member sequences were assigned cap-types. The resulting dataset afforded insight into HADSF evolution. Notably, a similar distribution of cap-type classes across different phyla was observed, indicating that all cap-types existed in the last universal common ancestor. In addition, comparative analyses of the predicted cap-type and functional assignments showed that different cap types carry out similar chemistries. Thus, while cap domains play a role in substrate recognition and chemical reactivity, cap-type does not strictly define functional class. Through this example, we have shown that CapPredictor is an effective new tool for the study of form and function in protein families where domain insertion occurs. © Proteins 2014;. © 2014 Wiley Periodicals, Inc.
    Proteins Structure Function and Bioinformatics 09/2014; 82(9). DOI:10.1002/prot.24543 · 2.92 Impact Factor
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    John A Latham, Danqi Chen, Karen N Allen, Debra Dunaway-Mariano
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    ABSTRACT: The work described in this paper, and its companion paper (Wu, R., Latham, J. A., Chen, D., Farelli, J., Zhao, H., Matthews, K. Allen, K. N., and Dunaway-Mariano, D. (2014) Structure and Catalysis in the Escherichia coli Hotdog-fold Thioesterase Paralogs YdiI and YbdB. Biochemistry, DOI: 10.1021/bi500334v), focuses on the evolution of a pair of paralogous hotdog-fold superfamily thioesterases of E. coil, YbdB and YdiI, which share a high level of sequence identity but perform different biological functions (viz., proofreader of 2,3-dihydroxybenzoyl-holoEntB in the enterobactin biosynthetic pathway and catalyst of the 1,4-dihydoxynapthoyl-CoA hydrolysis step in the menaquinone biosynthetic pathway, respectively). In vitro substrate activity screening of a library of thioester metabolites showed that YbdB displays high activity with benzoyl-holoEntB and benzoyl-CoA substrates, marginal activity with acyl-CoA thioesters, and no activity with 1,4-dihydoxynapthoyl-CoA. YdiI, on the other hand, showed a high level of activity with its physiological substrate, significant activity toward a wide range of acyl-CoA thioesters, and minimal activity toward benzoyl-ho/oEntB. These results were interpreted as evidence for substrate promiscuity that facilitates YbdB and YdiI evolvability, and divergence in substrate preference, which correlates with their assumed biological function. YdiI support of the menaquinone biosynthetic pathway was confirmed by demonstrating reduced anaerobic growth of the E. coil ydiI-knockout mutant (vs wild-type E. coil) on glucose in the presence of the electron acceptor fumarate. Bioinformatic analysis revealed that a small biological range exists for YbdB orthologs (i.e., limited to Enterobacteriales) relative to that of YdiI orthologs. The divergence in YbdB and YdiI substrate specificity detailed in this paper set the stage for their structural analyses reported in the companion paper.
    Biochemistry 07/2014; 53(29). DOI:10.1021/bi500333m · 3.01 Impact Factor
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    ABSTRACT: Herein, the structural determinants for substrate recognition and catalysis in two hotdog-fold thioesterase paralogs, YbdB and YdiI from Escherichia coli, are identified and analyzed to provide insight into the evolution of biological function in the hotdog-fold enzyme superfamily. The X-ray crystal structures of YbdB and YdiI, in complex with inert substrate analogs, determined in this study revealed the locations of the respective thioester substrate binding sites, and the identity of the residues positioned for substrate binding and catalysis. The importance of each of these residues was assessed through amino acid replacements followed by steady-state kinetic analyses of the corresponding site-directed mutants. Transient kinetic and solvent 18O-labeling studies were then carried out to provide insight into the role of Glu63 posited to function as the nucleophile or general base in catalysis. Finally, the structure-function-mechanism profiles of the two paralogs, along with that of a more distant homolog, were compared to identify conserved elements of substrate recognition and catalysis, which define the core traits of the hotdog-fold thioesterase family, as well as structural features that are unique to each thioesterase. Founded on the insight gained from this analysis, we conclude that the promiscuity revealed by in vitro substrate activity determinations, and posited to facilitate the evolution of new biological function, is the product of intrinsic plasticity in substrate binding as well as in the catalytic mechanism.
    Biochemistry 07/2014; 53(29). DOI:10.1021/bi500334v · 3.01 Impact Factor
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    ABSTRACT: Parasitic nematodes are responsible for devastating illnesses that plague many of the world's poorest populations indigenous to the tropical areas of developing nations. Among these diseases is lymphatic filariasis, a major cause of permanent and long-term disability. Proteins essential to nematodes that do not have mammalian counterparts represent targets for therapeutic inhibitor discovery. One promising target is trehalose-6-phosphate phosphatase (T6PP) from Brugia malayi. In the model nematode Caenorhabditis elegans, T6PP is essential for survival due to the toxic effect(s) of the accumulation of trehalose 6-phosphate. T6PP has also been shown to be essential in Mycobacterium tuberculosis. We determined the X-ray crystal structure of T6PP from B. malayi. The protein structure revealed a stabilizing N-terminal MIT-like domain and a catalytic C-terminal C2B-type HAD phosphatase fold. Structure-guided mutagenesis, combined with kinetic analyses using a designed competitive inhibitor, trehalose 6-sulfate, identified five residues important for binding and catalysis. This structure-function analysis along with computational mapping provided the basis for the proposed model of the T6PP-trehalose 6-phosphate complex. The model indicates a substrate-binding mode wherein shape complementarity and van der Waals interactions drive recognition. The mode of binding is in sharp contrast to the homolog sucrose-6-phosphate phosphatase where extensive hydrogen-bond interactions are made to the substrate. Together these results suggest that high-affinity inhibitors will be bi-dentate, taking advantage of substrate-like binding to the phosphoryl-binding pocket while simultaneously utilizing non-native binding to the trehalose pocket. The conservation of the key residues that enforce the shape of the substrate pocket in T6PP enzymes suggest that development of broad-range anthelmintic and antibacterial therapeutics employing this platform may be possible.
    PLoS Pathogens 07/2014; 10(7):e1004245. DOI:10.1371/journal.ppat.1004245 · 8.14 Impact Factor
  • Debra Dunaway-Mariano, Hazel M. Holden, Frank M. Raushel
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    ABSTRACT: Professor W. Wallace Cleland, the architect of modern steady-state enzyme kinetics, died on March 6, 2013, from injuries sustained in a fall outside of his home. He will be most remembered for giving the enzyme community Ping-Pong kinetics and the invention of dithiothreitol (DTT). He pioneered the utilization of heavy atom isotope effects for the elucidation of the chemical mechanisms of enzyme-catalyzed reactions. His favorite research journal was Biochemistry, in which he published more than 135 papers beginning in 1964 with the disclosure of DTT.
    Biochemistry 12/2013; 52(51):9092–9096. DOI:10.1021/bi4015709 · 3.01 Impact Factor
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    ABSTRACT: The enzyme 2-keto-3-deoxy-9-O-phosphonononic acid phosphatase (KDN9P phosphatase) functions in the pathway for the production of 2-keto-3-deoxy-D-glycero-D-galacto-nononic acid, a sialic acid that is important for the survival of commensal bacteria in the human intestine. The enzyme is a member of the haloalkanoate dehalogenase superfamily and represents a good model for the active-site protonation state of family members. Crystals of approximate dimensions 1.5 × 1.0 × 1.0 mm were obtained in space group P21212, with unit-cell parameters a = 83.1, b = 108.9, c = 75.7 Å. A complete neutron data set was collected from a medium-sized H/D-exchanged crystal at BIODIFF at the Heinz Maier-Leibnitz Zentrum (MLZ), Garching, Germany in 18 d. Initial refinement to 2.3 Å resolution using only neutron data showed significant density for catalytically important residues.
    Acta Crystallographica Section F Structural Biology and Crystallization Communications 09/2013; 69(Pt 9):1015-9. DOI:10.1107/S1744309113021386 · 0.57 Impact Factor
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    ABSTRACT: To gain Information about how alkoxy substitution in arene rings of β-O-4 structural units within lignin governs the efficiencies/rates of radical cation C1-C2 bond cleavage reactions, single electron transfer (SET) photochemical and lignin peroxidase catalyzed oxidation reactions of dimeric/tetrameric model compounds have been explored. The results show that the radical cations derived from less alkoxy substituted dimeric β-O-4 models undergo more rapid C1-C2 bond cleavage than those of more alkoxy substituted analogs. These findings gained support from the results of DFT calculations, which demonstrate that C1-C2 bond dissociation energies of β-O-4 radical cations decrease as the degree of alkoxy substitution decreases. In SET reactions of tetrameric compounds consisting of two β-O-4 units, containing different degrees of alkoxy substitution, regioselective radical cation C-C bond cleavage was observed to occur in one case at the C1-C2 bond in the less alkoxy substituted β-O-4 moiety. However, regioselective C1-C2 cleavage in the more alkoxy substituted β-O-4 moiety was observed in another case, suggesting that other factors might participate in controlling this process. These observations show that lignins containing greater proportions of less rather than more alkoxylated rings as part of β-O-4 units would be more efficiently cleaved by SET mechanisms.
    The Journal of Organic Chemistry 08/2013; 78(18). DOI:10.1021/jo401680z · 4.64 Impact Factor
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    ABSTRACT: Although the universe of protein structures is vast, these innumerable structures can be categorized into a finite number of folds. New functions commonly evolve by elaboration of existing scaffolds, for example, via domain insertions. Thus, understanding structural diversity of a protein fold evolving via domain insertions is a fundamental challenge. The haloalkanoic dehalogenase superfamily serves as an excellent model system wherein a variable cap domain accessorizes the ubiquitous Rossmann-fold core domain. Here, we determine the impact of the cap-domain insertion on the sequence and structure divergence of the core domain. Through quantitative analysis on a unique dataset of 154 core-domain-only and cap-domain-only structures, basic principles of their evolution have been uncovered. The relationship between sequence and structure divergence of the core domain is shown to be monotonic and independent of the corresponding type of domain insert, reflecting the robustness of the Rossmann fold to mutation. However, core domains with the same cap type share greater similarity at the sequence and structure levels, suggesting interplay between the cap and core domains. Notably, results reveal that the variance in structure maps to α-helices flanking the central β-sheet and not to the domain-domain interface. Collectively, these results hint at intramolecular coevolution where the fold diverges differentially in the context of an accessory domain, a feature that might also apply to other multidomain superfamilies.
    Proceedings of the National Academy of Sciences 08/2013; 110(36). DOI:10.1073/pnas.1305519110 · 9.81 Impact Factor
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    ABSTRACT: The haloacid dehalogenase enzyme superfamily (HADSF) is largely composed of phosphatases, which, relative to members of other phosphatase families, have been particularly successful at adaptation to novel biological functions. Herein, we examine the structural basis for divergence of function in two bacterial homologs: 2-keto-3-deoxy-D-manno-octulosonate 8-phosphate phosphohydrolase (KDO8P phosphatase, KDO8PP) and 2-keto-3-deoxy-9-O-phosphonononic acid phosphohydrolase (KDN9P phosphatase, KDN9PP). KDO8PP and KDN9PP catalyze the final step of KDO and KDN synthesis, respectively, prior to transfer to CMP to form the activated sugar nucleotide. KDO8PP and KDN9PP orthologs derived from an evolutionarily diverse collection of bacterial species were subjected to steady-state kinetic analysis to determine their specificities towards catalyzed KDO8P and KDN9P hydrolysis. Although each enzyme was more active with its biological substrate, the degree of selectivity (as defined by the ratio of kcat/Km for KDO8P vs. KDN9P) varied significantly. High-resolution X-ray structure determination of Haemophilus influenzae KDO8PP bound to KDO/VO3(-) and Bacteriodes thetaiotaomicron KDN9PP bound to KDN/VO3(-) revealed the substrate-binding residues. Structures of the KDO8PP and KDN9PP orthologs were also determined to reveal the differences in active site structure that underlies the variation in substrate preference. Bioinformatic analysis was carried out to define the sequence divergence among KDN9PP and KDO8PP orthologs. The KDN9PP orthologs were found to exist as single domain proteins or fused with the pathway nucleotidyl transferases; fusion of KDO8PP with the transferase is rare. The KDO8PP and KDN9PP orthologs share a stringently conserved Arg residue, which forms a salt bridge with the substrate carboxylate group. The split of the KDN9PP lineage from the KDO8PP orthologs is easily tracked by the acquisition of a Glu/Lys pair that supports KDN9P binding. Moreover, independently evolved lineages of KDO8PP orthologs exist, separated by diffuse active-site sequence boundaries. We infer high tolerance of the KDO8PP catalytic platform to amino acid replacements that, in turn, influence substrate specificity changes and thereby facilitate divergence of biological function.
    Biochemistry 07/2013; 52(32). DOI:10.1021/bi400659k · 3.01 Impact Factor
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    ABSTRACT: The function of a Bacteroidetes menaquinone biosynthetic pathway fusion protein comprised of an N-terminal haloacid dehalogenase (HAD) family domain and a C-terminal hotdog-fold family domain is described. Whereas the thioesterase domain efficiently catalyzes 1,4-dihydroxynapthoyl-CoA hydrolysis, an intermediate step in the menaquinone pathway, the HAD domain is devoid of catalytic activity. In some Bacteroidetes a homologous, catalytically active 1,4-dihydroxynapthoyl-CoA thioesterase replaces the fusion protein. Following the gene fusion event, sequence divergence resulted in a HAD domain that functions solely as the oligomerization domain of an otherwise inactive thioesterase domain.
    FEBS letters 07/2013; 587(17). DOI:10.1016/j.febslet.2013.07.009 · 3.34 Impact Factor
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    ABSTRACT: Since the first genomes were sequenced, there has been an exponential increase of protein sequences deposited into databases worldwide. This increase in sequence data has allowed for dramatic improvements in our understanding of the metabolism of organisms. Unfortunately, as the number of protein sequences grows, the number of definitive functional assignments diminishes. Current methods involve comparison of sequence identity between known proteins and newly sequenced ones. As the sequence identity decreases, proteins are annotated as "hypothetical," and certainty in annotation shrinks. Thus a strategy for reliably determining protein function is necessary. The HADSF consists of Mg2+-dependent enzymes that catalyze a wide range of reactions including dehalogenation, phosphoryltransfer and dephosphorylation. Here we present the role that high-throughput screening (HTS) might play in functional discovery for families with members having common chemical function but divergence in physiological substrate (e.g., kinases). We highlight five areas of discovery enabled by the HTS-guided substrate profiling: 1) uncovering new metabolic pathways, 2) tracking of orthologs, 3) distribution of promiscuous versus specific family members, 4) annotation of hypothetical proteins, 5) functional assignment of proteins with known structures. This research is supported by the NIGMS (U54 GM093342).
    The FASEB Journal 04/2013; 27(571):1. · 5.48 Impact Factor
  • Chun Wu, Debra Dunaway-Mariano, Patrick S Mariano
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    ABSTRACT: Pyruvate phosphate dikinase (PPDK) catalyzes the phosphorylation reaction of pyruvate that forms phosphoenolpyruvate (PEP) via two partial reactions: PPDK + ATP + Pi --> PPDK-P + AMP + PPi and PPDK-P + pyruvate --> PEP + PPDK. Based on its role in the metabolism of microbial human pathogens, PPDK is a potential drug target. A screen of substances that bind to the PPDK ATP-grasp domain active site revealed that flavone analogs are potent inhibitors of the Clostridium symbiosum PPDK. In silico modeling studies suggested that placement of a 3-6 carbon tethered ammonium substituent at the 3'- or 4'-positions of 5,7-dihydroxyflavones would result in favorable electrostatic interactions with the PPDK Mg-ATP binding site. As a result, polymethylene-tethered amine derivatives of 5,7-dihydroxyflavones were prepared. Steady state kinetic analysis of these substances demonstrates that the 4'-aminohexyl-5,7-dyhydroxyflavone 11 is a potent competitive PPDK inhibitor (Ki = 1.6 +/- 0.1 micromolar). Single turnover experiments, conducted using 4'-aminopropyl-5,7-dihydroxyflavone 8 to show that this flavone specifically targets the ATP binding site and inhibits catalysis of only the PPDK + ATP + Pi --> PPDK-P + AMP PPi partial reaction. Finally, the 4'-aminopbutyl-5,7-dihydroxyflavone 9 displays selectivity for inhibition of PPDK versus other enzymes that utilize ATP and NAD.
    The Journal of Organic Chemistry 10/2012; 78(5). DOI:10.1021/jo3018473 · 4.64 Impact Factor
  • Rachel Gomez, Debra Dunaway-Mariano
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    ABSTRACT: Pseudomonas aeruginosa is a common bacterium that can cause infections in both animals and plants. The bacterium, an opportunistic pathogen with antibiotic resistance, is a large contributor to hospital infections, including those affecting patients with cystic fibrosis. P. aeruginosa is a member of the Pseudomonadacae family and can utilize a variety of organic compounds as carbon sources, including acyclic terpenes. One pathway found in P. aeruginosa is the Acyclic Terpene Utilization (Atu) pathway that is partly responsible for the metabolism of citronellol and geraniol into the downfield products acetyl-CoA and acetoactate. AtuH, annotated as a long chain acyl-CoA synthetase, is a member of the AMP-forming family of enzymes. In P. aeruginosa, AtuH is proposed to be responsible for the conversion of citronellate to cintronellyl-CoA through a two-step reaction involving the utilization of ATP to form an acyl-AMP intermediate before forming the acyl-CoA in the second step. The focus of this work is to characterize AtuH in terms of its biological function and range as well as its efficiency as an enzyme. This will include cloning, expression, and purification of the enzyme using various techniques of molecular biology followed by kinetic assays with a panel of carboxylic acid derivatives of various length, saturation and substitution. The results of this work will characterize AtuH, help prove or disprove its involvement in the Atu pathway, and expand our general knowledge of acyl-CoA synthetases and their importance in biological systems.
    2012 Society for Advancement of Hispanics/Chicanos and Native Americans in Science National Conference; 10/2012

Publication Stats

5k Citations
881.22 Total Impact Points

Institutions

  • 1999–2014
    • University of New Mexico
      • Department of Chemistry and Chemical Biology
      Albuquerque, New Mexico, United States
  • 1997–2012
    • Case Western Reserve University
      • Department of Biochemistry
      Cleveland, Ohio, United States
  • 2000–2006
    • Boston University
      Boston, Massachusetts, United States
  • 2004
    • University at Buffalo, The State University of New York
      Buffalo, New York, United States
  • 2003
    • Bielefeld University
      Bielefeld, North Rhine-Westphalia, Germany
  • 1981–2003
    • University of Wisconsin, Madison
      • Department of Biochemistry
      Madison, MS, United States
  • 1981–1998
    • University of Maryland, College Park
      • Department of Chemistry and Biochemistry
      College Park, MD, United States
  • 1992
    • University of California, San Francisco
      • Department of Pharmaceutical Chemistry
      San Francisco, California, United States
  • 1988–1989
    • Loyola University Maryland
      • Department of Chemistry
      Baltimore, Maryland, United States
  • 1987
    • Harvard Medical School
      Boston, Massachusetts, United States