Debra Dunaway-Mariano

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

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Publications (196)791.12 Total impact

  • [Show abstract] [Hide abstract]
    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; · 4.65 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; · 3.38 Impact Factor
  • 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. A., and Dunaway-Mariano, D. (2014) Structure and catalysis in the Escherichia coli hotdog-fold thioesterase paralogs YdiI and YbdB. Biochemistry, XX, XXXX (2)), focuses on the evolution of a pair of paralogous hotdog-fold superfamily thioesterases of E. coli, 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 towards a wide range of acyl-CoA thioesters, and minimal activity towards benzoyl-holoEntB. 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 growth of the E. coli ydiI-knockout mutant (vs wild-type E. coli) on fumarate. Bioinformatic analysis revealed that a small biological range exists for YbdB (i.e., limited to Enterobacteriales) relative to that of YdiI, and that not all of the bacterial genomes found to encode the enterobactin pathway enzymes, encode YbdB. Taken together, these findings indicate that YbdB evolved from an ancestral YdiI gene, to increase the efficiency of enterobactin synthesis. 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; · 3.38 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. · 8.14 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 02/2014; · 3.34 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. · 3.38 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. · 0.55 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; · 4.56 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; · 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; · 3.38 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; · 3.54 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; · 4.56 Impact Factor
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    ABSTRACT: The mammalian brown fat inducible thioesterase variant 2 (BFIT2), also known as ACOT11, is a multimodular protein containing two consecutive hotdog-fold domains and a C-terminal steroidogenic acute regulatory protein-related lipid transfer domain (StarD14). In this study, we demonstrate that the N-terminal region of human BFIT2 (hBFIT2) constitutes a mitochondrial location signal sequence, which undergoes mitochondrion-dependent posttranslational cleavage. The mature hBFIT2 is shown to be located in the mitochondrial matrix, whereas the paralog "cytoplasmic acetyl-CoA hydrolase" (CACH, also known as ACOT12) was found in the cytoplasm. In vitro activity analysis of full-length hBFIT2 isolated from stably transfected HEK293 cells demonstrates selective thioesterase activity directed toward long chain fatty acyl-CoA thioesters, thus distinguishing the catalytic function of BFIT2 from that of CACH. The results from a protein-lipid overlay test indicate that the hBFIT2 StarD14 domain binds phosphatidylinositol 4-phosphate.
    Biochemistry 08/2012; 51(35):6990-9. · 3.38 Impact Factor
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    ABSTRACT: The hotdog-fold enzyme 4-hydroxybenzoyl-coenzyme A (4-HB-CoA) thioesterase from Arthrobacter sp. strain AU catalyzes the hydrolysis of 4-HB-CoA to form 4-hydroxybenzoate (4-HB) and coenzyme A (CoA) in the final step of the 4-chlorobenzoate dehalogenation pathway. Guided by the published X-ray structures of the liganded enzyme (Thoden, J. B., Zhuang, Z., Dunaway-Mariano, D., and Holden H. M. (2003) J. Biol. Chem. 278, 43709-43716), a series of site-directed mutants were prepared for testing the roles of active site residues in substrate binding and catalysis. The mutant thioesterases were subjected to X-ray structure determination to confirm retention of the native fold, and in some cases, to reveal changes in the active site configuration. In parallel, the wild-type and mutant thioesterases were subjected to transient and steady-state kinetic analysis, and to (18)O-solvent labeling experiments. Evidence is provided that suggests that Glu73 functions in nucleophilic catalysis, that Gly65 and Gln58 contribute to transition-state stabilization via hydrogen bond formation with the thioester moiety and that Thr77 orients the water nucleophile for attack at the 4-hydroxybenzoyl carbon of the enzyme-anhydride intermediate. The replacement of Glu73 with Asp was shown to switch the function of the carboxylate residue from nucleophilic catalysis to base catalysis and thus, the reaction from a two-step process involving a covalent enzyme intermediate to a single-step hydrolysis reaction. The E73D/T77A double mutant regained most of the catalytic efficiency lost in the E73D single mutant. The results from (31)P NMR experiments indicate that the substrate nucleotide unit is bound to the enzyme surface. Kinetic analysis of site-directed mutants was carried out to determine the contributions made by Arg102, Arg150, Ser120, and Thr121 in binding the nucleotide unit. Lastly, we show by kinetic and X-ray analyses of Asp31, His64, and Glu78 site-directed mutants that these three active site residues are important for productive binding of the substrate 4-hydroxybenzoyl ring.
    Biochemistry 08/2012; 51(35):7000-16. · 3.38 Impact Factor
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    ABSTRACT: Human THEM4 (hTHEM4) is comprised of a catalytically active hotdog-fold acyl-CoA thioesterase domain and an N-terminal domain of unknown fold and function. hTHEM4 has been linked to Akt1 regulation and cell apoptosis. Herein, we report the X-ray structure of hHTEM4 bound with undecan-2-one-CoA. Structure guided mutagenesis was carried out to confirm the catalytic residues. The N-terminal domain is shown to be partially comprised of irregular and flexible secondary structure, reminiscent of a protein-binding domain. We demonstrate direct hTHEM4-Akt1 binding by immunoprecipitation and by inhibition of Akt1 kinase activity, thus providing independent evidence that hTHEM4 is an Akt1 negative regulator.
    Biochemistry 08/2012; 51(33):6490-2. · 3.38 Impact Factor
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    Jian Dong, Zhihao Zhuang, Feng Song, Debra Dunaway-Mariano, Paul R Carey
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    ABSTRACT: 4-Hydroxybenzoyl-CoA (4-HB-CoA) thioesterase from Arthrobacter is the final enzyme catalyzing the hydrolysis of 4-HB-CoA to produce coenzyme A and 4-hydroxybenzoic acid in the bacterial 4-chlorobenzoate dehalogenation pathway. Using a mutation E73A that blocks catalysis, stable complexes of the enzyme and its substrate can be analyzed by Raman difference spectroscopy. Here we have used Raman difference spectroscopy, in the non-resonance regime, to characterize 4-HB-CoA bound in the active site of the E73A thioesterase. In addition we have characterized complexes of the wild-type enzyme complexed with the unreactive substrate analog 4-hydroxyphenacyl-CoA (4-HP-CoA). Both sets of complexes show evidence for two forms of the ligand in the active site, one population has the 4-hydroxy group protonated, 4-OH, while the second has the group as the hydroxide, 4-O(-). For bound 4-HP-CoA X-ray data show that glutamate 78 is close to the 4-OH in the complex and it is likely that this is the proton acceptor for the 4-OH proton. Although the pK(a) of the 4-OH group on the free substrate in aqueous solution is 8.6, the relative populations of ionized and neutral 4-HB-CoA bound to E73A remain invariant between pH 7.3 and pH 9.8. The invariance with pH suggests that the 4-OH and the -COO(-) of E78 constitute a tightly coupled pair where their separate pK(a)s lose their individual qualities. Narrow band profiles are seen in the C=O double bond and C-S regions suggesting that the hydrolyzable thioester group is rigidly bound in the active site in a syn gauche conformation.
    Journal of Raman Spectroscopy 01/2012; 43(1):65-71. · 2.68 Impact Factor
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    ABSTRACT: The 4-hydroxybenzoyl-CoA (4-HB-CoA) thioesterase from Pseudomonas sp. strain CBS3 catalyzes the final step of the 4-chlorobenzoate degradation pathway, which is the hydrolysis of 4-HB-CoA to coenzyme A (CoA) and 4-hydroxybenzoate (4-HB). In previous work, X-ray structural analysis of the substrate-bound thioesterase provided evidence of the role of an active site Asp17 in nucleophilic catalysis [Thoden, J. B., Holden, H. M., Zhuang, Z., and Dunaway-Mariano, D. (2002) X-ray crystallographic analyses of inhibitor and substrate complexes of wild-type and mutant 4-hydroxybenzoyl-CoA thioesterase. J. Biol. Chem. 277, 27468-27476]. In the study presented here, kinetic techniques were used to test the catalytic mechanism that was suggested by the X-ray structural data. The time course for the multiple-turnover reaction of 50 μM [(14)C]-4-HB-CoA catalyzed by 10 μM thioesterase supported a two-step pathway in which the second step is rate-limiting. Steady-state product inhibition studies revealed that binding of CoA (K(is) = 250 ± 70 μM; K(ii) = 900 ± 300 μM) and 4-HB (K(is) = 1.2 ± 0.2 mM) is weak, suggesting that product release is not rate-limiting. A substantial D(2)O solvent kinetic isotope effect (3.8) on the steady-state k(cat) value (18 s(-1)) provided evidence that a chemical step involving proton transfer is the rate-limiting step. Taken together, the kinetic results support a two-chemical pathway. The microscopic rate constants governing the formation and consumption of the putative aspartyl 17-(4-hydroxybenzoyl)anhydride intermediate were determined by simulation-based fitting of a kinetic model to time courses for the substrate binding reaction (5.0 μM 4-HB-CoA and 0.54 μM thioesterase), single-turnover reaction (5 μM [(14)C]-4-HB-CoA catalyzed by 50 μM thioesterase), steady-state reaction (5.2 μM 4-HB-CoA catalyzed by 0.003 μM thioesterase), and transient-state multiple-turnover reaction (50 μM [(14)C]-4-HB-CoA catalyzed by 10 μM thioesterase). Together with the results obtained from solvent (18)O labeling experiments, the findings are interpreted as evidence of the formation of an aspartyl 17-(4-hydroxybenzoyl)anhydride intermediate that undergoes rate-limiting hydrolytic cleavage at the hydroxybenzoyl carbonyl carbon atom.
    Biochemistry 12/2011; 51(3):786-94. · 3.38 Impact Factor
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    ABSTRACT: The Enzyme Function Initiative (EFI) was recently established to address the challenge of assigning reliable functions to enzymes discovered in bacterial genome projects; in this Current Topic, we review the structure and operations of the EFI. The EFI includes the Superfamily/Genome, Protein, Structure, Computation, and Data/Dissemination Cores that provide the infrastructure for reliably predicting the in vitro functions of unknown enzymes. The initial targets for functional assignment are selected from five functionally diverse superfamilies (amidohydrolase, enolase, glutathione transferase, haloalkanoic acid dehalogenase, and isoprenoid synthase), with five superfamily specific Bridging Projects experimentally testing the predicted in vitro enzymatic activities. The EFI also includes the Microbiology Core that evaluates the in vivo context of in vitro enzymatic functions and confirms the functional predictions of the EFI. The deliverables of the EFI to the scientific community include (1) development of a large-scale, multidisciplinary sequence/structure-based strategy for functional assignment of unknown enzymes discovered in genome projects (target selection, protein production, structure determination, computation, experimental enzymology, microbiology, and structure-based annotation), (2) dissemination of the strategy to the community via publications, collaborations, workshops, and symposia, (3) computational and bioinformatic tools for using the strategy, (4) provision of experimental protocols and/or reagents for enzyme production and characterization, and (5) dissemination of data via the EFI's Website, http://enzymefunction.org. The realization of multidisciplinary strategies for functional assignment will begin to define the full metabolic diversity that exists in nature and will impact basic biochemical and evolutionary understanding, as well as a wide range of applications of central importance to industrial, medicinal, and pharmaceutical efforts.
    Biochemistry 11/2011; 50(46):9950-62. · 3.38 Impact Factor
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    Zhibing Lu, Debra Dunaway-Mariano, Karen N Allen
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    ABSTRACT: Analysis of the haloalkanoate dehalogenase superfamily (HADSF) has uncovered homologues occurring within the same organism that are found to possess broad, overlapping substrate specificities, and low catalytic efficiencies. Here we compare the HADSF phosphatase BT1666 from Bacteroides thetaiotaomicron VPI-5482 to a homologue with high sequence identity (40%) from the same organism BT4131, a known hexose-phosphate phosphatase. The goal is to find whether these enzymes represent duplicated versus paralogous activities. The X-ray crystal structure of BT1666 was determined to 1.82 Å resolution. Superposition of the BT1666 and BT4131 structures revealed a conserved fold and identical active sites suggestive of a common physiological substrate. The steady-state kinetic constants for BT1666 were determined for a diverse panel of phosphorylated metabolites to define its substrate specificity profile and overall level of catalytic efficiency. Whereas BT1666 and BT4131 are both promiscuous, their substrate specificity profiles are distinct. The catalytic efficiency of BT1666 (k(cat) /K(m) = 4.4 × 10(2) M(-1) s(-1) for the best substrate fructose 1,6-(bis)phosphate) is an order of magnitude less than that of BT4131 (k(cat) /K(m) = 6.7 × 10(3) M(-1) s(-1) for 2-deoxyglucose 6-phosphate). The seemingly identical active-site structures point to sequence variation outside the active site causing differences in conformational dynamics or subtle catalytic positioning effects that drive the divergence in catalytic efficiency and selectivity. The overlapping substrate profiles may be understood in terms of differential regulation of expression of the two enzymes or a conferred advantage in metabolic housekeeping functions by having a larger range of possible metabolites as substrates.
    Proteins Structure Function and Bioinformatics 11/2011; 79(11):3099-107. · 3.34 Impact Factor
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    ABSTRACT: The explosion of protein sequence information requires that current strategies for function assignment evolve to complement experimental approaches with computationally based function prediction. This necessitates the development of strategies based on the identification of sequence markers in the form of specificity determinants and a more informed definition of orthologues. Herein, we have undertaken the function assignment of the unknown haloalkanoate dehalogenase superfamily member BT2127 (Uniprot accession code Q8A5 V9) from Bacteroides thetaiotaomicron using an integrated bioinformatics-structure-mechanism approach. The substrate specificity profile and steady-state rate constants of BT2127 (with a k(cat)/K(m) value for pyrophosphate of ~1 × 10(5) M(-1) s(-1)), together with the gene context, support the assigned in vivo function as an inorganic pyrophosphatase. The X-ray structural analysis of wild-type BT2127 and several variants generated by site-directed mutagenesis shows that substrate discrimination is based, in part, on active site space restrictions imposed by the cap domain (specifically by residues Tyr76 and Glu47). Structure-guided site-directed mutagenesis coupled with kinetic analysis of the mutant enzymes identified the residues required for catalysis, substrate binding, and domain-domain association. On the basis of this structure-function analysis, the catalytic residues Asp11, Asp13, Thr113, and Lys147 as well the metal binding residues Asp171, Asn172, and Glu47 were used as markers to confirm BT2127 orthologues identified via sequence searches. This bioinformatic analysis demonstrated that the biological range of BT2127 orthologue is restricted to the phylum Bacteroidetes/Chlorobi. The key structural determinants in the divergence of BT2127 and its closest homologue, β-phosphoglucomutase, control the leaving group size (phosphate vs glucose phosphate) and the position of the Asp acid/base in the open versus closed conformations. HADSF pyrophosphatases represent a third mechanistic and fold type for bacterial pyrophosphatases.
    Biochemistry 09/2011; 50(41):8937-49. · 3.38 Impact Factor

Publication Stats

3k Citations
791.12 Total Impact Points

Institutions

  • 1999–2014
    • University of New Mexico
      • Department of Chemistry and Chemical Biology
      Albuquerque, New Mexico, United States
  • 2003–2013
    • Boston University
      • • Department of Chemistry
      • • Department of Physiology and Biophysics
      Boston, Massachusetts, United States
    • Bielefeld University
      Bielefeld, North Rhine-Westphalia, Germany
  • 2000–2012
    • Case Western Reserve University
      • Department of Biochemistry
      Cleveland, OH, United States
  • 2002–2010
    • University of Massachusetts Boston
      • Department of Chemistry
      Boston, Massachusetts, United States
    • University of Wisconsin, Madison
      • Department of Biochemistry
      Madison, MS, United States
    • National Institute of Standards and Technology
      Maryland, United States
  • 2004
    • University at Buffalo, The State University of New York
      Buffalo, New York, United States
  • 1983–1998
    • 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, CA, United States
  • 1978
    • Texas A&M University
      • Department of Chemistry
      College Station, TX, United States