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1.
ACS Cent Sci ; 1(2): 77-82, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26258173

RESUMO

Squalene (SQ) is an intermediate in the biosynthesis of sterols in eukaryotes and a few bacteria and of hopanoids in bacteria where they promote membrane stability and the formation of lipid rafts in their hosts. The genes for hopanoid biosynthesis are typically located on clusters that consist of four highly conserved genes-hpnC, hpnD, hpnE, and hpnF-for conversion of farnesyl diphosphate (FPP) to hopene or related pentacyclic metabolites. While hpnF is known to encode a squalene cyclase, the functions for hpnC, hpnD, and hpnE are not rigorously established. The hpnC, hpnD, and hpnE genes from Zymomonas mobilis and Rhodopseudomonas palustris were cloned into Escherichia coli, a bacterium that does not contain genes homologous to hpnC, hpnD, and hpnE, and their functions were established in vitro and in vivo. HpnD catalyzes formation of presqualene diphosphate (PSPP) from two molecules of FPP; HpnC converts PSPP to hydroxysqualene (HSQ); and HpnE, a member of the amine oxidoreductase family, reduces HSQ to SQ. Collectively the reactions catalyzed by these three enzymes constitute a new pathway for biosynthesis of SQ in bacteria.

2.
Proc Natl Acad Sci U S A ; 112(16): E1974-83, 2015 Apr 21.
Artigo em Inglês | MEDLINE | ID: mdl-25848029

RESUMO

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 substrate 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.


Assuntos
Família Multigênica , Monoéster Fosfórico Hidrolases/metabolismo , Ensaios de Triagem em Larga Escala , Cinética , Reprodutibilidade dos Testes , Especificidade por Substrato
3.
Proc Natl Acad Sci U S A ; 112(18): 5661-6, 2015 May 05.
Artigo em Inglês | MEDLINE | ID: mdl-25901324

RESUMO

Terpenoids are a large structurally diverse group of natural products with an array of functions in their hosts. The large amount of genomic information from recent sequencing efforts provides opportunities and challenges for the functional assignment of terpene synthases that construct the carbon skeletons of these compounds. Inferring function from the sequence and/or structure of these enzymes is not trivial because of the large number of possible reaction channels and products. We tackle this problem by developing an algorithm to enumerate possible carbocations derived from the farnesyl cation, the first reactive intermediate of the substrate, and evaluating their steric and electrostatic compatibility with the active site. The homology model of a putative pentalenene synthase (Uniprot: B5GLM7) from Streptomyces clavuligerus was used in an automated computational workflow for product prediction. Surprisingly, the workflow predicted a linear triquinane scaffold as the top product skeleton for B5GLM7. Biochemical characterization of B5GLM7 reveals the major product as (5S,7S,10R,11S)-cucumene, a sesquiterpene with a linear triquinane scaffold. To our knowledge, this is the first documentation of a terpene synthase involved in the synthesis of a linear triquinane. The success of our prediction for B5GLM7 suggests that this approach can be used to facilitate the functional assignment of novel terpene synthases.


Assuntos
Alquil e Aril Transferases/química , Streptomyces/enzimologia , Algoritmos , Carbono/química , Domínio Catalítico , Cátions , Análise por Conglomerados , Biologia Computacional , Simulação por Computador , Estrutura Terciária de Proteína , Software , Relação Estrutura-Atividade
4.
Biochemistry ; 54(3): 909-31, 2015 Jan 27.
Artigo em Inglês | MEDLINE | ID: mdl-25540822

RESUMO

The rate at which genome sequencing data is accruing demands enhanced methods for functional annotation and metabolism discovery. Solute binding proteins (SBPs) facilitate the transport of the first reactant in a metabolic pathway, thereby constraining the regions of chemical space and the chemistries that must be considered for pathway reconstruction. We describe high-throughput protein production and differential scanning fluorimetry platforms, which enabled the screening of 158 SBPs against a 189 component library specifically tailored for this class of proteins. Like all screening efforts, this approach is limited by the practical constraints imposed by construction of the library, i.e., we can study only those metabolites that are known to exist and which can be made in sufficient quantities for experimentation. To move beyond these inherent limitations, we illustrate the promise of crystallographic- and mass spectrometric-based approaches for the unbiased use of entire metabolomes as screening libraries. Together, our approaches identified 40 new SBP ligands, generated experiment-based annotations for 2084 SBPs in 71 isofunctional clusters, and defined numerous metabolic pathways, including novel catabolic pathways for the utilization of ethanolamine as sole nitrogen source and the use of d-Ala-d-Ala as sole carbon source. These efforts begin to define an integrated strategy for realizing the full value of amassing genome sequence data.


Assuntos
Proteínas de Transporte/metabolismo , Redes e Vias Metabólicas , Metaboloma , Metabolômica/métodos , Anotação de Sequência Molecular , Bacillus/metabolismo , Carboidratos/química , Clonagem Molecular , Cristalografia por Raios X , Fluorometria , Cinética , Ligantes , Reprodutibilidade dos Testes , Homologia de Sequência de Aminoácidos
5.
Biochemistry ; 53(20): 3357-66, 2014 May 27.
Artigo em Inglês | MEDLINE | ID: mdl-24831290

RESUMO

The l-lyxonate dehydratase (LyxD) in vitro enzymatic activity and in vivo metabolic function were assigned to members of an isofunctional family within the mandelate racemase (MR) subgroup of the enolase superfamily. This study combined in vitro and in vivo data to confirm that the dehydration of l-lyxonate is the biological role of the members of this family. In vitro kinetic experiments revealed catalytic efficiencies of ∼10(4) M(-1) s(-1) as previously observed for members of other families in the MR subgroup. Growth studies revealed that l-lyxonate is a carbon source for Pseudomonas aeruginosa PAO1; transcriptomics using qRT-PCR established that the gene encoding LyxD as well as several other conserved proximal genes were upregulated in cells grown on l-lyxonate. The proximal genes were shown to be involved in a pathway for the degradation of l-lyxonate, in which the first step is dehydration by LyxD followed by dehydration of the 2-keto-3-deoxy-l-lyxonate product by 2-keto-3-deoxy-l-lyxonate dehydratase to yield α-ketoglutarate semialdehyde. In the final step, α-ketoglutarate semialdehyde is oxidized by a dehydrogenase to α-ketoglutarate, an intermediate in the citric acid cycle. An X-ray structure for the LyxD from Labrenzia aggregata IAM 12614 with Mg(2+) in the active site was determined that confirmed the expectation based on sequence alignments that LyxDs possess a conserved catalytic His-Asp dyad at the end of seventh and sixth ß-strands of the (ß/α)7ß-barrel domain as well as a conserved KxR motif at the end of second ß-strand; substitutions for His 316 or Arg 179 inactivated the enzyme. This is the first example of both the LyxD function in the enolase superfamily and a pathway for the catabolism of l-lyxonate.


Assuntos
Proteínas de Bactérias/química , Pseudomonas aeruginosa/química , Transdução de Sinais/fisiologia , Açúcares Ácidos/química , Sequência de Aminoácidos , Proteínas de Bactérias/genética , Proteínas de Bactérias/fisiologia , Dados de Sequência Molecular , Estrutura Secundária de Proteína , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/fisiologia , Transdução de Sinais/genética , Açúcares Ácidos/metabolismo , Transcriptoma
6.
Biochemistry ; 53(16): 2722-31, 2014 Apr 29.
Artigo em Inglês | MEDLINE | ID: mdl-24697546

RESUMO

The continued increase in the size of the protein sequence databases as a result of advances in genome sequencing technology is overwhelming the ability to perform experimental characterization of function. Consequently, functions are assigned to the vast majority of proteins via automated, homology-based methods, with the result that as many as 50% are incorrectly annotated or unannotated ( Schnoes et al. PLoS Comput. Biol. 2009 , 5 ( 12 ), e1000605 ). This manuscript describes a study of the D-mannonate dehydratase (ManD) subgroup of the enolase superfamily (ENS) to investigate how function diverges as sequence diverges. Previously, one member of the subgroup had been experimentally characterized as ManD [dehydration of D-mannonate to 2-keto-3-deoxy-D-mannonate (equivalently, 2-keto-3-deoxy-D-gluconate)]. In this study, 42 additional members were characterized to sample sequence-function space in the ManD subgroup. These were found to differ in both catalytic efficiency and substrate specificity: (1) high efficiency (kcat/KM = 10(3) to 10(4) M(-1) s(-1)) for dehydration of D-mannonate, (2) low efficiency (kcat/KM = 10(1) to 10(2) M(-1) s(-1)) for dehydration of d-mannonate and/or D-gluconate, and 3) no-activity with either D-mannonate or D-gluconate (or any other acid sugar tested). Thus, the ManD subgroup is not isofunctional and includes D-gluconate dehydratases (GlcDs) that are divergent from the GlcDs that have been characterized in the mandelate racemase subgroup of the ENS (Lamble et al. FEBS Lett. 2004 , 576 , 133 - 136 ) (Ahmed et al. Biochem. J. 2005 , 390 , 529 - 540 ). These observations signal caution for functional assignment based on sequence homology and lay the foundation for the studies of the physiological functions of the GlcDs and the promiscuous ManDs/GlcDs.


Assuntos
Hidroliases/química , Hidroliases/metabolismo , Fosfopiruvato Hidratase/metabolismo , Domínio Catalítico , Cristalografia por Raios X , Gluconatos/metabolismo , Hidroliases/genética , Cinética , Dados de Sequência Molecular , Mutação , Fosfopiruvato Hidratase/química , Conformação Proteica , Relação Estrutura-Atividade , Especificidade por Substrato , Açúcares Ácidos/metabolismo
7.
Nature ; 502(7473): 698-702, 2013 Oct 31.
Artigo em Inglês | MEDLINE | ID: mdl-24056934

RESUMO

Assigning valid functions to proteins identified in genome projects is challenging: overprediction and database annotation errors are the principal concerns. We and others are developing computation-guided strategies for functional discovery with 'metabolite docking' to experimentally derived or homology-based three-dimensional structures. Bacterial metabolic pathways often are encoded by 'genome neighbourhoods' (gene clusters and/or operons), which can provide important clues for functional assignment. We recently demonstrated the synergy of docking and pathway context by 'predicting' the intermediates in the glycolytic pathway in Escherichia coli. Metabolite docking to multiple binding proteins and enzymes in the same pathway increases the reliability of in silico predictions of substrate specificities because the pathway intermediates are structurally similar. Here we report that structure-guided approaches for predicting the substrate specificities of several enzymes encoded by a bacterial gene cluster allowed the correct prediction of the in vitro activity of a structurally characterized enzyme of unknown function (PDB 2PMQ), 2-epimerization of trans-4-hydroxy-L-proline betaine (tHyp-B) and cis-4-hydroxy-D-proline betaine (cHyp-B), and also the correct identification of the catabolic pathway in which Hyp-B 2-epimerase participates. The substrate-liganded pose predicted by virtual library screening (docking) was confirmed experimentally. The enzymatic activities in the predicted pathway were confirmed by in vitro assays and genetic analyses; the intermediates were identified by metabolomics; and repression of the genes encoding the pathway by high salt concentrations was established by transcriptomics, confirming the osmolyte role of tHyp-B. This study establishes the utility of structure-guided functional predictions to enable the discovery of new metabolic pathways.


Assuntos
Bactérias , Enzimas/química , Enzimas/genética , Genoma Bacteriano/genética , Redes e Vias Metabólicas/genética , Anotação de Sequência Molecular/métodos , Homologia Estrutural de Proteína , Bactérias/enzimologia , Bactérias/genética , Bactérias/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Enzimas/metabolismo , Perfilação da Expressão Gênica , Genes Bacterianos/genética , Glicólise , Cinética , Metabolismo , Metabolômica , Modelos Moleculares , Família Multigênica/genética , Óperon , Especificidade por Substrato
8.
Curr Opin Struct Biol ; 23(3): 335-44, 2013 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-23642905

RESUMO

Despite a multitude of recent technical breakthroughs speeding high-resolution structural analysis of biological macromolecules, production of sufficient quantities of well-behaved, active protein continues to represent the rate-limiting step in many structure determination efforts. These challenges are only amplified when considered in the context of ongoing structural genomics efforts, which are now contending with multi-domain eukaryotic proteins, secreted proteins, and ever-larger macromolecular assemblies. Exciting new developments in eukaryotic expression platforms, including insect and mammalian-based systems, promise enhanced opportunities for structural approaches to some of the most important biological problems. Development and implementation of automated eukaryotic expression techniques promises to significantly improve production of materials for structural, functional, and biomedical research applications.


Assuntos
Células Eucarióticas/metabolismo , Genômica , Proteínas Recombinantes/biossíntese , Animais , Biologia Computacional , Células Eucarióticas/química , Expressão Gênica , Ensaios de Triagem em Larga Escala , Conformação Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação
9.
Proc Natl Acad Sci U S A ; 110(13): E1196-202, 2013 Mar 26.
Artigo em Inglês | MEDLINE | ID: mdl-23493556

RESUMO

The number of available protein sequences has increased exponentially with the advent of high-throughput genomic sequencing, creating a significant challenge for functional annotation. Here, we describe a large-scale study on assigning function to unknown members of the trans-polyprenyl transferase (E-PTS) subgroup in the isoprenoid synthase superfamily, which provides substrates for the biosynthesis of the more than 55,000 isoprenoid metabolites. Although the mechanism for determining the product chain length for these enzymes is known, there is no simple relationship between function and primary sequence, so that assigning function is challenging. We addressed this challenge through large-scale bioinformatics analysis of >5,000 putative polyprenyl transferases; experimental characterization of the chain-length specificity of 79 diverse members of this group; determination of 27 structures of 19 of these enzymes, including seven cocrystallized with substrate analogs or products; and the development and successful application of a computational approach to predict function that leverages available structural data through homology modeling and docking of possible products into the active site. The crystallographic structures and computational structural models of the enzyme-ligand complexes elucidate the structural basis of specificity. As a result of this study, the percentage of E-PTS sequences similar to functionally annotated ones (BLAST e-value ≤ 1e(-70)) increased from 40.6 to 68.8%, and the percentage of sequences similar to available crystal structures increased from 28.9 to 47.4%. The high accuracy of our blind prediction of newly characterized enzymes indicates the potential to predict function to the complete polyprenyl transferase subgroup of the isoprenoid synthase superfamily computationally.


Assuntos
Alquil e Aril Transferases/genética , Carbono-Carbono Ligases/genética , Bases de Dados de Proteínas , Simulação de Acoplamento Molecular/métodos , Análise de Sequência de Proteína/métodos , Alquil e Aril Transferases/metabolismo , Carbono-Carbono Ligases/metabolismo , Cristalografia por Raios X
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