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1.
ACS Cent Sci ; 8(4): 430-440, 2022 Apr 27.
Artigo em Inglês | MEDLINE | ID: mdl-35505869

RESUMO

The considerable utility of glycoside phosphorylases (GPs) has led to substantial efforts over the past two decades to expand the breadth of known GP activities. Driven largely by the increase of available genomic DNA sequence data, the gap between the number of sequences in the carbohydrate active enzyme database (CAZy DB) and its functionally characterized members continues to grow. This wealth of sequence data presented an exciting opportunity to explore the ever-expanding CAZy DB to discover new GPs with never-before-described functionalities. Utilizing an in silico sequence analysis of CAZy family GH94, we discovered and then functionally and structurally characterized the new GP ß-1,3-N-acetylglucosaminide phosphorylase. This new GP was sourced from the genome of the cell-wall-less Mollicute bacterium, Acholeplasma laidlawii and was found to synthesize ß-1,3-linked N-acetylglucosaminide linkages. The resulting poly-ß-1,3-N-acetylglucosamine represents a new, previously undescribed biopolymer that completes the set of possible ß-linked GlcNAc homopolysaccharides together with chitin (ß-1,4) and PNAG (poly-ß-1,6-N-acetylglucosamine). The new biopolymer was denoted acholetin, a combination of the genus Acholeplasma and the polysaccharide chitin, and the new GP was thus denoted acholetin phosphorylase (AchP). Use of the reverse phosphorolysis action of AchP provides an efficient method to enzymatically synthesize acholetin, which is a new biodegradable polymeric material.

2.
Process Biochem ; 114: 185-192, 2022 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-35462854

RESUMO

Chromohalobacter salixigens contains a uronate dehydrogenase termed CsUDH that can convert uronic acids to their corresponding C1,C6-dicarboxy aldaric acids, an important enzyme reaction applicable for biotechnological use of sugar acids. To increase the thermal stability of this enzyme for biotechnological processes, directed evolution using gene family shuffling was applied, and the hits selected from 2-tier screening of a shuffled gene family library contained in total 16 mutations, only some of which when examined individually appreciably increased thermal stability. Most mutations, while having minimal or no effect on thermal stability when tested in isolation, were found to exhibit synergy when combined; CsUDH-inc containing all 16 mutations had ΔK t 0.5 +18 °C, such that k cat was unaffected by incubation for 1 hr at ~70 °C. X-ray crystal structure of CsUDH-inc showed tight packing of the mutated residue side-chains, and comparison of rescaled B-values showed no obvious differences between wild type and mutant structures. Activity of CsUDH-inc was severely depressed on glucuronic and galacturonic acids. Combining select combinations of only three mutations resulted in good or comparable activity on these uronic acids, while maintaining some improved thermostability with ΔK t 0.5 ~+ 10 °C, indicating potential to further thermally optimize CsUDH for hyperthermophilic reaction environments.

3.
Science ; 373(6557): 871-876, 2021 08 20.
Artigo em Inglês | MEDLINE | ID: mdl-34282049

RESUMO

DeepMind presented notably accurate predictions at the recent 14th Critical Assessment of Structure Prediction (CASP14) conference. We explored network architectures that incorporate related ideas and obtained the best performance with a three-track network in which information at the one-dimensional (1D) sequence level, the 2D distance map level, and the 3D coordinate level is successively transformed and integrated. The three-track network produces structure predictions with accuracies approaching those of DeepMind in CASP14, enables the rapid solution of challenging x-ray crystallography and cryo-electron microscopy structure modeling problems, and provides insights into the functions of proteins of currently unknown structure. The network also enables rapid generation of accurate protein-protein complex models from sequence information alone, short-circuiting traditional approaches that require modeling of individual subunits followed by docking. We make the method available to the scientific community to speed biological research.


Assuntos
Aprendizado Profundo , Conformação Proteica , Dobramento de Proteína , Proteínas/química , Proteínas ADAM/química , Sequência de Aminoácidos , Simulação por Computador , Microscopia Crioeletrônica , Cristalografia por Raios X , Bases de Dados de Proteínas , Proteínas de Membrana/química , Modelos Moleculares , Complexos Multiproteicos/química , Redes Neurais de Computação , Subunidades Proteicas/química , Proteínas/fisiologia , Receptores Acoplados a Proteínas G/química , Esfingosina N-Aciltransferase/química
4.
Nat Plants ; 6(9): 1158-1166, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32868887

RESUMO

Rubisco sustains the biosphere through the fixation of CO2 into biomass. In plants and cyanobacteria, form I Rubisco is structurally comprised of large and small subunits, whereas all other Rubisco forms lack small subunits. The rise of the form I complex through the innovation of small subunits represents a key, yet poorly understood, transition in Rubisco's evolution. Through metagenomic analyses, we discovered a previously uncharacterized clade sister to form I Rubisco that evolved without small subunits. This clade diverged before the evolution of cyanobacteria and the origin of the small subunit; thus, it provides a unique reference point to advance our understanding of form I Rubisco evolution. Structural and kinetic data presented here reveal how a proto-form I Rubisco assembled and functioned without the structural stability imparted from small subunits. Our findings provide insight into a key evolutionary transition of the most abundant enzyme on Earth and the predominant entry point for nearly all global organic carbon.


Assuntos
Cianobactérias/genética , Cianobactérias/metabolismo , Estrutura Molecular , Fotossíntese/genética , Fotossíntese/fisiologia , Fenômenos Fisiológicos Vegetais/genética , Ribulose-Bifosfato Carboxilase/genética , Ribulose-Bifosfato Carboxilase/metabolismo , Filogenia
5.
Biochemistry ; 59(16): 1630-1639, 2020 04 28.
Artigo em Inglês | MEDLINE | ID: mdl-32250597

RESUMO

α/ß hydrolases make up a large and diverse protein superfamily. In natural product biosynthesis, cis-acting thioesterase α/ß hydrolases can terminate biosynthetic assembly lines and release products by hydrolyzing or cyclizing the biosynthetic intermediate. Thioesterases can also act in trans, removing aberrant intermediates and restarting stalled biosynthesis. Knockout of this "editing" function leads to reduced product titers. The borrelidin biosynthetic gene cluster from Streptomyces parvulus Tü4055 contains a hitherto uncharacterized stand-alone thioesterase, borB. In this work, we demonstrate that purified BorB cleaves acyl substrates with a preference for propionate, which supports the hypothesis that it is also an editing thioesterase. The crystal structure of BorB shows a wedgelike hydrophobic substrate binding crevice that limits substrate length. To investigate the structure-function relationship, we made chimeric BorB variants using loop regions from characterized homologues with different specificities. BorB chimeras slightly reduced activity, arguing that the modified region is a not major determinant of substrate preference. The structure-function relationships described here contribute to the process of elimination for understanding thioesterase specificity and, ultimately, engineering and applying trans-acting thioesterases in biosynthetic assembly lines.


Assuntos
Proteínas de Bactérias/química , Ácido Graxo Sintases/química , Streptomyces/enzimologia , Tioléster Hidrolases/química , Proteínas de Bactérias/genética , Domínio Catalítico , Ácido Graxo Sintases/genética , Cinética , Família Multigênica , Engenharia de Proteínas , Especificidade por Substrato , Tioléster Hidrolases/genética
6.
J Ind Microbiol Biotechnol ; 46(8): 1225-1235, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31115703

RESUMO

Engineered polyketide synthases (PKSs) are promising synthetic biology platforms for the production of chemicals with diverse applications. The dehydratase (DH) domain within modular type I PKSs generates an α,ß-unsaturated bond in nascent polyketide intermediates through a dehydration reaction. Several crystal structures of DH domains have been solved, providing important structural insights into substrate selection and dehydration. Here, we present two DH domain structures from two chemically diverse PKSs. The first DH domain, isolated from the third module in the borrelidin PKS, is specific towards a trans-cyclopentane-carboxylate-containing polyketide substrate. The second DH domain, isolated from the first module in the fluvirucin B1 PKS, accepts an amide-containing polyketide intermediate. Sequence-structure analysis of these domains, in addition to previously published DH structures, display many significant similarities and key differences pertaining to substrate selection. The two major differences between BorA DH M3, FluA DH M1 and other DH domains are found in regions of unmodeled residues or residues containing high B-factors. These two regions are located between α3-ß11 and ß7-α2. From the catalytic Asp located in α3 to a conserved Pro in ß11, the residues between them form part of the bottom of the substrate-binding cavity responsible for binding to acyl-ACP intermediates.


Assuntos
Policetídeo Sintases/química , Sítios de Ligação , Álcoois Graxos/química , Álcoois Graxos/metabolismo , Modelos Moleculares , Policetídeo Sintases/metabolismo , Estrutura Terciária de Proteína , Especificidade por Substrato
7.
Nat Commun ; 9(1): 4596, 2018 10 30.
Artigo em Inglês | MEDLINE | ID: mdl-30375402

RESUMO

In the original version of this Article, an incorrect URL was provided in the Data Availability Statement regarding the deposition of plasmids listed in Supplementary Table 4. The correct URL is https://public-registry.jbei.org/folders/378 . This error has been corrected in both the PDF and HTML versions of the Article.

8.
Nat Commun ; 9(1): 3617, 2018 09 06.
Artigo em Inglês | MEDLINE | ID: mdl-30190458

RESUMO

Tightly regulated promoters are essential for numerous biological applications, where strong inducibility, portability, and scalability are desirable. Current systems are often incompatible with large-scale fermentations due to high inducer costs and strict media requirements. Here, we describe the bottom-up engineering of 'Jungle Express', an expression system that enables efficient gene regulation in diverse proteobacteria. This system is guided by EilR, a multidrug-binding repressor with high affinity to its optimized operator and cationic dyes that act as powerful inducers at negligible costs. In E. coli, the engineered promoters exhibit minimal basal transcription and are inducible over four orders of magnitude by 1 µM crystal violet, reaching expression levels exceeding those of the strongest current bacterial systems. Further, we provide molecular insights into specific interactions of EilR with its operator and with two inducers. The versatility of Jungle Express opens the way for tightly controlled and efficient gene expression that is not restricted to host organism, substrate, or scale.


Assuntos
Proteínas de Bactérias/genética , Escherichia coli/genética , Engenharia Genética/métodos , Regiões Operadoras Genéticas , Proteínas Repressoras/genética , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Cristalografia por Raios X , Escherichia coli/efeitos dos fármacos , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Violeta Genciana/farmacologia , Sequências Repetidas Invertidas , Regiões Promotoras Genéticas , Proteobactérias/efeitos dos fármacos , Proteobactérias/genética , Proteínas Repressoras/metabolismo , Corantes de Rosanilina/farmacologia , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Transcrição Genética
9.
Acta Crystallogr D Struct Biol ; 74(Pt 7): 702-710, 2018 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-29968680

RESUMO

The development of robust enzymes, in particular cellulases, is a key step in the success of biological routes to `second-generation' biofuels. The typical sources of the enzymes used to degrade biomass include mesophilic and thermophilic organisms. The endoglucanase J30 from glycoside hydrolase family 9 was originally identified through metagenomic analyses of compost-derived bacterial consortia. These studies, which were tailored to favor growth on targeted feedstocks, have already been shown to identify cellulases with considerable thermal tolerance. The amino-acid sequence of J30 shows comparably low identity to those of previously analyzed enzymes. As an enzyme that combines a well measurable activity with a relatively low optimal temperature (50°C) and a modest thermal tolerance, it offers the potential for structural optimization aimed at increased stability. Here, the crystal structure of wild-type J30 is presented along with that of a designed triple-mutant variant with improved characteristics for industrial applications. Through the introduction of a structural Zn2+ site, the thermal tolerance was increased by more than 10°C and was paralleled by an increase in the catalytic optimum temperature by more than 5°C.


Assuntos
Glicosídeo Hidrolases/química , Engenharia de Proteínas/métodos , Zinco/química , Biocatálise , Cristalografia por Raios X , Estabilidade Enzimática , Proteínas Mutantes , Ligação Proteica , Temperatura
10.
Nat Chem Biol ; 14(5): 451-457, 2018 05.
Artigo em Inglês | MEDLINE | ID: mdl-29556105

RESUMO

Microbial toluene biosynthesis was reported in anoxic lake sediments more than three decades ago, but the enzyme catalyzing this biochemically challenging reaction has never been identified. Here we report the toluene-producing enzyme PhdB, a glycyl radical enzyme of bacterial origin that catalyzes phenylacetate decarboxylation, and its cognate activating enzyme PhdA, a radical S-adenosylmethionine enzyme, discovered in two distinct anoxic microbial communities that produce toluene. The unconventional process of enzyme discovery from a complex microbial community (>300,000 genes), rather than from a microbial isolate, involved metagenomics- and metaproteomics-enabled biochemistry, as well as in vitro confirmation of activity with recombinant enzymes. This work expands the known catalytic range of glycyl radical enzymes (only seven reaction types had been characterized previously) and aromatic-hydrocarbon-producing enzymes, and will enable first-time biochemical synthesis of an aromatic fuel hydrocarbon from renewable resources, such as lignocellulosic biomass, rather than from petroleum.


Assuntos
Bactérias/enzimologia , Microbiota , Tolueno/metabolismo , Acidobacteria/enzimologia , Acidobacteria/genética , Acidobacteria/isolamento & purificação , Anaerobiose , Bactérias/genética , Biomassa , Carboxiliases/metabolismo , Catálise , Genes Bacterianos , Sedimentos Geológicos/microbiologia , Lagos/microbiologia , Lignina/química , Funções Verossimilhança , Metagenômica , Fenilacetatos/química , Filogenia , Proteômica , Proteínas Recombinantes/metabolismo , Esgotos/microbiologia
11.
J Appl Crystallogr ; 50(Pt 5): 1352-1358, 2017 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-29021733

RESUMO

Using statistical analysis of the Biological Macromolecular Crystallization Database, combined with previous knowledge about crystallization reagents, a crystallization screen called the Berkeley Screen has been created. Correlating crystallization conditions and high-resolution protein structures, it is possible to better understand the influence that a particular solution has on protein crystal formation. Ions and small molecules such as buffers and precipitants used in crystallization experiments were identified in electron density maps, highlighting the role of these chemicals in protein crystal packing. The Berkeley Screen has been extensively used to crystallize target proteins from the Joint BioEnergy Institute and the Collaborative Crystallography program at the Berkeley Center for Structural Biology, contributing to several Protein Data Bank entries and related publications. The Berkeley Screen provides the crystallographic community with an efficient set of solutions for general macromolecular crystallization trials, offering a valuable alternative to the existing commercially available screens.

13.
Sci Rep ; 7(1): 3673, 2017 06 16.
Artigo em Inglês | MEDLINE | ID: mdl-28623285

RESUMO

The human chaperonin TRiC consists of eight non-identical subunits, and its protein-folding activity is critical for cellular health. Misfolded proteins are associated with many human diseases, such as amyloid diseases, cancer, and neuropathies, making TRiC a potential therapeutic target. A detailed structural understanding of its ATP-dependent folding mechanism and substrate recognition is therefore of great importance. Of particular health-related interest is the mutation Histidine 147 to Arginine (H147R) in human TRiC subunit 5 (CCT5), which has been associated with hereditary sensory neuropathy. In this paper, we describe the crystal structures of CCT5 and the CCT5-H147R mutant, which provide important structural information for this vital protein-folding machine in humans. This first X-ray crystallographic study of a single human CCT subunit in the context of a hexadecameric complex can be expanded in the future to the other 7 subunits that form the TRiC complex.


Assuntos
Chaperonina com TCP-1/química , Chaperonina com TCP-1/genética , Suscetibilidade a Doenças , Neuropatias Hereditárias Sensoriais e Autônomas/genética , Difosfato de Adenosina/química , Difosfato de Adenosina/metabolismo , Sequência de Aminoácidos , Humanos , Modelos Moleculares , Mutação , Conformação Proteica , Multimerização Proteica , Subunidades Proteicas/química , Subunidades Proteicas/genética , Relação Estrutura-Atividade
14.
Proc Natl Acad Sci U S A ; 113(50): 14324-14329, 2016 12 13.
Artigo em Inglês | MEDLINE | ID: mdl-27911781

RESUMO

Stilbenes are diphenyl ethene compounds produced naturally in a wide variety of plant species and some bacteria. Stilbenes are also derived from lignin during kraft pulping. Stilbene cleavage oxygenases (SCOs) cleave the central double bond of stilbenes, forming two phenolic aldehydes. Here, we report the structure of an SCO. The X-ray structure of NOV1 from Novosphingobium aromaticivorans was determined in complex with its substrate resveratrol (1.89 Å), its product vanillin (1.75 Å), and without any bound ligand (1.61 Å). The enzyme is a seven-bladed ß-propeller with an iron cofactor coordinated by four histidines. In all three structures, dioxygen is observed bound to the iron in a side-on fashion. These structures, along with EPR analysis, allow us to propose a mechanism in which a ferric-superoxide reacts with substrate activated by deprotonation of a phenol group at position 4 of the substrate, which allows movement of electron density toward the central double bond and thus facilitates reaction with the ferric superoxide electrophile. Correspondingly, NOV1 cleaves a wide range of other stilbene-like compounds with a 4'-OH group, offering potential in processing some solubilized fragments of lignin into monomer aromatic compounds.


Assuntos
Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Dioxigenases/química , Dioxigenases/metabolismo , Estilbenos/metabolismo , Proteínas de Bactérias/genética , Domínio Catalítico , Cristalografia por Raios X , Dioxigenases/genética , Espectroscopia de Ressonância de Spin Eletrônica , Modelos Moleculares , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Resveratrol , Sphingomonadaceae/enzimologia , Sphingomonadaceae/genética , Especificidade por Substrato
15.
Plant Cell Physiol ; 57(3): 568-79, 2016 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-26858288

RESUMO

Lignin poses a major challenge in the processing of plant biomass for agro-industrial applications. For bioengineering purposes, there is a pressing interest in identifying and characterizing the enzymes responsible for the biosynthesis of lignin. Hydroxycinnamoyl-CoA:shikimate hydroxycinnamoyl transferase (HCT; EC 2.3.1.133) is a key metabolic entry point for the synthesis of the most important lignin monomers: coniferyl and sinapyl alcohols. In this study, we investigated the substrate promiscuity of HCT from a bryophyte (Physcomitrella) and from five representatives of vascular plants (Arabidopsis, poplar, switchgrass, pine and Selaginella) using a yeast expression system. We demonstrate for these HCTs a conserved capacity to acylate with p-coumaroyl-CoA several phenolic compounds in addition to the canonical acceptor shikimate normally used during lignin biosynthesis. Using either recombinant HCT from switchgrass (PvHCT2a) or an Arabidopsis stem protein extract, we show evidence of the inhibitory effect of these phenolics on the synthesis of p-coumaroyl shikimate in vitro, which presumably occurs via a mechanism of competitive inhibition. A structural study of PvHCT2a confirmed the binding of a non-canonical acceptor in a similar manner to shikimate in the active site of the enzyme. Finally, we exploited in Arabidopsis the substrate flexibility of HCT to reduce lignin content and improve biomass saccharification by engineering transgenic lines that overproduce one of the HCT non-canonical acceptors. Our results demonstrate conservation of HCT substrate promiscuity and provide support for a new strategy for lignin reduction in the effort to improve the quality of plant biomass for forage and cellulosic biofuels.


Assuntos
Ácidos Cumáricos/metabolismo , Lignina/metabolismo , Plantas/enzimologia , Ácido Chiquímico/metabolismo , Aciltransferases/antagonistas & inibidores , Aciltransferases/química , Aciltransferases/metabolismo , Arabidopsis/enzimologia , Arabidopsis/genética , Sítios de Ligação , Biomassa , Vias Biossintéticas , Metabolismo dos Carboidratos , Hidroxibenzoatos/metabolismo , Modelos Moleculares , Oxirredução , Plantas Geneticamente Modificadas , Proteínas Recombinantes/metabolismo , Especificidade por Substrato
16.
ChemMedChem ; 9(10): 2286-93, 2014 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-25047814

RESUMO

The actin filament-binding and filament-severing activities of the aplyronine, kabiramide, and reidispongiolide families of marine macrolides are located within the hydrophobic tail region of the molecule. Two synthetic tail analogues of aplyronine C (SF-01 and GC-04) are shown to bind to G-actin with dissociation constants of (285±33) and (132±13) nM, respectively. The crystal structures of actin complexes with GC-04, SF-01, and kabiramide C reveal a conserved mode of tail binding within the cleft that forms between subdomains (SD) 1 and 3. Our studies support the view that filament severing is brought about by specific binding of the tail region to the SD1/SD3 cleft on the upper protomer, which displaces loop-D from the lower protomer on the same half-filament. With previous studies showing that the GC-04 analogue can sever actin filaments, it is argued that the shorter complex lifetime of tail analogues with F-actin would make them more effective at severing filaments compared with plasma gelsolin. Structure-based analyses are used to suggest more reactive or targetable forms of GC-04 and SF-01, which may serve to boost the capacity of the serum actin scavenging system, to generate antibody conjugates against tumor cell antigens, and to decrease sputum viscosity in children with cystic fibrosis.


Assuntos
Actinas/química , Macrolídeos/química , Actinas/metabolismo , Animais , Cristalografia por Raios X , Modelos Moleculares , Coelhos
17.
Structure ; 22(7): 1028-36, 2014 Jul 08.
Artigo em Inglês | MEDLINE | ID: mdl-24954619

RESUMO

The archaeal enzyme geranylgeranyl reductase (GGR) catalyzes hydrogenation of carbon-carbon double bonds to produce the saturated alkyl chains of the organism's unusual isoprenoid-derived cell membrane. Enzymatic reduction of isoprenoid double bonds is of considerable interest both to natural products researchers and to synthetic biologists interested in the microbial production of isoprenoid drug or biofuel molecules. Here we present crystal structures of GGR from Sulfolobus acidocaldarius, including the structure of GGR bound to geranylgeranyl pyrophosphate (GGPP). The structures are presented alongside activity data that depict the sequential reduction of GGPP to H6GGPP via the intermediates H2GGPP and H4GGPP. We then modified the enzyme to generate sequence variants that display increased rates of H6GGPP production or are able to halt the extent of reduction at H2GGPP and H4GGPP. Crystal structures of these variants not only reveal the structural bases for their altered activities; they also shed light onto the catalytic mechanism employed.


Assuntos
Proteínas Arqueais/química , Oxirredutases/química , Estrutura Terciária de Proteína , Terpenos/química , Proteínas Arqueais/genética , Proteínas Arqueais/metabolismo , Cristalografia por Raios X , Cinética , Modelos Moleculares , Estrutura Molecular , Mutação , Oxirredutases/genética , Oxirredutases/metabolismo , Fosfatos de Poli-Isoprenil/química , Fosfatos de Poli-Isoprenil/metabolismo , Ligação Proteica , Especificidade por Substrato , Sulfolobus acidocaldarius/enzimologia , Sulfolobus acidocaldarius/genética , Sulfolobus acidocaldarius/metabolismo , Terpenos/metabolismo
18.
Appl Environ Microbiol ; 80(2): 497-505, 2014 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-24212572

RESUMO

Major efforts in bioenergy research have focused on producing fuels that can directly replace petroleum-derived gasoline and diesel fuel through metabolic engineering of microbial fatty acid biosynthetic pathways. Typically, growth and pathway induction are conducted under aerobic conditions, but for operational efficiency in an industrial context, anaerobic culture conditions would be preferred to obviate the need to maintain specific dissolved oxygen concentrations and to maximize the proportion of reducing equivalents directed to biofuel biosynthesis rather than ATP production. A major concern with fermentative growth conditions is elevated NADH levels, which can adversely affect cell physiology. The purpose of this study was to identify homologs of Escherichia coli FabG, an essential reductase involved in fatty acid biosynthesis, that display a higher preference for NADH than for NADPH as a cofactor. Four potential NADH-dependent FabG variants were identified through bioinformatic analyses supported by crystallographic structure determination (1.3- to 2.0-Å resolution). In vitro assays of cofactor (NADH/NADPH) preference in the four variants showed up to ≈ 35-fold preference for NADH, which was observed with the Cupriavidus taiwanensis FabG variant. In addition, FabG homologs were overexpressed in fatty acid- and methyl ketone-overproducing E. coli host strains under anaerobic conditions, and the C. taiwanensis variant led to a 60% higher free fatty acid titer and 75% higher methyl ketone titer relative to the titers of the control strains. With further engineering, this work could serve as a starting point for establishing a microbial host strain for production of fatty acid-derived biofuels (e.g., methyl ketones) under anaerobic conditions.


Assuntos
Oxirredutases do Álcool/química , Oxirredutases do Álcool/metabolismo , Escherichia coli/metabolismo , Ácidos Graxos/biossíntese , NAD/metabolismo , Proteínas Recombinantes/metabolismo , Oxirredutases do Álcool/genética , Sequência de Aminoácidos , Anaerobiose , Sítios de Ligação , Cristalografia por Raios X , Escherichia coli/genética , Escherichia coli/crescimento & desenvolvimento , Ácidos Graxos/metabolismo , Microbiologia Industrial/métodos , Dados de Sequência Molecular , NADP/metabolismo , Conformação Proteica , Engenharia de Proteínas/métodos , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Homologia de Sequência de Aminoácidos
19.
PLoS One ; 8(11): e79725, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-24244549

RESUMO

Ionic liquid pretreatment of biomass has been shown to greatly reduce the recalcitrance of lignocellulosic biomass, resulting in improved sugar yields after enzymatic saccharification. However, even under these improved saccharification conditions the cost of enzymes still represents a significant proportion of the total cost of producing sugars and ultimately fuels from lignocellulosic biomass. Much of the high cost of enzymes is due to the low catalytic efficiency and stability of lignocellulolytic enzymes, especially cellulases, under conditions that include high temperatures and the presence of residual pretreatment chemicals, such as acids, organic solvents, bases, or ionic liquids. Improving the efficiency of the saccharification process on ionic liquid pretreated biomass will facilitate reduced enzyme loading and cost. Thermophilic cellulases have been shown to be stable and active in ionic liquids but their activity is typically at lower levels. Cel5A_Tma, a thermophilic endoglucanase from Thermotoga maritima, is highly active on cellulosic substrates and is stable in ionic liquid environments. Here, our motivation was to engineer mutants of Cel5A_Tma with higher activity on 1-ethyl-3-methylimidazolium acetate ([C2mim][OAc]) pretreated biomass. We developed a robotic platform to screen a random mutagenesis library of Cel5A_Tma. Twelve mutants with 25-42% improvement in specific activity on carboxymethyl cellulose and up to 30% improvement on ionic-liquid pretreated switchgrass were successfully isolated and characterized from a library of twenty thousand variants. Interestingly, most of the mutations in the improved variants are located distally to the active site on the protein surface and are not directly involved with substrate binding.


Assuntos
Celulase/metabolismo , Líquidos Iônicos , Poaceae , Thermotoga maritima/enzimologia , Biomassa , Celulase/química , Celulase/genética , Ativação Enzimática , Ensaios de Triagem em Larga Escala , Modelos Moleculares , Mutagênese , Conformação Proteica , Thermotoga maritima/genética
20.
Acta Crystallogr D Biol Crystallogr ; 68(Pt 10): 1320-8, 2012 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-22993086

RESUMO

Micrococcus luteus is a Gram-positive bacterium that produces iso- and anteiso-branched alkenes by the head-to-head condensation of fatty-acid thioesters [coenzyme A (CoA) or acyl carrier protein (ACP)]; this activity is of interest for the production of advanced biofuels. In an effort to better understand the control of the formation of branched fatty acids in M. luteus, the structure of FabH (MlFabH) was determined. FabH, or ß-ketoacyl-ACP synthase III, catalyzes the initial step of fatty-acid biosynthesis: the condensation of malonyl-ACP with an acyl-CoA. Analysis of the MlFabH structure provides insights into its substrate selectivity with regard to length and branching of the acyl-CoA. The most structurally divergent region of FabH is the L9 loop region located at the dimer interface, which is involved in the formation of the acyl-binding channel and thus limits the substrate-channel size. The residue Phe336, which is positioned near the catalytic triad, appears to play a major role in branched-substrate selectivity. In addition to structural studies of MlFabH, transcriptional studies of M. luteus were also performed, focusing on the increase in the ratio of anteiso:iso-branched alkenes that was observed during the transition from early to late stationary phase. Gene-expression microarray analysis identified two genes involved in leucine and isoleucine metabolism that may explain this transition.


Assuntos
Acetil-CoA C-Acetiltransferase/química , Proteínas de Bactérias/química , Ácidos Graxos/química , Micrococcus luteus/enzimologia , Acetil-CoA C-Acetiltransferase/metabolismo , Proteínas de Bactérias/metabolismo , Domínio Catalítico , Sequência Conservada , Cristalografia por Raios X , Ácidos Graxos/classificação , Ácidos Graxos/metabolismo , Interações Hidrofóbicas e Hidrofílicas , Micrococcus luteus/metabolismo , Especificidade por Substrato
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