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1.
Nat Commun ; 12(1): 4174, 2021 07 07.
Artigo em Inglês | MEDLINE | ID: mdl-34234105

RESUMO

The folding of ß-barrel outer membrane proteins (OMPs) in Gram-negative bacteria is catalysed by the ß-barrel assembly machinery (BAM). How lateral opening in the ß-barrel of the major subunit BamA assists in OMP folding, and the contribution of membrane disruption to BAM catalysis remain unresolved. Here, we use an anti-BamA monoclonal antibody fragment (Fab1) and two disulphide-crosslinked BAM variants (lid-locked (LL), and POTRA-5-locked (P5L)) to dissect these roles. Despite being lethal in vivo, we show that all complexes catalyse folding in vitro, albeit less efficiently than wild-type BAM. CryoEM reveals that while Fab1 and BAM-P5L trap an open-barrel state, BAM-LL contains a mixture of closed and contorted, partially-open structures. Finally, all three complexes globally destabilise the lipid bilayer, while BamA does not, revealing that the BAM lipoproteins are required for this function. Together the results provide insights into the role of BAM structure and lipid dynamics in OMP folding.


Assuntos
Proteínas da Membrana Bacteriana Externa/metabolismo , Proteínas de Escherichia coli/metabolismo , Hidrolases/metabolismo , Lipossomos/metabolismo , Dobramento de Proteína , Proteínas da Membrana Bacteriana Externa/genética , Proteínas da Membrana Bacteriana Externa/isolamento & purificação , Proteínas da Membrana Bacteriana Externa/ultraestrutura , Microscopia Crioeletrônica , Difusão Dinâmica da Luz , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/isolamento & purificação , Proteínas de Escherichia coli/ultraestrutura , Hidrolases/genética , Hidrolases/isolamento & purificação , Hidrolases/ultraestrutura , Metabolismo dos Lipídeos , Lipossomos/ultraestrutura , Simulação de Dinâmica Molecular , Conformação Proteica em Folha beta , Proteolipídeos/metabolismo , Proteolipídeos/ultraestrutura , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/ultraestrutura
2.
FEBS J ; 288(19): 5768-5780, 2021 10.
Artigo em Inglês | MEDLINE | ID: mdl-33843134

RESUMO

Mycophenolic acid (MPA) is a fungal natural product and first-line immunosuppressive drug for organ transplantations and autoimmune diseases. In the compartmentalized biosynthesis of MPA, the acyl-coenzyme A (CoA) hydrolase MpaH' located in peroxisomes catalyzes the highly specific hydrolysis of MPA-CoA to produce the final product MPA. The strict substrate specificity of MpaH' not only averts undesired hydrolysis of various cellular acyl-CoAs, but also prevents MPA-CoA from further peroxisomal ß-oxidation catabolism. To elucidate the structural basis for this important property, in this study, we solve the crystal structures of the substrate-free form of MpaH' and the MpaH'S139A mutant in complex with the product MPA. The MpaH' structure reveals a canonical α/ß-hydrolase fold with an unusually large cap domain and a rare location of the acidic residue D163 of catalytic triad after strand ß6. MpaH' also forms an atypical dimer with the unique C-terminal helices α13 and α14 arming the cap domain of the other protomer and indirectly participating in the substrate binding. With these characteristics, we propose that MpaH' and its homologs form a new subfamily of α/ß hydrolase fold protein. The crystal structure of MpaH'S139A /MPA complex and the modeled structure of MpaH'/MPA-CoA, together with the structure-guided mutagenesis analysis and isothermal titration calorimetry (ITC) measurements, provide important mechanistic insights into the high substrate specificity of MpaH'.


Assuntos
Acil Coenzima A/química , Hidrolases/ultraestrutura , Ácido Micofenólico/metabolismo , Peroxissomos/ultraestrutura , Sequência de Aminoácidos/genética , Domínio Catalítico/genética , Hidrolases/química , Hidrolases/genética , Ácido Micofenólico/química , Penicillium/genética , Penicillium/ultraestrutura , Peroxissomos/enzimologia , Estrutura Secundária de Proteína/genética , Especificidade por Substrato/genética
3.
J Biol Chem ; 295(27): 9087-9104, 2020 07 03.
Artigo em Inglês | MEDLINE | ID: mdl-32404365

RESUMO

When faced with increased osmolarity in the environment, many bacterial cells accumulate the compatible solute ectoine and its derivative 5-hydroxyectoine. Both compounds are not only potent osmostress protectants, but also serve as effective chemical chaperones stabilizing protein functionality. Ectoines are energy-rich nitrogen and carbon sources that have an ecological impact that shapes microbial communities. Although the biochemistry of ectoine and 5-hydroxyectoine biosynthesis is well understood, our understanding of their catabolism is only rudimentary. Here, we combined biochemical and structural approaches to unravel the core of ectoine and 5-hydroxy-ectoine catabolisms. We show that a conserved enzyme bimodule consisting of the EutD ectoine/5-hydroxyectoine hydrolase and the EutE deacetylase degrades both ectoines. We determined the high-resolution crystal structures of both enzymes, derived from the salt-tolerant bacteria Ruegeria pomeroyi and Halomonas elongata These structures, either in their apo-forms or in forms capturing substrates or intermediates, provided detailed insights into the catalytic cores of the EutD and EutE enzymes. The combined biochemical and structural results indicate that the EutD homodimer opens the pyrimidine ring of ectoine through an unusual covalent intermediate, N-α-2 acetyl-l-2,4-diaminobutyrate (α-ADABA). We found that α-ADABA is then deacetylated by the zinc-dependent EutE monomer into diaminobutyric acid (DABA), which is further catabolized to l-aspartate. We observed that the EutD-EutE bimodule synthesizes exclusively the α-, but not the γ-isomers of ADABA or hydroxy-ADABA. Of note, α-ADABA is known to induce the MocR/GabR-type repressor EnuR, which controls the expression of many ectoine catabolic genes clusters. We conclude that hydroxy-α-ADABA might serve a similar function.


Assuntos
Diamino Aminoácidos/metabolismo , Osmorregulação/fisiologia , Proteínas de Bactérias/metabolismo , Domínio Catalítico , Regulação Bacteriana da Expressão Gênica/genética , Halomonas/metabolismo , Histona Desacetilases/metabolismo , Histona Desacetilases/ultraestrutura , Hidrolases/metabolismo , Hidrolases/ultraestrutura , Chaperonas Moleculares/metabolismo , Família Multigênica , Rhodobacteraceae/metabolismo
4.
J Biol Chem ; 295(7): 2113-2124, 2020 02 14.
Artigo em Inglês | MEDLINE | ID: mdl-31914412

RESUMO

A recently discovered ornithine-ammonia cycle (OAC) serves as a conduit in the nitrogen storage and remobilization machinery in cyanobacteria. The OAC involves an arginine catabolic reaction catalyzed by the arginine dihydrolase ArgZ whose catalytic mechanism is unknown. Here we determined the crystal structures at 1.2-3.0 Å of unliganded ArgZ from the cyanobacterium Synechocystis sp. PCC6803 and of ArgZ complexed with its substrate arginine, a covalently linked reaction intermediate, or the reaction product ornithine. The structures reveal that a key residue, Asn71, in the ArgZ active center functions as the determinant distinguishing ArgZ from other members of the guanidino group-modifying enzyme superfamily. The structures, along with biochemical evidence from enzymatic assays coupled with electrospray ionization MS techniques, further suggest that ArgZ-catalyzed conversion of arginine to ornithine, ammonia, and carbon dioxide consists of two successive cycles of amine hydrolysis. Finally, we show that arginine dihydrolases are broadly distributed among bacteria and metazoans, suggesting that the OAC may be frequently used for redistribution of nitrogen from arginine catabolism or nitrogen fixation.


Assuntos
Catálise , Hidrolases/ultraestrutura , Conformação Proteica , Synechocystis/ultraestrutura , Amônia/química , Arginina/química , Dióxido de Carbono/metabolismo , Cristalografia por Raios X , Hidrolases/química , Hidrolases/genética , Nitrogênio/química , Ornitina/química , Synechocystis/enzimologia
5.
Nat Commun ; 10(1): 3868, 2019 08 27.
Artigo em Inglês | MEDLINE | ID: mdl-31455765

RESUMO

Nonribosomal peptide synthetases (NRPSs) underlie the biosynthesis of many natural products that have important medicinal utility. Protection of the NRPS peptide products from proteolysis is critical to these pathways and is often achieved by structural modification, principally the introduction of D-amino acid residues into the elongating peptide. These amino acids are generally formed in situ from their L-stereoisomers by epimerization domains or dual-function condensation/epimerization domains. In singular contrast, the thioesterase domain of nocardicin biosynthesis mediates both the effectively complete L- to D-epimerization of its C-terminal amino acid residue (≥100:1) and hydrolytic product release. We report herein high-resolution crystal structures of the nocardicin thioesterase domain in ligand-free form and reacted with a structurally precise fluorophosphonate substrate mimic that identify the complete peptide binding pocket to accommodate both stereoisomers. These structures combined with additional functional studies provide detailed mechanistic insight into this unique dual-function NRPS domain.


Assuntos
Isomerases de Aminoácido/metabolismo , Proteínas de Bactérias/metabolismo , Hidrolases/metabolismo , Lactamas/metabolismo , Peptídeo Sintases/metabolismo , Isomerases de Aminoácido/ultraestrutura , Proteínas de Bactérias/ultraestrutura , Cristalografia por Raios X , Hidrolases/ultraestrutura , Modelos Moleculares , Nocardia/enzimologia , Organofosfonatos/metabolismo , Peptídeo Sintases/ultraestrutura , Peptídeos/metabolismo , Estrutura Secundária de Proteína , Estereoisomerismo , Especificidade por Substrato
6.
Sci Rep ; 7: 44542, 2017 03 15.
Artigo em Inglês | MEDLINE | ID: mdl-28295028

RESUMO

(+)-γ-lactamase catalyzes the specific hydrolysis of (+)-γ-lactam out of the racemic γ-lactam (2-Azabicyclo[2.2.1]hept-5-en-3-one) to leave optically pure (-)-γ-lactam, which is the key building block of antiviral drugs such as carbovir and abacavir. However, no structural data has been reported on how the enzymes bind the γ-lactams and achieve their enantioselectivities. We previously identified an isochorismatase-like hydrolase (IHL, Mh33H4-5540) with (+)-γ-lactamase activity, which constitutes a novel family of γ-lactamase. Here, we first discovered that this enzyme actually hydrolyzed both (+)- and (-)-γ-lactam, but with apparently different specificities. We determined the crystal structures of the apo-form, (+)-γ-lactam bound, and (-)-γ-lactam bound forms of the enzyme. The structures showed that the binding sites of both (+) and (-)-γ-lactam resemble those of IHLs, but the "cover" loop conserved in IHLs is lacking in the enzyme, probably resulting in its incomplete enantioselectivity. Structural, biochemical, and molecular dynamics simulation studies demonstrated that the steric clash caused by the binding-site residues, especially the side-chain of Cys111 would reduce the binding affinity of (-)-γ-lactam and possibly the catalytic efficiency, which might explain the different catalytic specificities of the enantiomers of γ-lactam. Our results would facilitate the directed evolution and application of Mh33H4-5540 in antiviral drug synthesis.


Assuntos
Amidoidrolases/química , Hidrolases/química , Lactamas/química , Actinomycetales/enzimologia , Amidoidrolases/ultraestrutura , Sítios de Ligação , Hidrolases/ultraestrutura , Hidrólise , Modelos Moleculares , Simulação de Dinâmica Molecular , Estereoisomerismo , Especificidade por Substrato
7.
Biochem Biophys Res Commun ; 461(1): 122-7, 2015 May 22.
Artigo em Inglês | MEDLINE | ID: mdl-25858319

RESUMO

Proteins in the haloalkaloic acid dehalogenase (HAD) superfamily, which is one of the largest enzyme families, is generally composed of a catalytic core domain and a cap domain. Although proteins in this family show broad substrate specificities, the mechanisms of their substrate recognition are not well understood. In this study, we identified a new substrate binding motif of HAD proteins from structural and functional analyses, and propose that this motif might be crucial for interacting with hydrophobic rings of substrates. The crystal structure of TON_0338, one of the 17 putative HAD proteins identified in a hyperthermophilic archaeon, Thermococcus onnurineus NA1, was determined as an apo-form at 2.0 Å resolution. In addition, we determined the crystal structure TON_0338 in complex with Mg(2+) or N-cyclohexyl-2-aminoethanesulfonic acid (CHES) at 1.7 Å resolution. Examination of the apo-form and CHES-bound structures revealed that CHES is sandwiched between Trp58 and Trp61, suggesting that this Trp sandwich might function as a substrate recognition motif. In the phosphatase assay, TON_0338 was shown to have high activity for flavin mononucleotide (FMN), and the docking analysis suggested that the flavin of FMN may interact with Trp58 and Trp61 in a way similar to that observed in the crystal structure. Moreover, the replacement of these tryptophan residues significantly reduced the phosphatase activity for FMN. Our results suggest that WxxW may function as a substrate binding motif in HAD proteins, and expand the diversity of their substrate recognition mode.


Assuntos
Hidrolases/química , Hidrolases/ultraestrutura , Modelos Químicos , Simulação de Acoplamento Molecular , Monoéster Fosfórico Hidrolases/química , Monoéster Fosfórico Hidrolases/ultraestrutura , Thermococcus/enzimologia , Sequência de Aminoácidos , Sítios de Ligação , Simulação por Computador , Ativação Enzimática , Estabilidade Enzimática , Hidrolases/isolamento & purificação , Conformação Molecular , Dados de Sequência Molecular , Monoéster Fosfórico Hidrolases/isolamento & purificação , Ligação Proteica , Especificidade da Espécie , Relação Estrutura-Atividade , Especificidade por Substrato , Thermococcus/classificação
8.
J Biomol NMR ; 61(3-4): 249-60, 2015 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-25430058

RESUMO

NMR structural studies on membrane proteins are often complicated by their large size, taking into account the contribution of the membrane mimetic. Therefore, classical resonance assignment approaches often fail. The large size of phospholipid nanodiscs, a detergent-free phospholipid bilayer mimetic, prevented their use in high-resolution solution-state NMR spectroscopy so far. We recently introduced smaller nanodiscs that are suitable for NMR structure determination. However, side-chain assignments of a membrane protein in nanodiscs still remain elusive. Here, we utilized a NOE-based approach to assign (stereo-) specifically labeled Ile, Leu, Val and Ala methyl labeled and uniformly (15)N-Phe and (15)N-Tyr labeled OmpX and calculated a refined high-resolution structure. In addition, we were able to obtain residual dipolar couplings (RDCs) of OmpX in nanodiscs using Pf1 phage medium for the induction of weak alignment. Back-calculated NOESY spectra of the obtained NMR structures were compared to experimental NOESYs in order to validate the quality of these structures. We further used NOE information between protonated lipid head groups and side-chain methyls to determine the position of OmpX in the phospholipid bilayer. These data were verified by paramagnetic relaxation enhancement (PRE) experiments obtained with Gd(3+)-modified lipids. Taken together, this study emphasizes the need for the (stereo-) specific labeling of membrane proteins in a highly deuterated background for high-resolution structure determination, particularly in large membrane mimicking systems like phospholipid nanodiscs. Structure validation by NOESY back-calculation will be helpful for the structure determination and validation of membrane proteins where NOE assignment is often difficult. The use of protein to lipid NOEs will be beneficial for the positioning of a membrane protein in the lipid bilayer without the need for preparing multiple protein samples.


Assuntos
Proteínas da Membrana Bacteriana Externa/ultraestrutura , Proteínas de Escherichia coli/ultraestrutura , Hidrolases/ultraestrutura , Bicamadas Lipídicas/química , Ressonância Magnética Nuclear Biomolecular/métodos , Fosfolipídeos/química , Aminoácidos/química , Proteínas da Membrana Bacteriana Externa/química , Bacteriófago Pf1 , Escherichia coli/metabolismo , Proteínas de Escherichia coli/química , Hidrolases/química , Metilação , Micelas , Modelos Moleculares , Isótopos de Nitrogênio , Estrutura Terciária de Proteína
9.
J Membr Biol ; 247(9-10): 883-95, 2014 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-24930025

RESUMO

Amphipathic polymers known as "amphipols" provide a highly stabilizing environment for handling membrane proteins in aqueous solutions. A8-35, an amphipol with a polyacrylate backbone and hydrophobic grafts, has been extensively characterized and widely employed for structural and functional studies of membrane proteins using biochemical and biophysical approaches. Given the sensitivity of membrane proteins to their environment, it is important to examine what effects amphipols may have on the structure and dynamics of the proteins they complex. Here we present the first molecular dynamics study of an amphipol-stabilized membrane protein, using Escherichia coli OmpX as a model. We begin by describing the structure of the complexes formed by supplementing OmpX with increasing amounts of A8-35, in order to determine how the amphipol interacts with the transmembrane and extramembrane surfaces of the protein. We then compare the dynamics of the protein in either A8-35, a detergent, or a lipid bilayer. We find that protein dynamics on all accessible length scales is restrained by A8-35, which provides a basis to understanding some of the stabilizing and functional effects of amphipols that have been experimentally observed.


Assuntos
Proteínas da Membrana Bacteriana Externa/química , Proteínas da Membrana Bacteriana Externa/ultraestrutura , Membrana Celular/química , Membrana Celular/ultraestrutura , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/ultraestrutura , Hidrolases/química , Hidrolases/ultraestrutura , Simulação de Dinâmica Molecular , Polímeros/química , Propilaminas/química , Tensoativos/química , Simulação por Computador , Interações Hidrofóbicas e Hidrofílicas , Bicamadas Lipídicas/química , Modelos Biológicos , Modelos Químicos , Conformação Proteica , Solubilidade , Soluções , Propriedades de Superfície , Água/química
10.
J Membr Biol ; 247(9-10): 965-70, 2014 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-24668145

RESUMO

Amphipathic polymers called amphipols provide a valuable alternative to detergents for keeping integral membrane proteins soluble in aqueous buffers. Here, we characterize spatial contacts of amphipol A8-35 with membrane proteins from two architectural classes: The 8-stranded ß-barrel outer membrane protein OmpX and the α-helical protein bacteriorhodopsin. OmpX is well structured in A8-35, with its barrel adopting a fold closely similar to that in dihexanoylphosphocholine micelles. The accessibility of A8-35-trapped OmpX by a water-soluble paramagnetic molecule is highly similar to that in detergent micelles and resembles the accessibility in the natural membrane. For the α-helical protein bacteriorhodopsin, previously shown to keep its fold and function in amphipols, NMR data show that the imidazole protons of a polyhistidine tag at the N-terminus of the protein are exchange protected in the presence of detergent and lipid bilayer nanodiscs, but not in amphipols, indicating the absence of an interaction in the latter case. Overall, A8-35 exhibits protein interaction properties somewhat different from detergents and lipid bilayer nanodiscs, while maintaining the structure of solubilized integral membrane proteins.


Assuntos
Proteínas da Membrana Bacteriana Externa/química , Proteínas da Membrana Bacteriana Externa/ultraestrutura , Bacteriorodopsinas/química , Bacteriorodopsinas/ultraestrutura , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/ultraestrutura , Hidrolases/química , Hidrolases/ultraestrutura , Polímeros/química , Propilaminas/química , Solventes/química , Tensoativos/química , Interações Hidrofóbicas e Hidrofílicas , Ligação Proteica , Conformação Proteica , Mapeamento de Interação de Proteínas/métodos , Estrutura Terciária de Proteína , Solubilidade
11.
Proteins ; 82(4): 695-700, 2014 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-24115105

RESUMO

The protein Smu.1393c from Streptococcus mutans is annotated as a putative α/ß hydrolase, but it has low sequence identity to the structure-known α/ß hydrolases. Here we present the crystal structure of Smu.1393c at 2.0 Å resolution. Smu.1393c has a fully open alkaline substrate pocket, whose conformation is unique among other similar hydrolase structures. Three residues, Ser101, His251, and Glu125, were identified as the active center of Smu.1393c. By screening a series of artificial hydrolase substrates, we demonstrated Smu.1393c had low carboxylesterase activity towards short-chain carboxyl esters, which provided a clue for exploring the in vivo function of Smu.1393c.


Assuntos
Hidrolases/química , Hidrolases/ultraestrutura , Streptococcus mutans/enzimologia , Proteínas de Bactérias/química , Proteínas de Bactérias/ultraestrutura , Hidrolases de Éster Carboxílico , Domínio Catalítico , Cristalização , Cristalografia por Raios X
12.
Appl Environ Microbiol ; 78(15): 5463-5, 2012 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-22636008

RESUMO

In the course of screening for virus-host systems in extreme thermal environments, we have isolated a strain of the hyperthermophilic archaeaon Acidianus hospitalis producing unusual filamentous particles with a zipper-like appearance. The particles were shown to represent a secreted form of a genuine cellular enzyme, tetrathionate hydrolase, involved in sulfur metabolism.


Assuntos
Acidianus/enzimologia , Hidrolases/química , Modelos Moleculares , Complexos Multiproteicos/química , Conformação Proteica , Dimerização , Eletroforese em Gel de Poliacrilamida , Hidrolases/isolamento & purificação , Hidrolases/ultraestrutura , Microscopia Eletrônica , Mitomicina , Complexos Multiproteicos/isolamento & purificação , Complexos Multiproteicos/ultraestrutura
13.
Proc Natl Acad Sci U S A ; 103(15): 5652-7, 2006 Apr 11.
Artigo em Inglês | MEDLINE | ID: mdl-16585518

RESUMO

The recent discovery and characterization of silicatein, a mineral-synthesizing enzyme that assembles to form the filamentous organic core of the glassy skeletal elements (spicules) of a marine sponge, has led to the development of new low-temperature synthetic routes to metastable semiconducting metal oxides. These protein filaments were shown in vitro to catalyze the hydrolysis and structurally direct the polycondensation of metal oxides at neutral pH and low temperature. Based on the confirmation of the catalytic mechanism and the essential participation of specific serine and histidine residues (presenting a nucleophilic hydroxyl and a nucleophilicity-enhancing hydrogen-bonding imidazole nitrogen) in silicatein's catalytic active site, we therefore sought to develop a synthetic mimic that provides both catalysis and the surface determinants necessary to template and structurally direct heterogeneous nucleation through condensation. Using lithographically patterned poly(dimethylsiloxane) stamps, bifunctional self-assembled monolayer surfaces containing the essential catalytic and templating elements were fabricated by using alkane thiols microcontact-printed on gold substrates. The interface between chemically distinct self-assembled monolayer domains provided the necessary juxtaposition of nucleophilic (hydroxyl) and hydrogen-bonding (imidazole) agents to catalyze the hydrolysis of a gallium oxide precursor and template the condensed product to form gallium oxohydroxide (GaOOH) and the defect spinel, gamma-gallium oxide (gamma-Ga(2)O(3)). Using this approach, the production of patterned substrates for catalytic synthesis and templating of semiconductors for device applications can be envisioned.


Assuntos
Semicondutores , Domínio Catalítico , Ligação de Hidrogênio , Hidrolases/química , Hidrolases/ultraestrutura , Metais , Microscopia Eletrônica , Modelos Moleculares , Óxidos , Dióxido de Silício , Propriedades de Superfície
14.
J Biol Chem ; 271(34): 20322-30, 1996 Aug 23.
Artigo em Inglês | MEDLINE | ID: mdl-8702766

RESUMO

L-2-Haloacid dehalogenase catalyzes the hydrolytic dehalogenation of L-2-haloalkanoic acids to yield the corresponding D-2-hydroxyalkanoic acids. The crystal structure of the homodimeric enzyme from Pseudomonas sp. YL has been determined by a multiple isomorphous replacement method and refined at 2.5 A resolution to a crystallographic R-factor of 19.5%. The subunit consists of two structurally distinct domains: the core domain and the subdomain. The core domain has an alpha/beta structure formed by a six-stranded parallel beta-sheet flanked by five alpha-helices. The subdomain inserted into the core domain has a four helix bundle structure providing the greater part of the interface for dimer formation. There is an active site cavity between the domains. An experimentally identified nucleophilic residue, Asp-10, is located on a loop following the amino-terminal beta-strand in the core domain, and other functional residues, Thr-14, Arg-41, Ser-118, Lys-151, Tyr-157, Ser-175, Asn-177, and Asp-180, detected by a site-directed mutagenesis experiment, are arranged around the nucleophile in the active site. Although the enzyme is an alpha/beta-type hydrolase, it does not belong to the alpha/beta hydrolase fold family, from the viewpoint of the topological feature and the position of the nucleophile.


Assuntos
Hidrolases/ultraestrutura , Pseudomonas/enzimologia , Sequência de Aminoácidos , Sítios de Ligação , Ácidos Carboxílicos/metabolismo , Hidrocarbonetos Halogenados/metabolismo , Ligação de Hidrogênio , Modelos Moleculares , Dados de Sequência Molecular , Conformação Proteica , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Difração de Raios X
16.
J Bacteriol ; 175(19): 6105-12, 1993 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-8407782

RESUMO

A cyanide-degrading enzyme from Bacillus pumilus C1 has been purified and characterized. This enzyme consisted of three polypeptides of 45.6, 44.6, and 41.2 kDa; the molecular mass by gel filtration was 417 kDa. Electron microscopy revealed a multimeric, rod-shaped protein approximately 9 by 50 nm. Cyanide was rapidly degraded to formate and ammonia. Enzyme activity was optimal at 37 degrees C and pH 7.8 to 8.0. Activity was enhanced by Sc3+, Cr3+, Fe3+, and Tb3+; enhancement was independent of metal ion concentration at concentrations above 5 microM. Reversible enhancement of enzymatic activity by azide was maximal at 4.5 mM azide and increased with time. No activity was recorded with the cyanide substrate analogs CNO-, SCN-, CH3CN, and N3- and the possible degradation intermediate HCONH2. Kinetic studies indicated a Km of 2.56 +/- 0.48 mM for cyanide and a Vmax of 88.03 +/- 4.67 mmol of cyanide per min/mg/liter. The Km increased approximately twofold in the presence of 10 microM Cr3+ to 5.28 +/- 0.38 mM for cyanide, and the Vmax increased to 197.11 +/- 8.51 mmol of cyanide per min/mg/liter. We propose naming this enzyme cyanide dihydratase.


Assuntos
Bacillus/enzimologia , Hidrolases/metabolismo , Cátions Bivalentes/farmacologia , Centrifugação com Gradiente de Concentração , Cromatografia DEAE-Celulose , Cromatografia em Gel , Eletroforese em Gel de Poliacrilamida , Hidrolases/isolamento & purificação , Hidrolases/ultraestrutura , Cinética , Substâncias Macromoleculares , Manganês/farmacologia , Microscopia Eletrônica , Peso Molecular , Cianeto de Potássio/metabolismo , Termodinâmica
17.
EMBO J ; 10(6): 1297-302, 1991 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-2026135

RESUMO

Haloalkane dehalogenase from Xanthobacter autotrophicus GJ10 converts 1-haloalkanes to the corresponding alcohols and halide ions with water as the sole cosubstrate and without any need for oxygen or cofactors. The three-dimensional structure has been determined by multiple isomorphous replacement techniques using three heavy atom derivatives. The structure has been refined at 2.4 A resolution to an R-factor of 17.9%. The monomeric enzyme is a spherical molecule and is composed to two domains: domain I has an alpha/beta type structure with a central eight-stranded mainly parallel beta-sheet. Domain II lies like a cap on top of domain I and consists of alpha-helices connected by loops. Except for the cap domain the structure resembles that of the dienelactone hydrolase in spite of any significant sequence homology. The putative active site is completely buried in an internal hydrophobic cavity which is located between the two domains. From the analysis of the structure it is suggested that Asp124 is the nucleophilic residue essential for the catalysis. It interacts with His289 which is hydrogen-bonded to Asp260.


Assuntos
Bactérias/enzimologia , Hidrolases/ultraestrutura , Sequência de Aminoácidos , Sítios de Ligação , Gráficos por Computador , Cristalografia , Modelos Moleculares , Dados de Sequência Molecular , Difração de Raios X
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