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1.
Curr Opin Struct Biol ; 73: 102342, 2022 04.
Artigo em Inglês | MEDLINE | ID: mdl-35240455

RESUMO

The selective insertion of oxygen into non-activated organic molecules has to date been considered of utmost importance to synthesize existing and next generation industrial chemicals or pharmaceuticals. In this respect, the minimal requirements and high activity of fungal unspecific peroxygenases (UPOs) situate them as the jewel in the crown of C-H oxyfunctionalization biocatalysts. Although their limited availability and development has hindered their incorporation into industry, the conjunction of directed evolution and computational design is approaching UPOs to practical applications. In this review, we will address the most recent advances in UPO engineering, both of the long and short UPO families, while discussing the future prospects in this fast-moving field of research.


Assuntos
Oxigenases de Função Mista , Engenharia de Proteínas , Humanos , Oxigenases de Função Mista/química
2.
ACS Omega ; 4(6): 10593-10598, 2019 Jun 30.
Artigo em Inglês | MEDLINE | ID: mdl-31460157

RESUMO

An efficient multienzyme system for the preparative synthesis of d-xylonate, a chemical with versatile industrial applications, is described. The multienzyme system is based on d-xylose oxidation catalyzed by the xylose dehydrogenase from Calulobacter crescentus and the use of catalytic amounts of NAD+. The cofactor is regenerated in situ by coupling the reduction of acetaldehyde into ethanol catalyzed by alcohol dehydrogenase from Clostridium kluyveri. Excellent conversions (>95%) were obtained in a process that allows easy product isolation by simple evaporation of the volatile buffer and byproducts.

3.
Molecules ; 24(13)2019 Jun 28.
Artigo em Inglês | MEDLINE | ID: mdl-31261738

RESUMO

We have cloned, overexpressed, purified, and characterized a 2-ketogluconate kinase (2-dehydrogluconokinase, EC 2.7.1.13) from Cupriavidus necator (Ralstonia eutropha) H16. Exploration of its substrate specificity revealed that three ketoacids (2-keto-3-deoxy-d-gluconate, 2-keto-d-gulonate, and 2-keto-3-deoxy-d-gulonate) with structures close to the natural substrate (2-keto-d-gluconate) were successfully phosphorylated at an efficiency lower than or comparable to 2-ketogluconate, as depicted by the measured kinetic constant values. Eleven aldo and keto monosaccharides of different chain lengths and stereochemistries were also assayed but not found to be substrates. 2-ketogluconate-6-phosphate was synthesized at a preparative scale and was fully characterized for the first time.


Assuntos
Cupriavidus necator/enzimologia , Proteínas Quinases/genética , Proteínas Quinases/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Clonagem Molecular , Gluconatos/metabolismo , Fosforilação , Proteínas Quinases/química , Estabilidade Proteica , Especificidade por Substrato
4.
ACS Catal ; 8(9): 8804-8809, 2018 Sep 07.
Artigo em Inglês | MEDLINE | ID: mdl-30221031

RESUMO

Asymmetric aldol addition of simple aldehydes and ketones to electrophiles is a cornerstone reaction for the synthesis of unusual sugars and chiral building blocks. We investigated d-fructose-6-phosphate aldolase from E. coli (FSA) D6X variants as catalysts for the aldol additions of ethanal and nonfunctionalized linear and cyclic aliphatic ketones as nucleophiles to nonphosphorylated hydroxyaldehydes. Thus, addition of propanone, cyclobutanone, cyclopentanone, or ethanal to 3-hydroxypropanal or (S)- or (R)-3-hydroxybutanal catalyzed by FSA D6H and D6Q variants furnished rare deoxysugars in 8-77% isolated yields with high stereoselectivity (97:3 dr and >95% ee).

5.
Biomed Res Int ; 2017: 8421418, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-29147660

RESUMO

Hypolactasia, or intestinal lactase deficiency, affects more than half of the world population. Currently, xylose quantification in urine after gaxilose oral administration for the noninvasive diagnosis of hypolactasia is performed with the hand-operated nonautomatable phloroglucinol reaction. This work demonstrates that a new enzymatic xylose quantification method, based on the activity of xylose dehydrogenase from Caulobacter crescentus, represents an excellent alternative to the manual phloroglucinol reaction. The new method is automatable and facilitates the use of the gaxilose test for hypolactasia diagnosis in the clinical practice. The analytical validation of the new technique was performed in three different autoanalyzers, using buffer or urine samples spiked with different xylose concentrations. For the comparison between the phloroglucinol and the enzymatic assays, 224 urine samples of patients to whom the gaxilose test had been prescribed were assayed by both methods. A mean bias of -16.08 mg of xylose was observed when comparing the results obtained by both techniques. After adjusting the cut-off of the enzymatic method to 19.18 mg of xylose, the Kappa coefficient was found to be 0.9531, indicating an excellent level of agreement between both analytical procedures. This new assay represents the first automatable enzymatic technique validated for xylose quantification in urine.


Assuntos
Proteínas de Bactérias/química , Desidrogenases de Carboidrato/química , Caulobacter crescentus/enzimologia , Intolerância à Lactose/urina , Xilose/urina , Feminino , Humanos , Masculino
6.
Chemistry ; 23(21): 5005-5009, 2017 Apr 11.
Artigo em Inglês | MEDLINE | ID: mdl-28266745

RESUMO

d-Fructose-6-phosphate aldolase (FSA) was probed for extended nucleophile promiscuity by using a series of fluorogenic substrates to reveal retro-aldol activity. Four nucleophiles ethanal, propanone, butanone, and cyclopentanone were subsequently confirmed to be non-natural substrates in the synthesis direction using the wild-type enzyme and its D6H variant. This exceptional widening of the nucleophile substrate scope offers a rapid entry, in good yields and high stereoselectivity, to less oxygenated alkyl ketones and aldehydes, which was hitherto impossible.


Assuntos
Aldeído Liases/metabolismo , Aldeídos/química , Frutose-Bifosfato Aldolase/metabolismo , Frutosefosfatos/química , Cetonas/química , Aldeído Liases/química , Catálise , Frutose-Bifosfato Aldolase/química , Estrutura Molecular , Estereoisomerismo
7.
J Biotechnol ; 234: 50-57, 2016 Sep 20.
Artigo em Inglês | MEDLINE | ID: mdl-27480343

RESUMO

The gene xylB from Caulobacter crescentus has been cloned and expressed in Escherichia coli providing a high yield of xylose dehydrogenase (XylB) production and excellent purity (97%). Purified recombinant XylB showed an absolute dependence on the cofactor NAD(+) and a strong preference for d-xylose against other assayed mono and disaccharides. Additionally, XylB showed strong stability when stored as freeze-dried powder at least 250days both at 4°C and room temperature. In addition, more than 80% of the initial activity of rehydrated freeze-dried enzyme remained after 150days of incubation at 4°C. Based on these characteristics, the capability of XylB in d-xylose detection and quantification was studied. The linearity of the method was maintained up to concentrations of d-xylose of 10mg/dL and the calculated limits of detection (LoD) and quantification (LoQ) of xylose in buffer were 0.568mg/dL and 1.89mg/dL respectively. Thus, enzymatic detection was found to be an excellent method for quantification of d-xylose in both buffer and urine samples. This method can easily be incorporated in a new test for the diagnosis of hypolactasia through the measurement of intestinal lactase activity.


Assuntos
Oxirredutases do Álcool/metabolismo , Caulobacter crescentus/enzimologia , Xilose/urina , Oxirredutases do Álcool/biossíntese , Oxirredutases do Álcool/genética , Oxirredutases do Álcool/isolamento & purificação , Caulobacter crescentus/genética , Ativação Enzimática , Estabilidade Enzimática , Escherichia coli/genética , Humanos , Cinética , Limite de Detecção , Espectrometria de Massas , NAD/metabolismo , Oligossacarídeos/análise , Proteínas Recombinantes/análise , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/isolamento & purificação
8.
Int J Mol Sci ; 16(11): 27835-49, 2015 Nov 24.
Artigo em Inglês | MEDLINE | ID: mdl-26610480

RESUMO

Dihydroxyacetone (DHA) kinase from Citrobacter freundii provides an easy entry for the preparation of DHA phosphate; a very important C3 building block in nature. To modify the phosphoryl donor specificity of this enzyme from ATP to inorganic polyphosphate (poly-P); a directed evolution program has been initiated. In the first cycle of evolution, the native enzyme was subjected to one round of error-prone PCR (EP-PCR) followed directly (without selection) by a round of DNA shuffling. Although the wild-type DHAK did not show activity with poly-P, after screening, sixteen mutant clones showed an activity with poly-phosphate as phosphoryl donor statistically significant. The most active mutant presented a single mutation (Glu526Lys) located in a flexible loop near of the active center. Interestingly, our theoretical studies, based on molecular dynamics simulations and hybrid Quantum Mechanics/Molecular Mechanics (QM/MM) optimizations, suggest that this mutation has an effect on the binding of the poly-P favoring a more adequate position in the active center for the reaction to take place.


Assuntos
Trifosfato de Adenosina/química , Modelos Moleculares , Fosfotransferases (Aceptor do Grupo Álcool)/química , Polifosfatos/química , Trifosfato de Adenosina/metabolismo , Sítios de Ligação , Biblioteca Gênica , Conformação Molecular , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Mutação , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Polifosfatos/metabolismo , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Relação Estrutura-Atividade , Especificidade por Substrato
9.
Appl Microbiol Biotechnol ; 99(7): 3057-68, 2015 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-25324130

RESUMO

The TM1072 gene from Thermotoga maritima codifies for a putative form of a rhamnulose-1-phosphate aldolase (Rha-1PA Tm). To investigate this enzyme further, its gene was cloned and expressed in Escherichia coli. The purified enzyme was activated by Co(2+) as a divalent metal ion cofactor, instead of Zn(2+) as its E. coli homologue, and exhibited a maximum of activity at 95 °C. Furthermore, the enzyme displayed a high stability against extreme reaction conditions, retaining 90 % of its activity in the presence of 40 % of acetonitrile and showing a half-life greater than 3 h at 115 °C. The kinetic parameters at room temperature (R/T) were also studied; the K M was calculated to be 3.6 ± 0.33 mM, while k cat/K M was found to be 0.7 × 10(3) s(-1) M(-1). Given these characteristics, Rha-1PA Tm is an attractive enzyme for use as a biocatalyst for industrial applications, offering intriguing possibilities for practical biocatalysis.


Assuntos
Aldeído Liases/genética , Aldeído Liases/metabolismo , Thermotoga maritima/enzimologia , Aldeído Liases/química , Sequência de Aminoácidos , Catálise , Clonagem Molecular , Estabilidade Enzimática , Escherichia coli/genética , Meia-Vida , Concentração de Íons de Hidrogênio , Cinética , Dados de Sequência Molecular , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Temperatura , Thermotoga maritima/genética
10.
Chemistry ; 16(13): 4018-30, 2010 Apr 06.
Artigo em Inglês | MEDLINE | ID: mdl-20198665

RESUMO

A bifunctional aldolase/kinase enzyme named DLF has been constructed by gene fusion through overlap extension. This fusion enzyme consists of monomeric fructose-1,6-bisphosphate aldolase (FBPA) from Staphylococcus carnosus and the homodimeric dihydroxyacetone kinase (DHAK) from Citrobacter freundii CECT 4626 with an intervening linker of five amino acid residues. The fusion protein was expressed soluble and retained both kinase and aldolase activities. The secondary structures of the bifunctional enzyme and the parental enzymes were analyzed by circular dichroism (CD) spectroscopy to study the effect of the covalent coupling of the two parent proteins on the structure of the fused enzyme. Because S. carnosus FBPA is a thermostable protein, the effect of the fusion on the thermal stability of the bifunctional enzyme has also been studied. The proximity of the active centers in the fused enzyme promotes a kinetic advantage as the 20-fold increment in the initial velocity of the overall aldol reaction indicates. Experimental evidence supports that this increase in the reaction rate can be explained in terms of substrate channeling.


Assuntos
Aldeído Liases/química , Fosfotransferases/química , Staphylococcus/química , Aldeído Liases/metabolismo , Sequência de Aminoácidos , Sítios de Ligação , Biocatálise , Dicroísmo Circular , Cristalografia por Raios X , Modelos Moleculares , Dados de Sequência Molecular , Fosfotransferases/metabolismo , Engenharia de Proteínas , Estereoisomerismo
11.
Chem Commun (Camb) ; (13): 1721-3, 2009 Apr 07.
Artigo em Inglês | MEDLINE | ID: mdl-19294274

RESUMO

A new bifunctional enzyme that displays both aldolase and kinase activities has been designed and successfully used in the synthesis of aldol adducts, employing DHA as initial donor, with an increase in the reaction rate of 20-fold over the parent enzymes, which can be interpreted in terms of substrate channelling.


Assuntos
Aldeído Liases/metabolismo , Fosfotransferases/metabolismo , Catálise
13.
Chem Commun (Camb) ; (14): 1634-5, 2004 Jul 21.
Artigo em Inglês | MEDLINE | ID: mdl-15263954

RESUMO

A multienzyme system composed by recombinant dihydroxyacetone kinase from Citrobacter freundii, fuculose-1-phosphate aldolase and acetate kinase, allows a practical one-pot C-C bond formation catalysed by dihydroxyacetone phosphate-dependent aldolases from dihydroxyacetone and an aldehyde.


Assuntos
Fosfato de Di-Hidroxiacetona/química , Di-Hidroxiacetona/química , Frutose-Bifosfato Aldolase/química , Complexos Multienzimáticos/metabolismo , Catálise , Escherichia coli/enzimologia , Frutose-Bifosfato Aldolase/metabolismo , Cinética , Estrutura Molecular , Fosfotransferases (Aceptor do Grupo Álcool)/genética
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