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1.
Int J Biol Macromol ; 147: 170-176, 2020 Jan 07.
Artigo em Inglês | MEDLINE | ID: mdl-31923511

RESUMO

Bacterial UDP-N-acetyl-d-glucosamine:heparosan alpha-1, 4-N-acetyl-d-glucosaminyltransferases (KfiAs) are in high demand for the development of animal-free heparin (HP) production. Until now, EcKfiA from Escherichia coli O10:K5:H4 was the sole identified member of this family. The lack of known members has limited research into molecular structure and catalytic mechanism of the KfiA superfamily, and restricted its application in enzymatic glycan synthesis. Herein, we report the identification and characterization of Gallibacterium anatis GaKfiA, doubling the number of known members of the KfiA family. GaKfiA is a monofunctional enzyme that transfers N-acetyl-d-glucosamine (GlcNAc) residues from their nucleotide forms to the nonreducing ends of saccharide chains structurally equivalent to the backbone of HP. The catalytic efficiency of GaKfiA is lower than that of EcKfiA. However, a single mutation of GaKfiA, N56D, resulted in a drastic increase in kcat/Km compared with wild-type GaKfiA. These data once again indicate the key role of a complete DXD motif for the catalytic efficiency of glycosyltransferases. This study deepens understanding of the mechanism of KfiA, and will assist in research into animal-free HP production.

2.
Mol Biotechnol ; 61(10): 791-800, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31444737

RESUMO

Glycosaminoglycans (GAGs) and their low-molecular weight derivates have received considerable interest in terms of their potential clinical applications, and display a wide variety of pharmacological and pharmacokinetic properties. Structurally distinct GAG chains can be prepared by enzymatic depolymerization. A variety of bacterial chondroitin sulfate (CS) lyases have been identified, and have been widely used as catalysts in this process. Here, we identified a putative chondroitin AC exolyase gene, AschnAC, from an Arthrobacter sp. strain found in a CS manufacturing workshop. We expressed the enzyme, AsChnAC, recombinantly in Escherichia coli, then purified and characterized it in vitro. The enzyme indeed displayed exolytic cleavage activity toward HA and various CSs. Removing the putative N-terminal secretion signal peptide of AsChnAC improved its expression level in E. coli while maintaining chondroitin AC exolyase activity. This novel catalyst exhibited its optimal activity in the absence of added metal ions. AsChnAC has potential applications in preparation of low-molecular weight GAGs, making it an attractive catalyst for further investigation.


Assuntos
Arthrobacter/enzimologia , Condroitina Liases/genética , Condroitina Liases/metabolismo , Arthrobacter/genética , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Biocatálise , Clonagem Molecular , Escherichia coli/genética , Glicosaminoglicanos/química , Glicosaminoglicanos/metabolismo , Peso Molecular , Proteínas Recombinantes/metabolismo
3.
Microb Cell Fact ; 18(1): 118, 2019 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-31262296

RESUMO

BACKGROUND: Enzymatic glycan synthesis has leapt forward in recent years and a number of glucuronosyltransferase (EC 2.4.1.17) have been identified and prepared, which provides a guide to an efficient approach to prepare glycans containing glucuronic acid (GlcA) residues. The uridine 5'-diphosphate (UDP) activated form, UDP-GlcA, is the monosaccharide donor for these glucuronidation reactions. RESULTS: To produce UDP-GlcA in a cost-effective way, an efficient three-step cascade route was developed using whole cells expressing hyperthermophilic enzymes to afford UDP-GlcA from starch. By coupling a coenzyme regeneration system with an appropriate expression level with UDP-glucose 6-dehydrogenase in a single strain, the cells were able to meet NAD+ requirements. Without addition of exogenous NAD+, the reaction produced 1.3 g L-1 UDP-GlcA, representing 100% and 46% conversion of UDP-Glc and UTP respectively. Finally, an anion exchange chromatography purification method was developed. UDP-GlcA was successfully obtained from the cascade system. The yield of UDP-GlcA during purification was about 92.0%. CONCLUSIONS: This work built a de novo hyperthermophilic biosynthetic cascade into E. coli host cells, with the cells able to meet NAD+ cofactor requirements and act as microbial factories for UDP-GlcA synthesis, which opens a door to large-scale production of cheaper UDP-GlcA.


Assuntos
Escherichia coli/metabolismo , Engenharia Metabólica/métodos , Uridina Difosfato Ácido Glucurônico/biossíntese , Vias Biossintéticas , Escherichia coli/genética , Glucuronatos/biossíntese , Glucuronosiltransferase/metabolismo
4.
Metab Eng ; 49: 212-219, 2018 09.
Artigo em Inglês | MEDLINE | ID: mdl-30125674

RESUMO

The development of D-glucaric acid (GA) production in recombinant cells has leapt forward in recent years, and higher throughput screening and selection of better-performing recombinant cells or biocatalysts is in current demand. A biosensor system which converts GA concentration into fluorescence signal in Escherichia coli was developed in 2016, but its application has rarely been reported. Herein, an effective high-throughput screening approach independent of special-purpose devices such as microfluidic platforms was established and tentatively applied. In this one-pot two-strain system, GA producers-bacterial or yeast cells containing the GA biosynthetic pathway-were sorted with the help of another E. coli strain acting as a GA biosensor. The identification of highly active mutants of myo-inositol oxygenase through this system validates its effectiveness in sorting E. coli cells. Subsequently, accurate ranking of the GA synthesis capacity of a small library of Saccharomyces cerevisiae strains containing distinct GA synthesis pathways demonstrated that this optimized one-pot two-strain system may also be used for eukaryotic producer strains. These results will assist in research into metabolic engineering for GA production and development of biosensor applications.


Assuntos
Técnicas Biossensoriais , Escherichia coli , Glutaratos , Inositol Oxigenase , Mutação , Saccharomyces cerevisiae , Escherichia coli/genética , Escherichia coli/metabolismo , Glutaratos/análise , Glutaratos/metabolismo , Inositol Oxigenase/genética , Inositol Oxigenase/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo
5.
Appl Microbiol Biotechnol ; 102(11): 4785-4797, 2018 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-29610966

RESUMO

Avibacterium paragallinarum is a Gram-negative bacterium that causes infectious coryza in chicken. It was reported that the capsule polysaccharides extracted from Av. paragallinarum genotype A contained chondroitin. Chondroitin synthase of Av. paragallinarum (ApCS) encoded by one gene within the presumed capsule biosynthesis gene cluster exhibited considerable homology to identified bacterial chondroitin synthases. Herein, we report the identification and characterization of ApCS. This enzyme indeed displays chondroitin synthase activity involved in the biosynthesis of the capsule. ApCS is a bifunctional protein catalyzing the elongation of the chondroitin chain by alternatively transferring the glucuronic acid (GlcA) and N-acetyl-D-galactosamine (GalNAc) residues from their nucleotide forms to the non-reducing ends of the saccharide chains. GlcA with a para-nitrophenyl group (pNP) could serve as the acceptor for ApCS; this enzyme shows a stringent donor tolerance when the acceptor is as small as this monosaccharide. Then, UDP-GalNAc and GlcA-pNP were injected sequentially through the chip-immobilized chondroitin synthases, and the surface plasmon resonance data demonstrated that the up-regulated extent caused by the binding of the donor is one possibly essential factor in successful polymerization reaction. This conclusion will, therefore, enhance the understanding of the mode of action of glycosyltransferase. Surprisingly, high activity at near-zero temperature as well as weak temperature dependence of this novel bacterial chondroitin synthase indicate that ApCS was a cold-active enzyme. From all accounts, ApCS becomes the fourth known bacterial chondroitin synthase, and the potential applications in artificial chondroitin sulfate and glycosaminoglycan synthetic approaches make it an attractive glycosyltransferase for further investigation.


Assuntos
Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Gammaproteobacteria/enzimologia , Gammaproteobacteria/genética , N-Acetilgalactosaminiltransferases/genética , N-Acetilgalactosaminiltransferases/metabolismo , Especificidade por Substrato
6.
Biochim Biophys Acta Gen Subj ; 1862(3): 547-556, 2018 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-29158133

RESUMO

BACKGROUND: The final structure of heparan sulfate chains is strictly regulated in vivo, though the biosynthesis is not guided by a template process. N-deacetylase/N-sulfotransferase (NDST) is the first modification enzyme in the HS biosynthetic pathway. The N-sulfo groups introduced by NDST are reportedly involved in determination of the susceptibility to subsequent processes catalyzed by C5-epimerse and 3-O-sulfotransferases. Understanding the substrate specificities of the four human NDST isoforms has become central to uncovering the regulatory mechanism of HS biosynthesis. METHODS: Highly-purified recombinant NDST-4 (rNDST-4) and a selective library of structurally-defined oligosaccharides were employed to determine the substrate specificity of rNDST-4. RESULTS: Full-length rNDST-4 lacks obvious N-deacetylase activity, and displays only N-sulfotransferase activity. Unlike NDST-1, NDST-4 did not show directional N-sulfotransferase activity while the N-deacetylase domain was inactive. CONCLUSION AND GENERAL SIGNIFICANCE: Individual NDST-4 could not effectively assume the key role in the distribution of N-S domains and N-Ac domains in HS biosynthesis in vivo.


Assuntos
Proteínas de Membrana/metabolismo , Oligossacarídeos/metabolismo , Sulfotransferases/metabolismo , Animais , Configuração de Carboidratos , Sequência de Carboidratos , Catálise , Glicosilação , Humanos , Oligossacarídeos/síntese química , Domínios Proteicos , Isoformas de Proteínas , Processamento de Proteína Pós-Traducional , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Bibliotecas de Moléculas Pequenas , Spodoptera , Especificidade por Substrato , Ressonância de Plasmônio de Superfície , Espectrometria de Massas em Tandem
7.
Appl Microbiol Biotechnol ; 102(2): 751-761, 2018 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-29159585

RESUMO

Capsule of Escherichia coli O5:K4:H4 is formed of a chondroitin-repeat disaccharide unit of glucuronic acid (GlcA)-N-acetylgalactosamine (GalNAc). This polysaccharide, commonly referred to as K4CP, is a potentially important source of precursors for chemoenzymatic or bioengineering synthesis of chondroitin sulfate. KfoA, encoded by a gene from region 2 of the K4 capsular gene cluster, shows high homology to the UDP-glucose-4-epimerase (GalE) from E. coli. KfoA is reputed to be responsible for uridine 5'-diphosphate-N-acetylgalactosamine (UDP-GalNAc) supply for K4CP biosynthesis in vivo, but it has not been biochemically characterized. Here, we probed the substrate specificity of KfoA by a capillary electrophoresis (CE)-based method. KfoA could epimerize both acetylated and non-acetylated substrates, but its k cat/K m value for UDP-GlcNAc was approximately 1300-fold that for UDP-Glc. Recombinant KfoA showed a strong preference for acetylated substrates in vitro. The conclusion that KfoA is a higher efficiency UDP-GalNAc provider than GalE was supported by a coupled assay developed based on the donor-acceptor combination specificity of E. coli K4 chondroitin polymerase (KfoC). Furthermore, residue Ser-301, located near the UDP-GlcNAc binding pocket, plays an important role in the determination of the conversion ratio of UDP-GlcNAc to UDP-GalNAc by KfoA. Our results deepen the understanding of the mechanism of KfoA and will assist in the research into the metabolic engineering for chondroitin sulfate production.


Assuntos
Sulfatos de Condroitina/biossíntese , Proteínas de Escherichia coli/metabolismo , Escherichia coli/enzimologia , UDPglucose 4-Epimerase/metabolismo , Acetilação , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Glucose/metabolismo , Cinética , Engenharia Metabólica , Especificidade por Substrato , UDPglucose 4-Epimerase/genética
8.
J Biol Chem ; 291(9): 4399-406, 2016 Feb 26.
Artigo em Inglês | MEDLINE | ID: mdl-26742844

RESUMO

Glycosaminoglycans (GAGs) are polysaccharides that play vital functional roles in numerous biological processes, and compounds belonging to this class have been implicated in a wide variety of diseases. Chondroitin AC lyase (ChnAC) (EC 4.2.2.5) catalyzes the degradation of various GAGs, including chondroitin sulfate and hyaluronic acid, to give the corresponding disaccharides containing an Δ(4)-unsaturated uronic acid at their non-reducing terminus. ChnAC has been isolated from various bacteria and utilized as an enzymatic tool for study and evaluating the sequencing of GAGs. Despite its substrate specificity and the fact that its crystal structure has been determined to a high resolution, the direction in which ChnAC catalyzes the cleavage of oligosaccharides remain unclear. Herein, we have determined the structural cues of substrate depolymerization and the cleavage direction of ChnAC using model substrates and recombinant ChnAC protein. Several structurally defined oligosaccharides were synthesized using a chemoenzymatic approach and subsequently cleaved using ChnAC. The degradation products resulting from this process were determined by mass spectrometry. The results revealed that ChnAC cleaved the ß1,4-glycosidic linkages between glucuronic acid and glucosamine units when these bonds were located on the reducing end of the oligosaccharide. In contrast, the presence of a GlcNAc-α-1,4-GlcA unit at the reducing end of the oligosaccharide prevented ChnAC from cleaving the GalNAc-ß1,4-GlcA moiety located in the middle or at the non-reducing end of the chain. These interesting results therefore provide direct proof that ChnAC cleaves oligosaccharide substrates from their reducing end toward their non-reducing end. This conclusion will therefore enhance our collective understanding of the mode of action of ChnAC.


Assuntos
Arthrobacter/enzimologia , Proteínas de Bactérias/metabolismo , Condroitina Liases/metabolismo , Oligossacarídeos/metabolismo , Resinas de Troca de Ânions , Proteínas de Bactérias/genética , Biocatálise , Sequência de Carboidratos , Condroitina Liases/genética , Cromatografia Líquida de Alta Pressão , Hidrólise , Oligossacarídeos/química , Proteínas Recombinantes/metabolismo , Espectrometria de Massas por Ionização por Electrospray , Especificidade por Substrato
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