RESUMO
Bacteria can be programmed to create engineered living materials (ELMs) with self-healing and evolvable functionalities. However, further development of ELMs is greatly hampered by the lack of engineerable nonpathogenic chassis and corresponding programmable endogenous biopolymers. Here, we describe a technological workflow for facilitating ELMs design by rationally integrating bioinformatics, structural biology and synthetic biology technologies. We first develop bioinformatics software, termed Bacteria Biopolymer Sniffer (BBSniffer), that allows fast mining of biopolymers and biopolymer-producing bacteria of interest. As a proof-of-principle study, using existing pathogenic pilus as input, we identify the covalently linked pili (CLP) biosynthetic gene cluster in the industrial workhorse Corynebacterium glutamicum. Genetic manipulation and structural characterization reveal the molecular mechanism of the CLP assembly, ultimately enabling a type of programmable pili for ELM design. Finally, engineering of the CLP-enabled living materials transforms cellulosic biomass into lycopene by coupling the extracellular and intracellular bioconversion ability.
Assuntos
Bactérias , Engenharia Metabólica , Fluxo de Trabalho , Licopeno , BiopolímerosRESUMO
An ongoing challenge in chemical research is to design catalysts that select the outcomes of the reactions of complex molecules. Chemists rely on organocatalysts or transition metal catalysts to control stereoselectivity, regioselectivity and periselectivity (selectivity among possible pericyclic reactions). Nature achieves these types of selectivity with a variety of enzymes such as the recently discovered pericyclases-a family of enzymes that catalyse pericyclic reactions1. Most characterized enzymatic pericyclic reactions have been cycloadditions, and it has been difficult to rationalize how the observed selectivities are achieved2-13. Here we report the discovery of two homologous groups of pericyclases that catalyse distinct reactions: one group catalyses an Alder-ene reaction that was, to our knowledge, previously unknown in biology; the second catalyses a stereoselective hetero-Diels-Alder reaction. Guided by computational studies, we have rationalized the observed differences in reactivities and designed mutant enzymes that reverse periselectivities from Alder-ene to hetero-Diels-Alder and vice versa. A combination of in vitro biochemical characterizations, computational studies, enzyme co-crystal structures, and mutational studies illustrate how high regioselectivity and periselectivity are achieved in nearly identical active sites.
Assuntos
Biocatálise , Reação de Cicloadição , Enzimas/metabolismo , Aspergillus/enzimologia , Aspergillus/genética , Produtos Biológicos/química , Produtos Biológicos/metabolismo , Domínio Catalítico , Enzimas/genética , Modelos MolecularesRESUMO
Zearalenone (ZEN) and its derivatives are estrogenic mycotoxins known to pose significant health threats to humans and animals. Especially, the derivative α-zearalanol (α-ZAL) is over 10 times more toxic than ZEN. Simultaneous degradation of ZEN and its derivatives, especially α-ZAL, using ZEN lactone hydrolases (ZHDs) is a promising solution to eliminate their potential hazards to food safety. However, most available ZHDs exhibit limited activity toward the more toxic α-ZAL compared to ZEN. Here, we identified a broad-substrate spectrum ZHD, named ZHDAY3, from Exophiala aquamarina CBS 119918, which could not only efficiently degrade ZEN but also exhibited 73% relative activity toward α-ZAL. Through rational design, we obtained the ZHDAY3(N153H) mutant, which exhibited the highest specific activity (253.3 ± 4.3 U/mg) reported so far for degrading α-ZAL. Molecular docking, structural comparative analysis, and kinetic analysis collectively suggested that the shorter distance between the side chain of the catalytic residue His242 and the lactone bond of α-ZAL and the increased binding affinity to the substrate were mainly responsible for the improved catalytic activity of ZHDAY3(N153H) mutant. This mechanism was further validated through additional molecular docking of 18 mutants and experimental verification of six mutants.IMPORTANCEThe mycotoxins zearalenone (ZEN) and its derivatives pose a significant threat to food safety. Here, we present a highly promising ZEN lactone hydrolase (ZHD), ZHDAY3, which is capable of efficiently degrading both ZEN and the more toxic derivative α-ZAL. Next, the ZHDAY3(N153H) mutant obtained by single-point mutation exhibited the highest specific activity for degrading α-ZAL reported thus far. We further elucidated the molecular mechanisms underlying the enhanced hydrolytic activity of ZHDAY3(N153H) toward α-ZAL. These findings represent the first investigation on the molecular mechanism of ZHDs against α-ZAL and are expected to provide a significant reference for further rational engineering of ZHDs, which will ultimately contribute to addressing the health risks and food safety issues posed by ZEN-like mycotoxins.
Assuntos
Micotoxinas , Zearalenona , Zeranol , Humanos , Animais , Zearalenona/química , Zearalenona/metabolismo , Zeranol/química , Zeranol/metabolismo , Lactonas , Mutação Puntual , Hidrolases/metabolismo , Simulação de Acoplamento Molecular , Cinética , Micotoxinas/metabolismoRESUMO
Bioactive natural products have evolved to inhibit specific cellular targets and have served as lead molecules for health and agricultural applications for the past century1-3. The post-genomics era has brought a renaissance in the discovery of natural products using synthetic-biology tools4-6. However, compared to traditional bioactivity-guided approaches, genome mining of natural products with specific and potent biological activities remains challenging4. Here we present the discovery and validation of a potent herbicide that targets a critical metabolic enzyme that is required for plant survival. Our approach is based on the co-clustering of a self-resistance gene in the natural-product biosynthesis gene cluster7-9, which provides insight into the potential biological activity of the encoded compound. We targeted dihydroxy-acid dehydratase in the branched-chain amino acid biosynthetic pathway in plants; the last step in this pathway is often targeted for herbicide development10. We show that the fungal sesquiterpenoid aspterric acid, which was discovered using the method described above, is a sub-micromolar inhibitor of dihydroxy-acid dehydratase that is effective as a herbicide in spray applications. The self-resistance gene astD was validated to be insensitive to aspterric acid and was deployed as a transgene in the establishment of plants that are resistant to aspterric acid. This herbicide-resistance gene combination complements the urgent ongoing efforts to overcome weed resistance11. Our discovery demonstrates the potential of using a resistance-gene-directed approach in the discovery of bioactive natural products.
Assuntos
Produtos Biológicos/metabolismo , Produtos Biológicos/farmacologia , Herbicidas/metabolismo , Herbicidas/farmacologia , Compostos Heterocíclicos com 3 Anéis/metabolismo , Compostos Heterocíclicos com 3 Anéis/farmacologia , Arabidopsis/efeitos dos fármacos , Arabidopsis/enzimologia , Arabidopsis/genética , Arabidopsis/crescimento & desenvolvimento , Produtos Biológicos/análise , Inibidores Enzimáticos/análise , Inibidores Enzimáticos/farmacologia , Resistência a Herbicidas/genética , Herbicidas/análise , Compostos Heterocíclicos com 3 Anéis/análise , Hidroliases/antagonistas & inibidores , Hidroliases/química , Hidroliases/metabolismo , Modelos Moleculares , Família Multigênica/genética , Reguladores de Crescimento de Plantas/análise , Reguladores de Crescimento de Plantas/farmacologia , Plantas Geneticamente Modificadas/genética , Transgenes/genéticaRESUMO
Cabotegravir is an integrase strand transfer inhibitor (INSTI) for HIV treatment and prevention. Cabotegravir-based long-acting pre-exposure prophylaxis (PrEP) presents an emerging paradigm for infectious disease control. In this scheme, a combination of a high efficacy and low solubility of anti-infection drugs permits the establishment of a pharmaceutical firewall in HIV-vulnerable groups over a long period. Although the structure-activity-relationship (SAR) of cabotegravir as an INSTI is known, the structural determinants of its low solubility have not been identified. In this work, we have integrated multiple experimental and computational methods, namely X-ray diffraction, solid-state NMR (SSNMR) spectroscopy, solution NMR spectroscopy, automated fragmentation (AF)-QM/MM and density functional theory (DFT) calculations, to address this question. The molecular organization of cabotegravir in crystal lattice has been determined. The combination of very-fast magic-angle-sample-spinning (VF MAS) SSNMR and solution NMR, as supported by AF-QM/MM and DFT calculations, permits the identification of structural factors that contribute to the low aqueous solubility of cabotegravir. Our study reveals the multitasking nature of pharmacophores in cabotegravir, which controls the drug solubility and, meanwhile, the biological activity. By unraveling these function-defining molecular features, our work could inspire further development of long-acting HIV PrEP drugs.
Assuntos
Infecções por HIV , Profilaxia Pré-Exposição , Piridonas , Humanos , Farmacóforo , Dicetopiperazinas , Infecções por HIV/prevenção & controleRESUMO
Stereoselective synthesis of cis-decalin structures using [4 + 2] cycloaddition is challenging. We explored the biosynthetic pathway of the fungal natural product fischerin (1) to identify a new pericyclase FinI that can catalyze such a reaction. The cocrystal structure of FinI, a predicted O-methyltransferase, with the product and SAM provides insight into cis-decalin formation in nature.
Assuntos
Produtos Biológicos , Biocatálise , Metiltransferases , CatáliseRESUMO
Azoxy compounds exhibit a wide array of biological activities and possess distinctive chemical properties. Although there has been considerable interest in the biosynthetic mechanisms of azoxy metabolites, the enzymatic basis responsible for azoxy bond formation has remained largely enigmatic. In this study, we unveil the enzyme cascade that constructs the azoxy bond in valanimycin biosynthesis. Our research demonstrates that a pair of metalloenzymes, comprising a membrane-bound hydrazine synthase and a nonheme diiron azoxy synthase, collaborate to convert an unstable pathway intermediate to an azoxy product through a hydrazine-azo-azoxy pathway. Additionally, by characterizing homologues of this enzyme pair from other azoxy metabolite pathways, we propose that this two-enzyme cascade could represent a conserved enzymatic strategy for azoxy bond formation in bacteria. These findings provide significant mechanistic insights into biological N-N bond formation and should facilitate the targeted isolation of bioactive azoxy compounds through genome mining.
Assuntos
Bactérias , HidrazinasRESUMO
The high-resolution X-ray crystal structure of the ternary complex FtmOx1 â 2OG â fumitremorgin B and the catalytic mechanism were recently reported by us (DOI 10.1002/anie.202112063). In their Correspondence, Zhang, Costello, Liu etâ al. criticize our work in several aspects. Herein, we address these questions one by one. These structural clarifications and new computational results further support the CarC-like mechanistic model.
Assuntos
Dioxigenases , Proteínas Fúngicas , Proteínas Fúngicas/química , Dioxigenases/química , CatáliseRESUMO
The biosynthesis of blasticidin S has drawn attention due to the participation of the radical S-adenosyl methionine (SAM) enzyme BlsE. The original assignment of BlsE as a radical-mediated, redox-neutral decarboxylase is unusual because this reaction appears to serve no biosynthetic purpose and would need to be reversed by a subsequent carboxylation step. Furthermore, with the exception of BlsE, all other radical SAM decarboxylases reported to date are oxidative in nature. Careful analysis of the BlsE reaction, however, demonstrates that BlsE is not a decarboxylase but instead a lyase that catalyzes the dehydration of cytosylglucuronic acid (CGA) to form cytosyl-4'-keto-3'-deoxy-d-glucuronic acid, which can rapidly decarboxylate nonenzymatically in vitro. Analysis of substrate isotopologs, fluorinated analogues, as well as computational models based on X-ray crystal structures of the BlsE·SAM (2.09 Å) and BlsE·SAM·CGA (2.62 Å) complexes suggests that BlsE catalysis likely proceeds via direct elimination of water from the CGA C4' α-hydroxyalkyl radical as opposed to 1,2-migration of the C3'-hydroxyl prior to dehydration. Biosynthetic and mechanistic implications of the revised assignment of BlsE are discussed.
Assuntos
Desidratação , S-Adenosilmetionina , Adenosilmetionina Descarboxilase , Humanos , Nucleosídeos , S-Adenosilmetionina/químicaRESUMO
Spectinomycin is a dioxane-bridged, tricyclic aminoglycoside produced by Streptomyces spectabilis ATCC 27741. While the spe biosynthetic gene cluster for spectinomycin has been reported, the chemistry underlying construction of the dioxane ring is unknown. The twitch radical SAM enzyme SpeY from the spe cluster is shown here to catalyze dehydrogenation of the C2' alcohol of (2'R,3'S)-tetrahydrospectinomycin to yield (3'S)-dihydrospectinomycin as a likely biosynthetic intermediate. This reaction is radical-mediated and initiated via H atom abstraction from C2' of the substrate by the 5'-deoxyadenosyl radical equivalent generated upon reductive cleavage of SAM. Crystallographic analysis of the ternary Michaelis complex places serine-183 adjacent to C2' of the bound substrate opposite C5' of SAM. Mutation of this residue to cysteine converts SpeY to the corresponding C2' epimerase mirroring the opposite phenomenon observed in the homologous twitch radical SAM epimerase HygY from the hygromycin B biosynthetic pathway. Phylogenetic analysis suggests a relatively recent evolutionary branching of putative twitch radical SAM epimerases bearing homologous cysteine residues to generate the SpeY clade of enzymes.
Assuntos
Racemases e Epimerases , Espectinomicina , Cisteína , Oxirredutases , Filogenia , Racemases e Epimerases/genética , S-Adenosilmetionina/metabolismoRESUMO
BACKGROUND: Tigecycline is a tetracycline derivative that constitutes one of the last-resort antibiotics used clinically to treat infections caused by both multiple drug-resistant (MDR) Gram-negative and Gram-positive bacteria. Resistance to this drug is often caused by chromosome-encoding mechanisms including over-expression of efflux pumps and ribosome protection. However, a number of variants of the flavin adenine dinucleotide (FAD)-dependent monooxygenase TetX, such as Tet(X4), emerged in recent years as conferring resistance to tigecycline in strains of Enterobacteriaceae, Acinetobacter sp., Pseudomonas sp., and Empedobacter sp. To date, mechanistic details underlying the improvement of catalytic activities of new TetX enzymes are not available. RESULTS: In this study, we found that Tet(X4) exhibited higher affinity and catalytic efficiency toward tigecycline when compared to Tet(X2), resulting in the expression of phenotypic tigecycline resistance in E. coli strains bearing the tet(X4) gene. Comparison between the structures of Tet(X4) and Tet(X4)-tigecycline complex and those of Tet(X2) showed that they shared an identical FAD-binding site and that the FAD and tigecycline adopted similar conformation in the catalytic pocket. Although the amino acid changes in Tet(X4) are not pivotal residues for FAD binding and substrate recognition, such substitutions caused the refolding of several alpha helixes and beta sheets in the secondary structure of the substrate-binding domain of Tet(X4), resulting in the formation of a larger number of loops in the structure. These changes in turn render the substrate-binding domain of Tet(X4) more flexible and efficient in capturing substrate molecules, thereby improving catalytic efficiency. CONCLUSIONS: Our works provide a better understanding of the molecular recognition of tigecycline by the TetX enzymes; these findings can help guide the rational design of the next-generation tetracycline antibiotics that can resist inactivation of the TetX variants.
Assuntos
Escherichia coli , Oxigenases de Função Mista , Antibacterianos/farmacologia , Escherichia coli/genética , Escherichia coli/metabolismo , Testes de Sensibilidade Microbiana , Oxigenases de Função Mista/genética , Oxigenases de Função Mista/metabolismo , Tigeciclina/metabolismo , Tigeciclina/farmacologiaRESUMO
The 2-oxoglutarate (2OG)-dependent non-heme enzyme FtmOx1 catalyzes the endoperoxide biosynthesis of verruculogen. Although several mechanistic studies have been carried out, the catalytic mechanism of FtmOx1 is not well determined owing to the lack of a reliable complex structure of FtmOx1 with fumitremorginâ B. Herein we provide the X-ray crystal structure of the ternary complex FtmOx1â 2OGâ fumitremorginâ B at a resolution of 1.22â Å. Our structures show that the binding of fumitremorginâ B induces significant compression of the active pocket and that Y68 is in close proximity to C26 of the substrate. Further MD simulation and QM/MM calculations support a CarC-like mechanism, in which Y68 acts as the Hâ atom donor for quenching the C26-centered substrate radical. Our results are consistent with all available experimental data and highlight the importance of accurate complex structures in the mechanistic study of enzymatic catalysis.
Assuntos
Dioxigenases , Catálise , Dioxigenases/química , Proteínas Fúngicas/química , Ácidos Cetoglutáricos/metabolismo , Teoria QuânticaRESUMO
The non-heme iron ergothioneine synthase (EgtB) is a sulfoxide synthase that catalyzes oxidative C-S bond formation in the synthesis of ergothioneine, which plays roles against oxidative stress in cells. Despite extensive experimental and computational studies of the catalytic mechanisms of EgtB, the root causes for the selective C-S bond formation remain elusive. Using quantum mechanics/molecular mechanics (QM/MM) calculations, we show herein that a coordination switch of the sulfoxide intermediate is involved in the catalysis of the non-heme iron EgtB. This coordination switch from the S to the O atom is driven by the S/π electrostatic interactions, which efficiently promotes the observed stereoselective C-S bond formation while bypassing cysteine dioxygenation. The present mechanism is in agreement with all available experimental data, including regioselectivity, stereoselectivity and KIE results. This match underscores the critical role of coordination switching in the catalysis of non-heme enzymes.
Assuntos
Ergotioneína , Catálise , Simulação de Dinâmica Molecular , Ferro , SulfóxidosRESUMO
Acetohydroxyacid synthase (AHAS) is the first enzyme in the branched-chain amino acid biosynthetic pathway and is a validated target for herbicide and fungicide development. Here we report harzianic acid (HA, 1) produced by the biocontrol fungus Trichoderma afroharzianum t-22 (Tht22) as a natural product inhibitor of AHAS. The biosynthetic pathway of HA was elucidated with heterologous reconstitution. Guided by a putative self-resistance enzyme in the genome, HA was biochemically demonstrated to be a selective inhibitor of fungal AHAS, including those from phytopathogenic fungi. In addition, HA can inhibit a common resistant variant of AHAS in which the active site proline is mutated. Structural analysis of AHAS complexed with HA revealed the molecular basis of competitive inhibition, which differs from all known commercial AHAS inhibitors. The alternative binding mode also rationalizes the selectivity of HA, as well as effectiveness toward resistant mutants. A proposed role of HA biosynthesis by Tht22 in the rhizosphere is discussed based on the data.
RESUMO
Medium-ring lactones are synthetically challenging due to unfavorable energetics involved in cyclization. We have discovered a thioesterase enzyme DcsB, from the decarestrictine C1 (1) biosynthetic pathway, that efficiently performs medium-ring lactonizations. DcsB shows broad substrate promiscuity toward linear substrates that vary in lengths and substituents, and is a potential biocatalyst for lactonization. X-ray crystal structure and computational analyses provide insights into the molecular basis of catalysis.
Assuntos
Lactonas/síntese química , Tioléster Hidrolases/química , Beauveria/enzimologia , Beauveria/genética , Biocatálise , Cristalografia por Raios X , Ciclização , Esterificação , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Lactonas/metabolismo , Simulação de Acoplamento Molecular , Família Multigênica , Ligação Proteica , Especificidade por Substrato , Tioléster Hidrolases/genética , Tioléster Hidrolases/metabolismoRESUMO
Amino acid dehydrogenases (AADHs) have shown considerable potential as biocatalysts in the asymmetric synthesis of chiral amino acids. However, compared to the widely studied α-AADHs, limited knowledge is available about ß-AADHs that enable the synthesis of ß-amino acids. Herein, we report the crystal structures of a l-erythro-3,5-diaminohexanoate dehydrogenase and its variants, the only known member of ß-AADH family. Crystal structure analysis, site-directed mutagenesis studies and quantum chemical calculations revealed the differences in the substrate binding and catalytic mechanism from α-AADHs. A number of rationally engineered variants were then obtained with improved activity (by 110-800 times) toward various aliphatic ß-amino acids without an enantioselectivity trade-off. Two ß-amino acids were prepared by using the outstanding variants with excellent enantioselectivity (>99 % ee) and high isolated yields (86-87 %). These results provide important insights into the molecular mechanism of 3,5-DAHDH, and establish a solid foundation for further design of ß-AADHs for the asymmetric synthesis of ß-amino acids.
Assuntos
Aminoácido Oxirredutases/metabolismo , Aminoácidos/biossíntese , Mycoplasma/enzimologia , Engenharia de Proteínas , Aminoácido Oxirredutases/química , Aminoácidos/química , Biocatálise , Cristalografia por Raios X , Modelos Moleculares , Estrutura MolecularRESUMO
N-alkylisonitrile, a precursor to isonitrile-containing lipopeptides, is biosynthesized by decarboxylation-assisted -N≡C group (isonitrile) formation by using N-alkylglycine as the substrate. This reaction is catalyzed by iron(II) and 2-oxoglutarate (Fe/2OG) dependent enzymes. Distinct from typical oxygenation or halogenation reactions catalyzed by this class of enzymes, installation of the isonitrile group represents a novel reaction type for Fe/2OG enzymes that involves a four-electron oxidative process. Reported here is a plausible mechanism of three Fe/2OG enzymes, Sav607, ScoE and SfaA, which catalyze isonitrile formation. The X-ray structures of iron-loaded ScoE in complex with its substrate and the intermediate, along with biochemical and biophysical data reveal that -N≡C bond formation involves two cycles of Fe/2OG enzyme catalysis. The reaction starts with an FeIV -oxo-catalyzed hydroxylation. It is likely followed by decarboxylation-assisted desaturation to complete isonitrile installation.
Assuntos
Glicina/síntese química , Ferro/química , Nitrilas/síntese química , Pró-Colágeno-Lisina 2-Oxoglutarato 5-Dioxigenase/química , Catálise , Glicina/análogos & derivados , Hidroxilação , Modelos Moleculares , Ressonância Magnética Nuclear Biomolecular , Oxirredução , Difração de Raios XRESUMO
Steroidal C7ß alcohols and their respective esters have shown significant promise as neuroprotective and anti-inflammatory agents to treat chronic neuronal damage like stroke, brain trauma, and cerebral ischemia. Since C7 is spatially far away from any functional groups that could direct C-H activation, these transformations are not readily accessible using modern synthetic organic techniques. Reported here are P450-BM3 mutants that catalyze the oxidative hydroxylation of six different steroids with pronounced C7 regioselectivities and ß stereoselectivities, as well as high activities. These challenging transformations were achieved by a focused mutagenesis strategy and application of a novel technology for protein library construction based on DNA assembly and USER (Uracil-Specific Excision Reagent) cloning. Upscaling reactions enabled the purification of the respective steroidal alcohols in moderate to excellent yields. The high-resolution X-ray structure and molecular dynamics simulations of the best mutant unveil the origin of regio- and stereoselectivity.
Assuntos
Sistema Enzimático do Citocromo P-450/química , Mutação , Esteroides/química , Sistema Enzimático do Citocromo P-450/genética , Ligação de Hidrogênio , Hidroxilação , Simulação de Dinâmica Molecular , Oxirredução , Estereoisomerismo , Especificidade por SubstratoRESUMO
Enzymatic stereodivergent synthesis to access all possible product stereoisomers bearing multiple stereocenters is relatively undeveloped, although enzymes are being increasingly used in both academic and industrial areas. When two stereocenters and thus four stereoisomeric products are involved, obtaining stereodivergent enzyme mutants for individually accessing all four stereoisomers would be ideal. Although significant success has been achieved in directed evolution of enzymes in general, stereodivergent engineering of one enzyme into four highly stereocomplementary variants for obtaining the full complement of stereoisomers bearing multiple stereocenters remains a challenge. Using Candida antarctica lipase B (CALB) as a model, we report the protein engineering of this enzyme into four highly stereocomplementary variants needed for obtaining all four stereoisomers in transesterification reactions between racemic acids and racemic alcohols in organic solvents. By generating and screening less than 25 variants of each isomer, we achieved >90% selectivity for all of the four possible stereoisomers in the model reaction. This difficult feat was accomplished by developing a strategy dubbed "focused rational iterative site-specific mutagenesis" (FRISM) at sites lining the enzyme's binding pocket. The accumulation of single mutations by iterative site-specific mutagenesis using a restricted set of rationally chosen amino acids allows the formation of ultrasmall mutant libraries requiring minimal screening for stereoselectivity. The crystal structure of all stereodivergent CALB variants, flanked by MD simulations, uncovered the source of selectivity.
Assuntos
Ésteres/química , Ésteres/metabolismo , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Lipase/genética , Lipase/metabolismo , Engenharia de Proteínas , Proteínas Fúngicas/química , Lipase/química , Simulação de Dinâmica Molecular , Mutação , Ligação Proteica , Conformação Proteica , EstereoisomerismoRESUMO
AgWH50C, an exo-ß-agarase of GH50 isolated from Agarivorans gilvus WH0801, plays a key role in the enzymatic production of neoagarobiose, which has great application prospect in the cosmetics and pharmaceutical industry. In contrast, the poor thermostability becomes the main obstructive factor of glycoside hydrolase (GH) family 50 agarases, including AgWH50C. Herein, based on the AgWH50C crystal structure, we designed several mutants by a multiple cross-linked rational design protocol used thermostability predicting softwares ETSS, PoPMuSiC, and HotMuSiC. To our surprise, the mutant K621F increased its relative activity by as much as 45% and the optimal temperature increased to 38 °C compared to that of wild-type, AgWH50C (30 °C). The thermostability of K621F also exhibited a substantial improvement. Considering that the gelling temperature of the agarose is higher than 35 °C, K621F can be used to hydrolyze agarose for neoagarobiose production.