RESUMO
Coenzyme B12-dependent glycerol dehydratase (GDHt) catalyzes the dehydration reaction of glycerol in the presence of adenosylcobalamin to yield 3-hydroxypropanal (3-HPA), which can be converted biologically to versatile platform chemicals such as 1,3-propanediol and 3-hydroxypropionic acid. Owing to the increased demand for biofuels, developing biological processes based on glycerol, which is a byproduct of biodiesel production, has attracted considerable attention recently. In this review, we will provide updates on the current understanding of the catalytic mechanism and structure of coenzyme B12-dependent GDHt, and then summarize the results of engineering attempts, with perspectives on future directions in its engineering.
RESUMO
Controlling the stable structures of metallic nanoparticles on mesoscopic and macroscopic length scales is of great interest in nanotechnology. Here, this task is accomplished using a synthetic biopolymer that is responsive to external stimuli and undergoes changes in secondary structure. Reversible aggregation of gold nanoparticles (GNP) is induced by pH dependent changes in a self-assembled monolayer of disulfide modified poly(L-glutamic acid) (SSPLGA) with M(w) approximately 27000. The disulfide anchoring group drives chemisorption onto the gold nanoparticles and leads to the formation of a self-assembled monolayer. Characterization of the modified GNP and its aggregation behavior is performed using dynamic light scattering (DLS), UV-vis and IR spectroscopy, and transmission electron microscopy (TEM). Experimental results show that decrease in pH near 5.5 leads to aggregation of the modified GNP. The change in aggregation behavior with pH occurs within minutes, is reversible, and happens within a narrow range of pH from about 4.5 to 5.5. Comparison with literature data on molar enthalpy of hydrogen bonding, specific optical rotation, and ionization for the helix-coil transition of PLGA indicates that the aggregation of the SSPLGA modified GNP corresponds to the transition in the secondary structure of the polyacid.
Assuntos
Ouro/química , Nanopartículas/química , Peptídeos/química , Dissulfetos/química , Concentração de Íons de Hidrogênio , Estrutura Molecular , Peso Molecular , Tamanho da Partícula , Propriedades de SuperfícieRESUMO
3-Hydroxypropionic acid (3-HP) can be produced via the biological route involving two enzymatic reactions: dehydration of glycerol to 3-hydroxypropanal (3-HPA) and then oxidation to 3-HP. However, commercial production of 3-HP using recombinant microorganisms has been hampered with several problems, some of which are associated with the toxicity of 3-HPA and the efficiency of NAD+ regeneration. We engineered α-ketoglutaric semialdehyde dehydrogenase (KGSADH) from Azospirillum brasilense for the second reaction to address these issues. The residues in the binding sites for the substrates, 3-HPA and NAD+, were randomized, and the resulting libraries were screened for higher activity. Isolated KGSADH variants had significantly lower Km values for both the substrates. The enzymes also showed higher substrate specificities for aldehyde and NAD+, less inhibition by NADH, and greater resistance to inactivation by 3-HPA than the wild-type enzyme. A recombinant Pseudomonas denitrificans strain with one of the engineered KGSADH variants exhibited less accumulation of 3-HPA, decreased levels of inactivation of the enzymes, and higher cell growth than that with the wild-type KGSADH. The flask culture of the P. denitrificans strain with the mutant KGSADH resulted in about 40% increase of 3-HP titer (53 mM) compared with that using the wild-type enzyme (37 mM).
Assuntos
Aldeído Oxirredutases/metabolismo , Azospirillum brasilense/enzimologia , Proteínas de Bactérias/metabolismo , Gliceraldeído/análogos & derivados , Ácido Láctico/análogos & derivados , NAD/metabolismo , Propano/metabolismo , Engenharia de Proteínas/métodos , Aldeído Oxirredutases/química , Gliceraldeído/metabolismo , Glicerol/metabolismo , Ácido Láctico/metabolismo , Conformação Proteica , Especificidade por SubstratoRESUMO
The development of novel anti-cancer drugs that induce apoptosis has long been a focus of drug discovery. Beta-lactam antibiotics have been used for over 60 years to fight bacterial infectious diseases with little or no side effects observed. Recently a new class of N-methylthiolated beta-lactams has been discovered that have potent activity against methicillin resistant Staphylococcus aureas. Most recently, we determined the potential effects of these N-thiolated beta-lactams on tumorigenic cell growth and found that they are apoptosis-inducers in human cancer cell lines. In the current study, we further determined the effects of the substitution of the O-methyl moiety on C3 and stereochemistry of the beta-lactams on the anti-proliferative and apoptosis-inducing abilities. We have found that lactam 18, in which C3 is substituted with an acrylate ester group, is a very effective proliferation inhibitor against human premalignant and malignant breast, leukemic, and simian virus 40-transformed fibroblast cells. Generally speaking, increasing the size of the moiety on C3 decreases its anti-proliferation potency, possibly indicating steric hindrance with the cellular target or decreased permeability through the cell membrane. We also found that the stereochemistry of the beta-lactams plays an important role in their potency. The 3S,4R isomers are more effective than their enantiomers (3R,4S), suggesting that 3S,4R configuration is more favorable for target interaction.
Assuntos
Antibacterianos/farmacologia , Apoptose/efeitos dos fármacos , beta-Lactamas/farmacologia , Antibacterianos/química , Humanos , Relação Estrutura-Atividade , Células Tumorais Cultivadas , beta-Lactamas/químicaRESUMO
This report describes the preparation of polyacrylate nanoparticles in which an N-thiolated beta-lactam antibiotic is covalently conjugated onto the polymer framework. These nanoparticles are formed in water by emulsion polymerization of an acrylated antibiotic pre-dissolved in a liquid acrylate monomer (or mixture of co-monomers) in the presence of sodium dodecyl sulfate as a surfactant and potassium persulfate as a radical initiator. Dynamic light scattering analysis and electron microscopy images of these emulsions show that the nanoparticles are approximately 40 nm in diameter. The emulsions have potent in vitro antibacterial properties against methicillin-resistant Staphylococcus aureus and have improved bioactivity relative to the non-polymerized form of the antibiotic. A unique feature of this methodology is the ability to incorporate water-insoluble drugs directly into the nanoparticle framework without the need for post-synthetic modification. Additionally, the antibiotic properties of the nanoparticles can be modulated by changing the length or location of the acrylate linker on the drug monomer.
Assuntos
Antibacterianos/síntese química , Resistência a Meticilina , Nanopartículas/química , Staphylococcus aureus/efeitos dos fármacos , beta-Lactamas/química , Resinas Acrílicas/química , Antibacterianos/química , Antibacterianos/farmacologia , Emulsões , Nanopartículas/ultraestrutura , Compostos de Potássio/química , Dodecilsulfato de Sódio/química , Sulfatos/química , Compostos de Sulfidrila/química , Água/química , beta-Lactamas/farmacologiaRESUMO
N-Thiolated beta-lactams are a new family of antibacterials that inhibit the growth of Staphylococcus bacteria. Unlike other beta-lactam drugs, these compounds retain their full antibacterial activity against methicillin-resistant Staphylococcus aureus (MRSA) strains and operate through a different mode of action. The structural features, which give these lactams their biological activity, have not yet been completely defined. Earlier efforts in our laboratory established that the N-organothio substituent is essential for antimicrobial activity while other groups at C(3) and C(4) on the lactam ring play a more subtle role. In this present study, we investigate these effects by varying the polar and steric nature of the ring substituents at these two centers. From the data presented herein, it appears that there is a need to balance the lipophilic character of the C(3)/C(4) groups to obtain an optimal anti-MRSA activity. The structure-bioactivity profiles more closely relate to the compound's ability to penetrate the bacterial cell membrane to sites of action within the cytoplasm rather than to any specific non-bonding interactions with a biological target. Based on these results, a model for the compounds' mode of action is presented.
Assuntos
Farmacorresistência Bacteriana/efeitos dos fármacos , Meticilina/farmacologia , Staphylococcus aureus/efeitos dos fármacos , Compostos de Sulfidrila/química , beta-Lactamas/química , beta-Lactamas/farmacologia , Antibacterianos/síntese química , Antibacterianos/química , Antibacterianos/farmacologia , Proliferação de Células/efeitos dos fármacos , Metilação , Estrutura Molecular , Relação Estrutura-Atividade , beta-Lactamas/síntese químicaRESUMO
In this report we describe a new family of N-thiolated beta-lactams that have antibacterial activity against methicillin-resistant Staphylococcus aureus (MRSA). The compounds show unprecedented structure-activity features and an unusual mode of action for a beta-lactam antibiotic.