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1.
Proc Natl Acad Sci U S A ; 115(28): 7308-7313, 2018 07 10.
Artigo em Inglês | MEDLINE | ID: mdl-29946033

RESUMO

Recently, heme proteins have been discovered and engineered by directed evolution to catalyze chemical transformations that are biochemically unprecedented. Many of these nonnatural enzyme-catalyzed reactions are assumed to proceed through a catalytic iron porphyrin carbene (IPC) intermediate, although this intermediate has never been observed in a protein. Using crystallographic, spectroscopic, and computational methods, we have captured and studied a catalytic IPC intermediate in the active site of an enzyme derived from thermostable Rhodothermus marinus (Rma) cytochrome c High-resolution crystal structures and computational methods reveal how directed evolution created an active site for carbene transfer in an electron transfer protein and how the laboratory-evolved enzyme achieves perfect carbene transfer stereoselectivity by holding the catalytic IPC in a single orientation. We also discovered that the IPC in Rma cytochrome c has a singlet ground electronic state and that the protein environment uses geometrical constraints and noncovalent interactions to influence different IPC electronic states. This information helps us to understand the impressive reactivity and selectivity of carbene transfer enzymes and offers insights that will guide and inspire future engineering efforts.


Assuntos
Proteínas de Bactérias/química , Evolução Molecular Direcionada , Metano/análogos & derivados , Porfirinas/química , Rhodothermus/enzimologia , Transferases/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Cristalografia por Raios X , Metano/química , Metano/metabolismo , Porfirinas/genética , Porfirinas/metabolismo , Rhodothermus/genética , Transferases/genética , Transferases/metabolismo
2.
Microb Cell Fact ; 16(1): 232, 2017 Dec 23.
Artigo em Inglês | MEDLINE | ID: mdl-29274637

RESUMO

BACKGROUND: The gene encoding a thermostable cellulase of family 12 was previously isolated from a Rhodothermus marinus through functional screening. CelA is a protein of 260 aminoacyl residues with a 28-residue amino-terminal signal peptide. Mature CelA was poorly synthesized in some Escherichia coli strains and not at all in others. Here we present an alternative approach for its heterologous production as a secreted polypeptide in Streptomyces. RESULTS: CelA was successfully over-expressed as a secreted polypeptide in Streptomyces lividans TK24. To this end, CelA was fused C-terminally to the secretory signal peptide of the subtilisin inhibitor protein (Sianidis et al. in J Biotechnol. 121: 498-507, 2006) from Streptomyces venezuelae and a new cloning strategy developed. Optimal growth media and conditions that stall biomass production promote excessive CelA secretion. Under optimal growth conditions in nutrient broth medium, significant amounts of mature CelA (50-90 mg/L or 100-120 mg/g of dry cell weight) are secreted in the spent growth media after 7 days. A protocol to rapidly purify CelA to homogeneity from culture supernatants was developed and specific anti-sera raised against it. Biophysical, biochemical and immmuno-detection analyses indicate that the enzyme is intact, stable and fully functional. CelA is the most thermostable heterologous polypeptide shown to be secreted from S. lividans. CONCLUSION: This study further validates and extends the use of the S. lividans platform for production of heterologous enzymes of industrial importance and extends it to active thermostable enzymes. This study contributes to developing a platform for poly-omics analysis of protein secretion in S. lividans.


Assuntos
Proteínas de Bactérias/metabolismo , Celulase/metabolismo , Expressão Gênica , Rhodothermus/enzimologia , Streptomyces lividans/genética , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Celulase/química , Celulase/genética , Estabilidade Enzimática , Temperatura Alta , Transporte Proteico , Rhodothermus/genética , Streptomyces lividans/metabolismo
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