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In nature, lipases (EC 3.1.1.3) catalyze the hydrolysis of triglycerides to form glycerol and fatty acids. Under the appropriate conditions, the reaction is reversible, and so biotechnological applications commonly make use of their capacity for esterification as well as for hydrolysis of a wide variety of compounds. In the present paper, we report the(More)
Oxidative stress has been implicated in the etiology of neurodegenerative disorders with α-synuclein pathology. Lipid peroxidation products such as 4-oxo-2-nonenal (ONE) and 4-hydroxy-2-nonenal (HNE) can covalently modify and structurally alter proteins. Herein, we have characterized ONE- or HNE-induced α-synuclein oligomers. Our results demonstrate that(More)
Inclusions of intraneuronal alpha-synuclein (α-synuclein) can be detected in brains of patients with Parkinson's disease and dementia with Lewy bodies. The aggregation of α-synuclein is a central feature of the disease pathogenesis. Among the different α-synuclein species, large oligomers/protofibrils have particular neurotoxic properties and should(More)
The presence of Lewy bodies, mainly consisting of aggregated α-synuclein, is a pathological hallmark of Parkinson's disease (PD) and dementia with Lewy bodies (DLB). The α-synuclein inclusions are predominantly found in neurons, but also appear frequently in astrocytes. However, the pathological significance of α-synuclein inclusions in astrocytes and the(More)
Here we report that an engineered microbial cytochrome P450 BM-3 (CYP102A subfamily) efficiently catalyzes the alpha-hydroxylation of phenylacetic acid esters. This P450 BM-3 variant also produces the authentic human metabolite of buspirone, R-6-hydroxybuspirone, with 99.5% ee.
We herein report the first directed evolution of Candida antarctica lipase A (CalA), employing a combinatorial active-site saturation test (CAST). Wild-type CalA has a modest E-value of 5.1 in kinetic resolution of 4-nitrophenyl 2-methylheptanoate. Enzyme variants were expressed in Pichia pastoris by using the episomal vector pBGP1 which allowed efficient(More)
A number of model structures of the CalA suggested by comparative modeling were tested by site-directed mutagenesis. Enzyme variants were created where amino acids predicted to play key roles for the lipase activity in the different models were replaced by an inert amino acid (alanine). The results from activity measurements of the overproduced and purified(More)
Ribonucleotide reductases are a family of essential enzymes that catalyze the reduction of ribonucleotides to their corresponding deoxyribonucleotides and provide cells with precursors for DNA synthesis. The different classes of ribonucleotide reductase are distinguished based on quaternary structures and enzyme activation mechanisms, but the components(More)
Aerobic ribonucleotide reductase from Escherichia coli regulates its level of activity by binding of effectors to an allosteric site in R1, located to the proposed interaction area of the two proteins that comprise the class I enzyme. Activity is increased by ATP binding and decreased by dATP binding. To study the mechanism governing this regulation, we(More)
The active site residue Asn-437 in protein R1 of the Escherichia coli ribonucleotide reductase makes a hydrogen bond to the 2'-OH group of the substrate. To elucidate its role(s) during catalysis, Asn-437 was engineered by site-directed mutagenesis to several other side chains (Ala, Ser, Asp, Gln). All mutant proteins were incapable of enzymatic turnover(More)