The Substrate-Bound Crystal Structure of a Baeyer–Villiger Monooxygenase Exhibits a Criegee-like Conformation

The Baeyer\u2013Villiger monooxygenases (BVMOs) are a family of bacterial flavoproteins that catalyze the synthetically useful Baeyer\u2013Villiger oxidation reaction. This involves the conversion of ketones into esters or cyclic ketones into lactones by introducing an oxygen atom adjacent to the carbonyl group. The BVMOs offer exquisite regio- and enantiospecificity while acting on a wide range of substrates. They use only NADPH and oxygen as cosubstrates, and produce only NADP+ and water as byproducts, making them environmentally attractive for industrial purposes. Here, we report the first crystal structure of a BVMO, cyclohexanone monooxygenase (CHMO) from Rhodococcus sp. HI-31 in complex with its substrate, cyclohexanone, as well as NADP+ and FAD, to 2.4 \uc5 resolution. This structure shows a drastic rotation of the NADP+ cofactor in comparison to previously reported NADP+-bound structures, as the nicotinamide moiety is no longer positioned above the flavin ring. Instead, the substrate, cyclohexanone, is found at this location, in an appropriate position for the formation of the Criegee intermediate. The rotation of NADP+ permits the substrate to gain access to the reactive flavin peroxyanion intermediate while preventing it from diffusing out of the active site. The structure thus reveals the conformation of the enzyme during the key catalytic step. CHMO is proposed to undergo a series of conformational changes to gradually move the substrate from the solvent, via binding in a solvent excluded pocket that dictates the enzyme\u2019s chemospecificity, to a location above the flavin\u2013peroxide adduct where catalysis occurs.Peer reviewed: YesNRC publication: Ye

NEA COMMITTEE ON SAFETY OF NUCLEAR INSTALLATIONS PRESSURISED WATER REACTOR MAIN STEAM LINE BREAK (MSLB) BENCHMARK Volume I: Final Specifications

designed: − to achieve the highest sustainable economic growth and employment and a rising standard of living in Member countries, while maintaining financial stability, and thus to contribute to the development of the world economy; − to contribute to sound economic expansion in Member as well as non-member countries in the process of economic development; and − to contribute to the expansion of world trade on a multilateral, non-discriminatory basis in accordance with international obligations

β Cell death and dysfunction during type 1 diabetes development in at-risk individuals

Role of the funding source: Funding from the NIH was used for support of the participating clinical centers and the coordinating center. The funding source did not participate in the collection or the analysis of the data. BACKGROUND. The β cell killing that characterizes type 1 diabetes (T1D) is thought to begin years before patients present clinically with metabolic decompensation; however, this primary pathologic process of the disease has not been measured. METHODS. Here, we measured β cell death with an assay that detects β cell–derived unmethylated insulin ( INS ) DNA. Using this assay, we performed an observational study of 50 participants from 2 cohorts at risk for developing T1D from the TrialNet Pathway to Prevention study and of 4 subjects who received islet autotransplants. RESULTS. In at-risk subjects, those who progressed to T1D had average levels of unmethylated INS DNA that were elevated modestly compared with those of healthy control subjects. In at-risk individuals that progressed to T1D, the observed increases in unmethylated INS DNA were associated with decreases in insulin secretion, indicating that the changes in unmethylated INS DNA are indicative of β cell killing. Subjects at high risk for T1D had levels of unmethylated INS DNA that were higher than those of healthy controls and higher than the levels of unmethylated INS DNA in the at-risk progressor and at-risk nonprogressor groups followed for 4 years. Evaluation of insulin secretory kinetics also distinguished high-risk subjects who progressed to overt disease from those who did not. CONCLUSION. We conclude that a blood test that measures unmethylated INS DNA serves as a marker of active β cell killing as the result of T1D-associated autoimmunity. Together, the data support the concept that β cell killing occurs sporadically during the years prior to diagnosis of T1D and is more intense in the peridiagnosis period. TRIAL REGISTRATION. Clinicaltrials.gov NCT00097292. FUNDING. Funding was from the NIH, the Juvenile Diabetes Research Foundation, and the American Diabetes Association