Mechanism of Rate Acceleration of Radical C-C Bond Formation Reaction by a Radical SAM GTP 3',8-Cyclase.

While the number of characterized radical S-adenosyl-l-methionine (SAM) enzymes is increasing, the roles of these enzymes in radical catalysis remain largely ambiguous. In radical SAM enzymes, the slow radical initiation step kinetically masks the subsequent steps, making it impossible to study the kinetics of radical chemistry. Due to this kinetic masking, it is unknown whether the subsequent radical reactions require rate acceleration by the enzyme active site. Here, we report the first evidence that a radical SAM enzyme MoaA accelerates the radical-mediated C-C bond formation. MoaA catalyzes an unprecedented 3',8-cyclization of GTP into 3',8-cyclo-7,8-dihydro-GTP (3',8-cH2GTP) during the molybdenum cofactor (Moco) biosynthesis. Through a series of EPR and biochemical characterizations, we found that MoaA catalyzes a shunt pathway in which an on-pathway intermediate, GTP C-3' radical, abstracts H-4' atom from (4'R)-5'-deoxyadenosine (5'-dA) to transiently generate 5'-deoxyadenos-4'-yl radical (5'-dA-C4'•) that is subsequently reduced stereospecifically to yield (4'S)-5'-dA. Detailed kinetic characterization of the shunt and the main pathways provided the comprehensive view of MoaA kinetics and determined the rate of the on-pathway 3',8-cyclization step as 2.7 ± 0.7 s-1. Together with DFT calculations, this observation suggested that the 3',8-cyclization by MoaA is accelerated by 6-9 orders of magnitude. Further experimental and theoretical characterizations suggested that the rate acceleration is achieved mainly by constraining the triphosphate and guanine base positions while leaving the ribose flexible, and a transition state stabilization through H-bond and electrostatic interactions with the positively charged R17 residue. This is the first evidence for rate acceleration of radical reactions by a radical SAM enzyme and provides insights into the mechanism by which radical SAM enzymes accelerate radical chemistry.

Paradigm Shift for Radical S-Adenosyl-l-methionine Reactions: The Organometallic Intermediate Ω Is Central to Catalysis.

Radical S-adenosyl-l-methionine (SAM) enzymes comprise a vast superfamily catalyzing diverse reactions essential to all life through homolytic SAM cleavage to liberate the highly reactive 5'-deoxyadenosyl radical (5'-dAdo·). Our recent observation of a catalytically competent organometallic intermediate Ω that forms during reaction of the radical SAM (RS) enzyme pyruvate formate-lyase activating-enzyme (PFL-AE) was therefore quite surprising, and led to the question of its broad relevance in the superfamily. We now show that Ω in PFL-AE forms as an intermediate under a variety of mixing order conditions, suggesting it is central to catalysis in this enzyme. We further demonstrate that Ω forms in a suite of RS enzymes chosen to span the totality of superfamily reaction types, implicating Ω as essential in catalysis across the RS superfamily. Finally, EPR and electron nuclear double resonance spectroscopy establish that Ω involves an Fe-C5' bond between 5'-dAdo· and the [4Fe-4S] cluster. An analogous organometallic bond is found in the well-known adenosylcobalamin (coenzyme B12) cofactor used to initiate radical reactions via a 5'-dAdo· intermediate. Liberation of a reactive 5'-dAdo· intermediate via homolytic metal-carbon bond cleavage thus appears to be similar for Ω and coenzyme B12. However, coenzyme B12 is involved in enzymes catalyzing only a small number (∼12) of distinct reactions, whereas the RS superfamily has more than 100 000 distinct sequences and over 80 reaction types characterized to date. The appearance of Ω across the RS superfamily therefore dramatically enlarges the sphere of bio-organometallic chemistry in Nature.

Advise and Assist: A Basic Medical Skills Course for Partner Forces.

BACKGROUND: Training partner forces in battlefield first-responder medical skills is an important component of US military advise-and-assist operations. We designed and executed a training curriculum focused on high-yield-based medical skills to prevent death on the battlefield for non-English speaking members of the Turkish, Azerbaijani, and Albanian militaries deployed to Afghanistan. METHODS: We designed a 2-hour training curriculum focusing on four basic medical skills: (1) assessment of scene safety; (2) limb tourniquet application; (3) wound bandaging; and (4) patient transportation via litter. Our combat medics delivered standardized training using both didactic and practicum components. Instructors made beforeand- after assessments of the proficiency of each participant for each skill in accordance with the Dreyfus model of skill acquisition. We also administered before-and-after, Likertscale- based surveys for training participants to report their self-assessed comfort level with each of the four skills. RESULTS: We delivered training to 187 participants over five classes. All 28 participants in the final teaching class completed the study. Instructors categorized each participant's skill level as novice before training for all four skills. After the training curriculum, all participants achieved a skill level consistent with advanced beginner for all four skills. Participants reported significant improvements in self-reported comfort levels for all taught procedures (ρ < .001 by Wilcoxon signed-rank test for all four skills). The largest reported increase in median comfort level was for tourniquet application: median pretraining comfort level, 4 (interquartile range [IQR], 0-6.25) versus 9.5 (IQR, 9-10) posttraining. CONCLUSION: Our curriculum resulted in significant improvements in instructor-assessed proficiency and self-reported comfort level for all four basic medical skills. Although our outcome measures have important limitations, this curriculum may be useful framework for future medics and physicians designing battlefield first-responder training curricula for members of foreign militaries.

Cardiovascular care facts: a report from the national cardiovascular data registry: 2011.

OBJECTIVES: The aim of this report was to characterize the patients, participating centers, and measures of quality of care and outcomes for 5 NCDR (National Cardiovascular Data Registry) programs: 1) ACTION (Acute Coronary Treatment and Intervention Outcomes Network) Registry-GWTG (Get With The Guidelines) for acute coronary syndromes; 2) CathPCI Registry for coronary angiography and percutaneous coronary intervention; 3) CARE (Carotid Artery Revascularization and Endarterectomy) Registry for carotid revascularization; 4) ICD Registry for implantable cardioverter defibrillators; and the 5) PINNACLE (Practice INNovation And CLinical Excellence) Registry for outpatients with cardiovascular disease (CVD). BACKGROUND: CVD is a leading cause of death and disability in the United States. The quality of care for patients with CVD is suboptimal. National registry programs, such as NCDR, permit assessments of the quality of care and outcomes for broad populations of patients with CVD. METHODS: For the year 2011, we assessed for each of the 5 NCDR programs: 1) demographic and clinical characteristics of enrolled patients; 2) key characteristics of participating centers; 3) measures of processes of care; and 4) patient outcomes. For selected variables, we assessed trends over time. RESULTS: In 2011 ACTION Registry-GWTG enrolled 119,967 patients in 567 hospitals; CathPCI enrolled 632,557 patients in 1,337 hospitals; CARE enrolled 4,934 patients in 130 hospitals; ICD enrolled 139,991 patients in 1,435 hospitals; and PINNACLE enrolled 249,198 patients (1,436,328 individual encounters) in 74 practices (1,222 individual providers). Data on performance metrics and outcomes, in some cases risk-adjusted with validated NCDR models, are presented. CONCLUSIONS: The NCDR provides a unique opportunity to understand the characteristics of large populations of patients with CVD, the centers that provide their care, quality of care provided, and important patient outcomes.