The enzyme YkvM from Bacillus subtilis was identified previously along with three other enzymes (YkvJKL) in a bioinformatics search for enzymes involved in the biosynthesis of queuosine, a 7-deazaguanine modified nucleoside found in tRNA GUN of Bacteria and Eukarya. Genetic analysis of ykvJKLM mutants in Acinetobacter confirmed that each was essential for queuosine biosynthesis, and the genes were renamed queCDEF. QueF exhibits significant homology to the type I GTP cyclohydrolases characterized by FolE. Given that GTP is the precursor to queuosine and that a cyclohydrolase-like reaction was postulated as the initial step in queuosine biosynthesis, QueF was proposed to be the putative cyclohydrolase-like enzyme responsible for this reaction. We have cloned the queF genes from B. subtilis and Escherichia coli and characterized the recombinant enzymes. Contrary to the predictions based on sequence analysis, we discovered that the enzymes, in fact, catalyze a mechanistically unrelated reaction, the NADPHdependentreductionof7-cyano-7-deazaguanineto7-aminomethyl-7-deazaguanine, a late step in the biosynthesis of queuosine. We report here in vitro and in vivo studies that demonstrate this catalytic activity, as well as preliminary biochemical and bioinformatics analysis that provide insight into the structure of this family of enzymes.tRNA ͉ modified base T he avalanche of new protein structures that have been reported over the last decade (see summary at www.rcsb. org͞pdb͞holdings.html) has made it clear that the number of scaffolds that are used to produce all of the proteins in a cell is surprisingly limited, with Ϸ80% of the proteins using one of the 400 structural folds identified to date (1, 2). Specific functions evolve by duplication, recombination, and divergence of this core repertoire (3). Analysis of the functions of the different members of a protein structural family reveal that, in general, catalytic mechanisms and chemistries are conserved in a given family whereas substrate specificity changes (4). Much rarer are the cases in which the reactions catalyzed differ among members of the family (3); the best characterized examples being the TIM barrel superfamilies (5) and the enolase superfamily (6). Understanding the molecular paths that lead to the evolution of one function from another in a given superfamily is one of the next challenges of structural biology, impacting not only our understanding of how proteins evolve, but also the task of correctly annotating the genes identified by whole-genome sequencing (7,8).We recently used comparative genomic techniques (9) to discover four previously uncharacterized bacterial genes families (queCDEF) involved in the biosynthesis of the modified nucleoside queuosine (10). Three of these families (queCDE) have homologs in Archaea and are therefore implicated in the biosynthesis of the related modified nucleoside archaeosine (Fig. 1). Both nucleosides share an unusual 7-deazaguanosine core structure but diverge in their phylogenetic distribution, location in the ...

QueF (MW = 19.4 kDa) is a recently characterized nitrile oxidoreductase which catalyzes the NADPH-dependent reduction of 7-cyano-7-deazaguanine (preQ 0 ) to 7-aminomethyl-7-deazaguanine, a late step in the biosynthesis of the modified tRNA nucleoside queuosine. Initial crystals of homododecameric Bacillus subtilis QueF diffracted poorly to 8.0 Å . A three-dimensional model based on homology with the tunnel-fold enzyme GTP cyclohydrolase I suggested catalysis at intersubunit interfaces and a potential role for substrate binding in quaternary structure stabilization. Guided by this insight, a second crystal form was grown that was strictly dependent on the presence of preQ 0 . This crystal form diffracted to 2.25 Å resolution.

Abstract-We describe the development, implementation, and evaluation of a standardized clinical pathway to facilitate safe discharge home at the earliest time after transfemoral transcatheter aortic valve replacement. Between May 2012 and October 2014, the Heart Team developed a clinical pathway suited to the unique requirements of transfemoral transcatheter aortic valve replacement in contemporary practice. The components included risk-stratified minimalist periprocedure approach, standardized postprocedure care with early mobilization and reconditioning, and criteria-driven discharge home. Our aim was to reduce variation in care, identify a subgroup of patients suitable for early discharge (≤48 hours), and decrease length of stay for all patients. We addressed barriers related to historical practices, complex multidisciplinary stakeholder engagement, and adoption of length of stay as a quality indicator. We retrospectively reviewed the experiences of 393 consecutive patients; 150 (38.2%) were discharged early. At baseline, early discharge patients had experienced less previous balloon aortic valvuloplasty, had higher left ventricular ejection fraction, better cognitive function, and were less frail than the standard discharge group (>48 hours). Early discharge was associated with the use of local anesthesia, implantation of balloon expandable device, avoidance of urinary catheter, and early removal of temporary pacemaker. Median length of stay was 1 day for early discharge and 3 days for other patients; 97.7% were discharged home. There were no differences in 30-day mortality (1.3%), disabling stroke (0.8%), or readmission (10.7%). The implementation of a transcatheter aortic valve replacement clinical pathway shifted the program's approach to combine standardized processes and individual risk stratification. The Vancouver transcatheter aortic valve replacement clinical pathway requires a rigorous assessment to determine its efficacy, safety, and reproducibility. Goals and Vision of the ProgramTranscatheter aortic valve replacement (TAVR) is a recommended treatment for inoperable and select higher surgical risk patients with severe aortic stenosis. [1][2][3] In the first decade of therapy development, careful case selection, increased periprocedural expertise, and enhanced technology have contributed to improved outcomes and patient access to TAVR. 4,5 Interest is shifting from "How we do TAVR" to "How we care for TAVR patients" to further optimize outcomes, reduce health service utilization, and contribute to the sustained success of transcatheter heart valve therapies. 6 Duration of hospital stay is an indicator of quality of care and a predictor of outcome in the elderly population. 7 There is a significant variation in reported postprocedure length of stay (LOS) after TAVR, ranging from 1 to >10 days.8-10 Similar variation exists in patients' disposition at the time of discharge; for example, the US Society of Thoracic Surgeons/American College of Cardiology Transcatheter Valve Therapy registry reported i...