Biochemical and Structural Characterization of the Tautomycetin Thioesterase: Analysis of a Stereoselective Polyketide Hydrolase

No AbstractPeer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/77955/1/5726_ftp.pd

Creative art and medical student development: a qualitative study

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/136052/1/medu13140.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/136052/2/medu13140_am.pd

Through another lens: the humanities and social sciences in the making of physicians

Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/148343/1/medu13817.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/148343/2/medu13817_am.pd

Evolution of eukaryal tRNA-guanine transglycosylase: insight gained from the heterocyclic substrate recognition by the wild-type and mutant human and Escherichia coli tRNA-guanine transglycosylases

The enzyme tRNA-guanine transglycosylase (TGT) is involved in the queuosine modification of tRNAs in eukarya and eubacteria and in the archaeosine modification of tRNAs in archaea. However, the different classes of TGTs utilize different heterocyclic substrates (and tRNA in the case of archaea). Based on the X-ray structural analyses, an earlier study [Stengl et al. (2005) Mechanism and substrate specificity of tRNA-guanine transglycosylases (TGTs): tRNA-modifying enzymes from the three different kingdoms of life share a common catalytic mechanism. Chembiochem, 6, 1926–1939] has made a compelling case for the divergent evolution of the eubacterial and archaeal TGTs. The X-ray structure of the eukaryal class of TGTs is not known. We performed sequence homology and phylogenetic analyses, and carried out enzyme kinetics studies with the wild-type and mutant TGTs from Escherichia coli and human using various heterocyclic substrates that we synthesized. Observations with the Cys145Val (E. coli) and the corresponding Val161Cys (human) TGTs are consistent with the idea that the Cys145 evolved in eubacterial TGTs to recognize preQ1 but not queuine, whereas the eukaryal equivalent, Val161, evolved for increased recognition of queuine and a concomitantly decreased recognition of preQ1. Both the phylogenetic and kinetic analyses support the conclusion that all TGTs have divergently evolved to specifically recognize their cognate heterocyclic substrates

SBSPKS: structure based sequence analysis of polyketide synthases

Polyketide synthases (PKSs) catalyze biosynthesis of a diverse family of pharmaceutically important secondary metabolites. Bioinformatics analysis of sequence and structural features of PKS proteins plays a crucial role in discovery of new natural products by genome mining, as well as in design of novel secondary metabolites by biosynthetic engineering. The availability of the crystal structures of various PKS catalytic and docking domains, and mammalian fatty acid synthase module prompted us to develop SBSPKS software which consists of three major components. Model_3D_PKS can be used for modeling, visualization and analysis of 3D structure of individual PKS catalytic domains, dimeric structures for complete PKS modules and prediction of substrate specificity. Dock_Dom_Anal identifies the key interacting residue pairs in inter-subunit interfaces based on alignment of inter-polypeptide linker sequences to the docking domain structure. In case of modular PKS with multiple open reading frames (ORFs), it can predict the cognate order of substrate channeling based on combinatorial evaluation of all possible interface contacts. NRPS–PKS provides user friendly tools for identifying various catalytic domains in the sequence of a Type I PKS protein and comparing them with experimentally characterized PKS/NRPS clusters cataloged in the backend databases of SBSPKS. SBSPKS is available at http://www.nii.ac.in/sbspks.html

CYP105-diverse structures, functions and roles in an intriguing family of enzymes in 'Streptomyces'

The cytochromes P450 (CYP or P450) are a large superfamily of haem-containing enzymes found in all domains of life. They catalyse a variety of complex reactions, predominantly mixed-function oxidations, often displaying highly regio- and/or stereospecific chemistry. In streptomycetes, they are predominantly associated with secondary metabolite biosynthetic pathways or with xenobiotic catabolism. Homologues of one family, CYP105, have been found in all Streptomyces species thus far sequenced. This review looks at the diverse biological functions of CYP105s and the biosynthetic/catabolic pathways they are associated with. Examples are presented showing a range of biotransformative abilities and different contexts. As biocatalysts capable of some remarkable chemistry, CYP105s have great biotechnological potential and merit detailed study. Recent developments in biotechnological applications which utilize CYP105s are described, alongside a brief overview of the benefits and drawbacks of using P450s in commercial applications. The role of CYP105s in vivo is in many cases undefined and provides a rich source for further investigation into the functions these enzymes fulfil and the metabolic pathways they participate in, in the natural environment

Degrees

https://stars.library.ucf.edu/bookartsexhibit-14thannual/1014/thumbnail.jp

An Exploration of Undeclared Students\u27 Expectations of Experiences for Faculty Interactions and Co-Curricular Involvement

Student persistence and achievement are areas of significant concern for institutions of higher education. With national college graduation rates hovering in the 50% range, it is important for colleges and universities to understand which student characteristics and campus environments lead to greater success, as well as the expectations students have of the college experience. Research on undeclared students is vast and dates back more than 70 years, and many of the seminal studies and respected research data have led to the perception that they are at higher risk of attrition and have lower levels of academic achievement than their declared peers. Research also shows that the two most important ways to help students connect to institutions is through faculty interactions and involvement in clubs and organizations. A new and growing body of research on student expectations posits that students who have unmet expectations of the college experience are also at higher risk of attrition. This study sought to integrate those three research concepts and analyze the expectations of undeclared students to determine if undeclared students had lower expectations of the college experience than their declared peers, specifically as those expectations relate to interactions with faculty and involvement in clubs and organizations. This study also sought to determine if undeclared students had lower levels of academic achievement or persistence than their declared peers. Using the College Student Expectations Questionnaire (CSXQ), this research analyzed the expectations of 3,219 first time in college (FTIC) students at a large, metropolitan, public university in the South who responded to the CSXQ during summer 2008 orientation. Results indicated that although differences were discovered between undeclared and declared students for expectations of student-faculty interaction and for expectations of involvement in clubs and organizations, the low effect sizes indicated that the differences could not be attributed directly to declaration of major. Results also indicated that undeclared students did not persist at rates statistically significantly different than their declared peers, nor did they achieve lower GPAs or fewer credit hours. While this study did not reveal statistically significant differences for any of the dependent variables, this research is beneficial in that these results contribute to the research findings that undeclared students are not attrition prone or less likely to achieve. More current research is needed on the population of undeclared students to determine if the perceptions are outdated and no longer generalizable to today\u27s generation of students. Additionally, more research is needed on the expectations of students, in general, to determine what impact, if any, those expectations have on student interactions with the college environment and on the outcomes of persistence, achievement, and graduation

Catalysis by tRNA -guanine transglycosylase from <italic>Escherichia coli</italic>.

The enzyme tRNA-guanine transglycosylase (TGT, EC 2.4.2.29) catalyzes a post-transcriptional transglycosylation reaction involved in the incorporation of the modified base queuine (7-[4, 5-cis-dihydroxy-2-cyclopenten-1-ylaminomethyl]-7-deazaguanine) into tRNA. Recently, this enzyme has been implicated in the virulence of Shigella flexneri, suggesting that it could be a target for antibiotic therapy. The crystal structure of the TGT from Zymomonas mobilis reveals a pair of aspartate residues (89 & 264, E. coli numbering), conserved in all known TGT sequences, that are separated in the active site by ca. 6 A. Based on their orientation in the active site, it has been hypothesized that these residues serve important roles in TGT catalysis. Aspartate 89 has been proposed to serve as an enzymic nucleophile that facilitates guanine displacement; however no solid data have been presented in support of this hypothesis. It is feasible that, in a dissociative mechanism, aspartate 89 might serve to stabilize an oxocarbenium ion transition state. Furthermore, we have proposed that aspartate 264 is involved in protonating the displaced guanine and deprotonating the incoming heterocycle. Site-directed mutagenesis was employed to probe the roles of each of these aspartates. The results of our biochemical and kinetic characterization of the resultant TGT mutants indicate that this pair of aspartates is critical for TGT catalysis, as only the conservative glutamate mutations at either position retained TGT activity. Contrary to our original hypothesis but consistent with our results, the recent TGT-RNA crystal structure indicates that aspartate 264 is the nucleophile responsible for guanine displacement and the covalent intermediate formation. Our results also suggest that aspartate 89 may be mediating critical proton transfer steps; however, this role is less definitive. To assess if the formation of the covalent intermediate is kinetically competent (i.e., rate of formation &ge;kcat), we used rapid quench techniques to measure the observed first-order rate constant of its formation. The results show that this rate constant is approximately 400-fold greater than kcat, indicating that it forms sufficiently rapidly to occur on the reaction pathway. This suggests that a step following intermediate formation (i.e., breakdown of the intermediate, product release) is rate-limiting in catalysis.Ph.D.BiochemistryPure SciencesUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/124459/2/3138201.pd

Urban fox squirrels exhibit tolerance to humans but respond to stimuli from natural predators

Animals in urban areas that experience frequent exposure to humans often behave differently than those in less urban areas, such as exhibiting less vigilance or anti‐predator behavior. These behavioral shifts may be an adaptive response to urbanization, but it may be costly if animals in urban areas also exhibit reduced anti‐predator behavior in the presence of natural predators. In trials with only a human observer as the stimulus, urban squirrels exhibited reduced vigilance and anti‐predator behavior compared to those in less urban areas. Next, we exposed squirrels in multiple urban and less urban sites to acoustic playbacks of a control stimulus (non‐predatory bird calls), a natural predator (hawk), and dogs and recorded their vigilance and three different anti‐predator behaviors when a human approached them while either broadcasting one of these three playbacks or no playbacks at all. Squirrels at urban sites also did not differ in their behavioral responses to the playbacks from possible predators (hawks or dogs) when they were compared to those at less urban sites exposed to these playbacks. Urban squirrels also exhibited increased vigilance and anti‐predator behavior when exposed to a human paired with hawk playbacks compared to the control playbacks. Together, our results indicate that urban squirrels did perceive and assess risk to the natural predator appropriately despite exhibiting increased tolerance to humans. These results provide little support for the hypothesis that increased tolerance to humans causes animals to lose their fear of natural predators.Animals in cities are often more tolerant of humans than their counterparts in rural areas. Whether or not animals in cities are also less fearful of natural predators is less certain. We show that fox squirrels in urban areas are more tolerant of humans compared to those in less urban areas but they still exhibit strong anti‐predator behavioral responses to stimuli from natural predators (hawks and dogs).Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/169334/1/eth13206.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/169334/2/eth13206_am.pd