Brent D. Feske

Using biocatalysis to integrate organic chemistry into a molecular biology laboratory course

Current cutting‐edge biomedical investigation requires that the researcher have an operational understanding of several diverse disciplines. Biocatalysis is a field of science that operates at the crossroads of organic chemistry, biochemistry, microbiology, and molecular biology, and provides an excellent model for interdisciplinary research. We have developed an inquiry‐based module that uses the mutagenesis of the yeast reductase, YDL124w, to study the bioorganic synthesis of the taxol side‐chain, a pharmacologically important molecule. Using related structures, students identify regions they think will affect enzyme stereoselective, design and generate site‐specific mutants, and then characterize the effect of these changes on enzyme activity. This laboratory activity gives our students experience, working in a scientific discipline outside of biology and exposes them to techniques and equipment they do not normally work with in a molecular biology course. These inter‐disciplinary experiences not only show the relevance of other sciences to biology, but also give our students the ability to communicate more effectively with scientists outside their discipline.

Stereochemical Insignificance Discovered in Acinetobacter baumannii Quorum Sensing

Stereochemistry is a key aspect of molecular recognition for biological systems. As such, receptors and enzymes are often highly stereospecific, only recognizing one stereoisomer of a ligand. Recently, the quorum sensing signaling molecules used by the nosocomial opportunistic pathogen, Acinetobacter baumannii, were identified, and the primary signaling molecule isolated from this species was N-(3-hydroxydodecanoyl)-l-homoserine lactone. A plethora of bacterial species have been demonstrated to utilize 3-hydroxy-acylhomoserine lactone autoinducers, and in virtually all cases, the (R)-stereoisomer was identified as the natural ligand and exhibited greater autoinducer activity than the corresponding (S)-stereoisomer. Using chemical synthesis and biochemical assays, we have uncovered a case of stereochemical insignificance in A. baumannii and provide a unique example where stereochemistry appears nonessential for acylhomoserine lactone-mediated quorum sensing signaling. Based on previously reported phylogenetic studies, we suggest that A. baumannii has evolutionarily adopted this unique, yet promiscuous quorum sensing system to ensure its survival, particularly in the presence of other proteobacteria.

Enantioselectivity and Enzyme-Substrate Docking Studies of a Ketoreductase from Sporobolomyces salmonicolor (SSCR) and Saccharomyces cerevisiae (YOL151w)

Models for two ketoreductases were created and used to predict the stereoselectivity of the enzymes. One was based on the crystal structure of Sporobolomyces salmonicolor. This model was used to predict the stereoselectivity for 46 ketone reductions using this enzyme; only 6 were incorrectly predicted. The stereochemistries of the products were compared to the experimental values found in the literature. The Prelog rules were also used to predict the stereoselectivity for this enzyme; however the Prelog rules seem to be highly substrate dependent. As a result, predicting stereoselectivity of KREDs is more complicated than is allowed for with just substrate size and geometry. This enzyme showed Prelog docking geometry for 13 substrates if the enzyme is assumed to prefer an anti-Prelog docking geometry. For SSCR the molecular modeling proved to be a better method for predicting stereoselectivity of the enzymes. The second model was a homology model for YOL151w based on the enzyme crystal structure of Sporobolomyces salmonicolor carbonyl reductase, SSCR. In this homology model, 14 compounds were docked and the predicted stereochemistry was compared to the literature values. Of these, 5 were incorrectly predicted.

Synthesis, stereochemical characterization, and antimicrobial evaluation of a potentially nonnephrotoxic 3′-C-acethydrazide puromycin analog

GRAPHICAL ABSTRACT ABSTRACT Puromycin is a peptidyl nucleoside endowed with significant antibiotic and anticancer properties, but also with an unfortunate nephrotoxic character that has hampered its use as a chemotherapeutic agent. Since hydrolysis of puromycins amide to puromycin aminonucleoside is the first metabolic step leading to nephrotoxicity, we designed a 3′-C-hydrazide analog where the nitrogen and carbon functionality around the amide carbonyl of puromycin are inverted. The title compound, synthesized in 11 steps from D-xylose, cannot be metabolized to the nephrotoxic aminonucleoside. Evaluation of the title compound on Staphylococcus epidermidis and multi-drug resistance Staphylococcus aureus did not show significant antimicrobial activity up to a 400 μM concentration.

Organic Synthesis with Amino Acid Dehydrogenases, Transaminases, Amine Oxidases, and Amine Dehydrogenases

Abstract This chapter describes various enzyme-catalyzed reactions for production of amino acids and chiral amines. Enantiomerically pure amino acids can be produced from keto acids via amino acid dehydrogenase (AADH) and transaminase (TA). Enantiomerically pure amines are available from prochiral ketones via catalysis with TA, amine oxidase (AO), and amine dehydrogenase (AmDH) enzymes.

Retraction: Stereochemical insignificance discovered in Acinetobacter baumannii quorum sensing.

The authors request the retraction of the manuscript, “Stereochemical Insignificance Discovered in Acinetobacter baumannii Quorum Sensing,” as the main conclusion reporting a case of stereochemical insignificance in A. baumannii is not supported by more recent data generated by our and another group. Subsequent to our publication, another laboratory reported the synthesis and biological evaluation of the acylhomoserine lactones (AHLs), N-((R)-3-hydroxydodecanoyl)-L-homoserine lactone (3a) and N-((S)-3-hydroxydodecanoyl)-L-homoserine lactone (3b), described in our manuscript (see Stacy, D. M.; Welsh, M. A.; Rather, P. N.; Blackwell, H. E. Attenuation of Quorum Sensing in the Pathogen Acinetobacter baumannii Using Non-native N-Acyl Homoserine Lactones. ACS Chem. Biol. 2012, 7, 1719-1728.). In our case, these AHLs were synthesized using CBS reduction methodology followed by HPLC purification to yield what we believed to be the pure diastereomers. During our biological characterization of these compounds, we found the AHLs to exhibit very similar autoinducer activities in Acinetobacter baumannii. In the report in ACS Chem. Biol., the authors prepared these same AHLs via Noyori reduction and in their hands, the 3b diastereomer exhibited a 40-fold decrease in autoinducer activity; thus, we sought to repeat our biological analyses to confirm our results. The diastereomeric AHLs prepared for our publication no longer existed in the laboratory and when we attempted to repeat the reported synthetic and purification methods, pure compounds could not be obtained, instead a mixture of 3a and 3b co-eluted as confirmed using chiral HPLC. Pure 3-hydroxy AHLs were obtained using the Noyori reduction conditions as reported by Blackwell and colleagues. To examine the effect of mixtures of 3a and 3b on autoinducer activity in Acinetobacter baumannii, we measured the EC50 values of pure AHLs prepared from the Noyori reduction methodology and varying mixtures of these diastereomers (1:1-1:9). Upon examination, little difference was found to exist between the EC50 values of pure 3a and a 1:1 mixture of 3a and 3b (0.173 mM and 0.498 mM, respectively; 2.9-fold difference). Moreover, our reported values of 0.67 mM and 0.82 mM for 3a and 3b, respectively, align with a ~1:1-1:2 mixture of the compounds. In the light of the results obtained, it is likely that the preparations employed for the work described in the PLOS ONE article included a mixture of the 3a and 3b diastereomers; the novel data do not support the conclusion that stereochemistry is not significant for the signaling activity of the two compounds. The attached figure http://www.plosone.org/attachments/pone.0037102.comment1.pdf reports the results of our further analyses showing how the stereochemistry and the mixture of the two stereoisomers affect the biological activity.