Browsing by Author "Cao, Hongnan"

Now showing 1 - 5 of 5
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    LucY: A Versatile New Fluorescent Reporter Protein
    (Public Library of Science, 2015) Auldridge, Michele E.; Cao, Hongnan; Sen, Saurabh; Franz, Laura P.; Bingman, Craig A.; Yennamalli, Ragothaman M.; Phillips, George N.Jr.; Mead, David; Steinmetz, Eric J.
    We report on the discovery, isolation, and use of a novel yellow fluorescent protein. Lucigen Yellow (LucY) binds one FAD molecule within its core, thus shielding it from water and maintaining its structure so that fluorescence is 10-fold higher than freely soluble FAD. LucY displays excitation and emission spectra characteristic of FAD, with 3 excitation peaks at 276 nm, 377 nm, and 460 nm and a single emission peak at 530 nm. These excitation and emission maxima provide the large Stokes shift beneficial to fluorescence experimentation. LucY belongs to the MurB family of UDP-N-acetylenolpyruvylglucosamine reductases. The high resolution crystal structure shows that in contrast to other structurally resolved MurB enzymes, LucY does not contain a potentially quenching aromatic residue near the FAD isoalloxazine ring, which may explain its increased fluorescence over related proteins. Using E. coli as a system in which to develop LucY as a reporter, we show that it is amenable to circular permutation and use as a reporter of protein-protein interaction. Fragmentation between its distinct domains renders LucY non-fluorescent, but fluorescence can be partially restored by fusion of the fragments to interacting protein domains. Thus, LucY may find application in Protein-fragment Complementation Assays for evaluating protein-protein interactions.
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    Structural Basis for the Stereochemical Control of Amine Installation in Nucleotide Sugar Aminotransferases
    (American Chemical Society, 2015) Wang, Fengbin; Singh, Shanteri; Xu, Weijun; Helmich, Kate E.; Miller, Mitchell D.; Cao, Hongnan; Bingman, Craig A.; Thorson, Jon S.; Phillips, George N.Jr.
    Sugar aminotransferases (SATs) are an important class of tailoring enzymes that catalyze the 5'-pyridoxal phosphate (PLP)-dependent stereo- and regiospecific installation of an amino group from an amino acid donor (typically L-Glu or L-Gln) to a corresponding ketosugar nucleotide acceptor. Herein we report the strategic structural study of two homologous C4 SATs (Micromonospora echinospora CalS13 and Escherichia coli WecE) that utilize identical substrates but differ in their stereochemistry of aminotransfer. This study reveals for the first time a new mode of SAT sugar nucleotide binding and, in conjunction with previously reported SAT structural studies, provides the basis from which to propose a universal model for SAT stereo- and regiochemical control of amine installation. Specifically, the universal model put forth highlights catalytic divergence to derive solely from distinctions within nucleotide sugar orientation upon binding within a relatively fixed SAT active site where the available ligand bound structures of the three out of four representative C3 and C4 SAT examples provide a basis for the overall model. Importantly, this study presents a new predictive model to support SAT functional annotation, biochemical study and rational engineering.
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    Structural dynamics of a methionine γ-lyase for calicheamicin biosynthesis: Rotation of the conserved tyrosine stacking with pyridoxal phosphate
    (AIP Publishing LLC, 2016) Cao, Hongnan; Tan, Kemin; Wang, Fengbin; Bigelow, Lance; Yennamalli, Ragothaman M.; Jedrzejczak, Robert; Babnigg, Gyorgy; Bingman, Craig A.; Joachimiak, Andrzej; Kharel, Madan K.; Singh, Shanteri; Thorson, Jon S.; Phillips, George N.Jr.
    CalE6 from Micromonospora echinospora is a (pyridoxal 5′ phosphate) PLP-dependent methionine γ-lyase involved in the biosynthesis of calicheamicins. We report the crystal structure of a CalE6 2-(N-morpholino)ethanesulfonic acid complex showing ligand-induced rotation of Tyr100, which stacks with PLP, resembling the corresponding tyrosine rotation of true catalytic intermediates of CalE6 homologs. Elastic network modeling and crystallographic ensemble refinement reveal mobility of the N-terminal loop, which involves both tetrameric assembly and PLP binding. Modeling and comparative structural analysis of PLP-dependent enzymes involved in Cys/Met metabolism shine light on the functional implications of the intrinsic dynamic properties of CalE6 in catalysis and holoenzyme maturation.
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    Structure and specificity of a permissive bacterial C-prenyltransferase
    (Springer Nature, 2017) Elshahawi, Sherif I.; Cao, Hongnan; Shaaban, Khaled A.; Ponomareva, Larissa V.; Subramanian, Thangaiah; Farman, Mark L.; Spielmann, H.Peter; Phillips, George N.Jr.; Thorson, Jon S.; Singh, Shanteri
    This study highlights the biochemical and structural characterization of the L-tryptophan C6 C-prenyltransferase (C-PT) PriB from Streptomyces sp. RM-5-8. PriB was found to be uniquely permissive to a diverse array of prenyl donors and acceptors including daptomycin. Two additional PTs also produced novel prenylated daptomycins with improved antibacterial activities over the parent drug.
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    Target highlights in CASP13: Experimental target structures through the eyes of their authors
    (Wiley, 2019) Lepore, Rosalba; Kryshtafovych, Andriy; Alahuhta, Markus; Veraszto, Harshul A.; Bomble, Yannick J.; Bufton, Joshua C.; Bullock, Alex N.; Caba, Cody; Cao, Hongnan; Davies, Owen R.; Desfosses, Ambroise; Dunne, Matthew; Fidelis, Krzysztof; Goulding, Celia W.; Gurusaran, Manickam; Gutsche, Irina; Harding, Christopher J.; Hartmann, Marcus D.; Hayes, Christopher S.; Joachimiak, Andrzej; Leiman, Petr G.; Loppnau, Peter; Lovering, Andrew L.; Lunin, Vladimir V.; Michalska, Karolina; Mir‐Sanchis, Ignacio; Mitra, A.K.; Moult, John; Phillips, George N.Jr.; Pinkas, Daniel M.; Rice, Phoebe A.; Tong, Yufeng; Topf, Maya; Walton, Jonathan D.; Schwede, Torsten
    The functional and biological significance of selected CASP13 targets are described by the authors of the structures. The structural biologists discuss the most interesting structural features of the target proteins and assess whether these features were correctly reproduced in the predictions submitted to the CASP13 experiment.