@article {1843, title = {Molecular basis for metabolite channeling in a ring opening enzyme of the phenylacetate degradation pathway.}, journal = {Nat Commun}, volume = {10}, year = {2019}, month = {2019 Sep 11}, pages = {4127}, abstract = {

Substrate channeling is a mechanism for the internal transfer of hydrophobic, unstable or toxic intermediates from the active site of one enzyme to another. Such transfer has previously been described to be mediated by a hydrophobic tunnel, the use of electrostatic highways or pivoting and by conformational changes. The enzyme PaaZ is used by many bacteria to degrade environmental pollutants. PaaZ is a bifunctional enzyme that catalyzes the ring opening of oxepin-CoA and converts it to 3-oxo-5,6-dehydrosuberyl-CoA. Here we report the structures of PaaZ determined by electron cryomicroscopy with and without bound ligands. The structures reveal that three domain-swapped dimers of the enzyme form a trilobed structure. A combination of small-angle X-ray scattering (SAXS), computational studies, mutagenesis and microbial growth experiments suggests that the key intermediate is transferred from one active site to the other by a mechanism of electrostatic pivoting of the CoA moiety, mediated by a set of conserved positively charged residues.

}, issn = {2041-1723}, doi = {10.1038/s41467-019-11931-1}, author = {Sathyanarayanan, Nitish and Cannone, Giuseppe and Gakhar, Lokesh and Katagihallimath, Nainesh and Sowdhamini, Ramanathan and Ramaswamy, Subramanian and Vinothkumar, Kutti R} } @article {1192, title = {Automation aided optimization of cloning, expression and purification of enzymes of the bacterial sialic acid catabolic and sialylation pathways enzymes for structural studies.}, journal = {Microb Biotechnol}, volume = {11}, year = {2018}, month = {2018 Mar}, pages = {420-428}, abstract = {

The process of obtaining a well-expressing, soluble and correctly folded constructs can be made easier and quicker by automating the optimization of cloning, expression and purification. While there are many semiautomated pipelines available for cloning, expression and purification, there is hardly any pipeline that involves complete automation. Here, we achieve complete automation of all the steps involved in cloning and in\ vivo expression screening. This is demonstrated using 18 genes involved in sialic acid catabolism and the surface sialylation pathway. Our main objective was to clone these genes into a His-tagged Gateway vector, followed by their small-scale expression optimization in\ vivo. The constructs that showed best soluble expression were then selected for purification studies and scaled up for crystallization studies. Our technique allowed us to quickly find conditions for producing significant quantities of soluble proteins in Escherichia coli, their large-scale purification and successful crystallization of a number of these proteins. The method can be implemented in other cases where one needs to screen a large number of constructs, clones and expression vectors for successful recombinant production of functional proteins.

}, issn = {1751-7915}, doi = {10.1111/1751-7915.13041}, author = {Bairy, Sneha and Gopalan, Lakshmi Narayanan and Setty, Thanuja Gangi and Srinivasachari, Sathya and Manjunath, Lavanyaa and Kumar, Jay Prakash and Guntupalli, Sai R and Bose, Sucharita and Nayak, Vinod and Ghosh, Swagatha and Sathyanarayanan, Nitish and Caing-Carlsson, Rhawnie and Wahlgren, Weixiao Yuan and Friemann, Rosmarie and Ramaswamy, S and Neerathilingam, Muniasamy} } @article {621, title = {Structure of a heterogeneous, glycosylated, lipid-bound, {\i}t in vivo-grown protein crystal at atomic resolution from the viviparous cockroach {\i}t Diploptera punctata}, journal = {IUCrJ}, volume = {3}, year = {2016}, month = {Jul}, pages = {282{\textendash}293}, abstract = {

Macromolecular crystals for X-ray diffraction studies are typically grown {\i}t in vitro} from pure and homogeneous samples; however, there are examples of protein crystals that have been identified {\i}t in vivo}. Recent developments in micro-crystallography techniques and the advent of X-ray free-electron lasers have allowed the determination of several protein structures from crystals grown {\i}t in cellulo}. Here, an atomic resolution (1.2{\AA}) crystal structure is reported of heterogeneous milk proteins grown inside a living organism in their functional niche. These {\i}t in vivo}-grown crystals were isolated from the midgut of an embryo within the only known viviparous cockroach, {\i}t Diploptera punctata}. The milk proteins crystallized in space group {\i}t P}1, and a structure was determined by anomalous dispersion from the native S atoms. The data revealed glycosylated proteins that adopt a lipocalin fold, bind lipids and organize to form a tightly packed crystalline lattice. A single crystal is estimated to contain more than three times the energy of an equivalent mass of dairy milk. This unique storage form of nourishment for developing embryos allows access to a constant supply of complete nutrients. Notably, the crystalline cockroach-milk proteins are highly heterogeneous with respect to amino-acid sequence, glycosylation and bound fatty-acid composition. These data present a unique example of protein heterogeneity within a single {\i}t in vivo}-grown crystal of a natural protein in its native environment at atomic resolution.

}, keywords = {glycosylation, protein heterogeneity, sulfur-SAD, viviparity in cockroach}, doi = {10.1107/S2052252516008903}, url = {http://dx.doi.org/10.1107/S2052252516008903}, author = {Banerjee, Sanchari and Coussens, Nathan P. and Gallat, Fran{\c c}ois-Xavier and Sathyanarayanan, Nitish and Srikanth, Jandhyam and Yagi, Koichiro J. and Gray, James S. S. and Tobe, Stephen S. and Stay, Barbara and Chavas, Leonard M. G. and Ramaswamy, Subramanian} }