%0 Journal Article %J Nat Commun %D 2019 %T Molecular basis for metabolite channeling in a ring opening enzyme of the phenylacetate degradation pathway. %A Sathyanarayanan, Nitish %A Cannone, Giuseppe %A Gakhar, Lokesh %A Katagihallimath, Nainesh %A Sowdhamini, Ramanathan %A Ramaswamy, Subramanian %A Vinothkumar, Kutti R %X

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.

%B Nat Commun %V 10 %P 4127 %8 2019 Sep 11 %G eng %N 1 %R 10.1038/s41467-019-11931-1 %0 Journal Article %J FEBS Lett %D 2019 %T Serotonin is essential for eye regeneration in planaria Schmidtea mediterranea. %A Sarkar, Arunabha %A Mukundan, Namita %A Sowndarya, Sai %A Dubey, Vinay Kumar %A Babu, Rosana %A Lakshmanan, Vairavan %A Rangiah, Kannan %A Panicker, Mitradas M %A Palakodeti, Dasaradhi %A Subramanian, Sabarinath Peruvemba %A Ramaswamy, Subramanian %X

Planaria is an ideal system to study factors involved in regeneration and tissue homeostasis. Little is known about the role of metabolites and small molecules in stem cell maintenance and lineage specification in planarians. Using liquid chromatography and mass spectrometry (LC-MS)-based quantitative metabolomics, we determined the relative levels of metabolites in stem cells, progenitors, and differentiated cells of the planarian Schmidtea mediterranea. Tryptophan and its metabolic product serotonin are significantly enriched in stem cells and progenitor population. Serotonin biosynthesis in these cells is brought about by a non-canonical enzyme, phenylalanine hydroxylase (PAH). Knockdown of Smed-pah leads to complete disappearance of eyes in regenerating planaria, while exogenous supply of serotonin and its precursor rescue the eyeless phenotype. Our results demonstrate a key role for serotonin in eye regeneration.

%B FEBS Lett %8 2019 Sep 17 %G eng %R 10.1002/1873-3468.13607 %0 Journal Article %J IUCrJ %D 2016 %T Structure of a heterogeneous, glycosylated, lipid-bound, ıt in vivo-grown protein crystal at atomic resolution from the viviparous cockroach ıt Diploptera punctata %A Banerjee, Sanchari %A Coussens, Nathan P. %A Gallat, François-Xavier %A Sathyanarayanan, Nitish %A Srikanth, Jandhyam %A Yagi, Koichiro J. %A Gray, James S. S. %A Tobe, Stephen S. %A Stay, Barbara %A Chavas, Leonard M. G. %A Ramaswamy, Subramanian %K glycosylation %K protein heterogeneity %K sulfur-SAD %K viviparity in cockroach %X

Macromolecular crystals for X-ray diffraction studies are typically grown ıt in vitro} from pure and homogeneous samples; however, there are examples of protein crystals that have been identified ıt 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 ıt 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 ıt in vivo}-grown crystals were isolated from the midgut of an embryo within the only known viviparous cockroach, ıt Diploptera punctata}. The milk proteins crystallized in space group ıt 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 ıt in vivo}-grown crystal of a natural protein in its native environment at atomic resolution.

%B IUCrJ %V 3 %P 282–293 %8 Jul %G eng %U http://dx.doi.org/10.1107/S2052252516008903 %R 10.1107/S2052252516008903