Biology subjects

Chiang, Y.-C.

Publications and source records attributed to Chiang, Y.-C..

3 recordsLinked to original sources

bioRxiv2018

Mechanistic basis for the evolution of chalcone synthase catalytic cysteine reactivity in land plants

Flavonoids are important polyphenolic natural products, ubiquitous in land plants, that play diverse functions in plants survival in their ecological niches, including UV protection, pigmentation for attracting pollinators, symbiotic nitrogen fixation, and defense against herbivores. Chalcone synthase (CHS) catalyzes the first committed step in plant flavonoid biosynthesis and is highly conserved in all land plants. In several previously reported crystal structures of flowering plant CHSs, the catalytic cysteine is oxidized to sulfinic acid, indicating enhanced nucleophilicity in this residue associated with its increased susceptibility to oxidation. In this study, we report a set of new crystal structures of CHSs representing all five major lineages of land plants. We reveal that the structures of CHS from a lycophyte and a moss species preserve the catalytic cysteine in a reduced state, in contrast to the cysteine sulfinic acid seen in all euphyllophyte CHS structures. In vivo complementation, in vitro biochemical and mutagenesis analyses, as well as molecular dynamics simulations identify a set of residues that differ between basal-plant and euphyllophyte CHSs and modulate catalytic cysteine reactivity. We propose that the CHS active-site environment has evolved in euphyllophytes to further enhance the nucleophilicity of the catalytic cysteine since the divergence of euphyllophytes from other vascular plant lineages 400 million years ago. These changes in CHS could have contributed to the diversification of flavonoid biosynthesis in euphyllophytes, which in turn contributed to their dominance in terrestrial ecosystems.

biochemistry↗

bioRxiv2018

Structural basis for independent origins of new catalytic machineries in plant AAAD proteins

Radiation of the plant pyridoxal 5-phosphate (PLP)-dependent aromatic L-amino acid decarboxylase (AAAD) family has yielded an array of paralogous enzymes exhibiting divergent substrate preferences and catalytic mechanisms. Plant AAADs catalyze either the decarboxylation or decarboxylation-dependent oxidative deamination of aromatic L-amino acids to produce aromatic monoamines or aromatic acetaldehydes, respectively. These compounds serve as key precursors for the biosynthesis of several important classes of plant natural products, including indole alkaloids, benzylisoquinoline alkaloids, hydroxycinnamic acid amides, phenylacetaldehyde-derived floral volatiles, and tyrosol derivatives. Here, we present the crystal structures of four functionally distinct plant AAAD paralogs. Through structural and functional analyses, we identify variable structural features of the substrate-binding pocket that underlie the divergent evolution of substrate selectivity toward indole, phenyl, or hydroxyphenyl amino acids in plant AAADs. Moreover, we describe two mechanistic classes of independently arising mutations in AAAD paralogs leading to the convergent evolution of the derived aldehyde synthase activity. Applying knowledge learned from this study, we successfully engineered a shortened benzylisoquinoline alkaloid pathway to produce (S)-norcoclaurine in yeast. This work highlights the pliability of the AAAD fold that allows change of substrate selectivity and access to alternative catalytic mechanisms with only a few mutations. SignificancePlants biosynthesize their own proteinogenic aromatic L-amino acids, namely L-phenylalanine, L-tyrosine and L-tryptophan, not only for building proteins but also for the production of a plethora of aromatic-amino-acid-derived natural products. Pyridoxal 5-phosphate (PLP)-dependent aromatic L-amino acid decarboxylase (AAAD) family enzymes play important roles in channeling various aromatic L-amino acids into diverse downstream specialized metabolic pathways. Through comparative structural analysis of four functionally divergent plant AAAD proteins together with biochemical characterization and molecular dynamics simulations, we reveal the structural and mechanistic basis for the rich divergent and convergent evolutionary development within the plant AAAD family. Knowledge learned from this study aids our ability to engineer high-value aromatic-L-amino-acid-derived natural product biosynthesis in heterologous chassis organisms.

biochemistry↗

bioRxiv2018

Spatiotemporal manipulation of ciliary glutamylation reveals its roles in intraciliary trafficking and Hedgehog signaling

Tubulin post-translational modifications (PTMs) occur spatiotemporally throughout cells and are suggested to be involved in a wide range of cellular activities. However, the complexity and dynamic distribution of tubulin PTMs within cells have hindered the understanding of their physiological roles in specific subcellular compartments. Here we develop a method to rapidly deplete tubulin glutamlyation inside the primary cilia, a microtubule-based sensory organelle protruding on the cell surface, by targeting an engineered deglutamylase to the cilia in minutes. This rapid deglutamylation quickly leads to altered ciliary functions such as kinesin-2-mediated anterograde intraflagellar transport and Hedgehog signaling, along with no apparent crosstalk to other PTMs such as acetylation and detyrosination. Our study offers a feasible approach to spatiotemporally manipulate tubulin PTMs in living cells. Future expansion of the repertoire of actuators that regulate PTMs may facilitate a comprehensive understanding of how diverse tubulin PTMs encode ciliary as well as cellular functions.

cell biology↗