Biology subjects

Washington, L. J.

Publications and source records attributed to Washington, L. J..

3 recordsLinked to original sources

bioRxiv2025

Small Extra-Large GTPase-like proteins influence rhizobial symbiosis in Lotus japonicus

Plants possess a unique class of heterotrimeric G subunits called extra-large GTPases (XLGs) which contribute to numerous developmental and stress responses. In addition to the canonical G domain, XLGs have an uncharacterized N-terminal domain and functions that are distinct from conventional G subunits. In this study, we identified homologs of XLG3 in Lotus japonicus responsive to rhizobial and mycorrhizal symbiosis. However, these proteins were approximately one-third the size of conventional XLGs and only aligned to the N-terminal domain, containing a putative nuclear localization signal and a cysteine-rich domain of unknown function. Multiple sequence alignment and phylogenetic analysis determined these small XLGs (SXLGs) did not share domains with other mono- or heterotrimeric G-protein classes and exhibited a pattern of duplication and neofunctionalization typical of genes involved in symbiotic signaling pathways. Transient expression of LjSXLGs in tobacco demonstrated their potential for localization to the plasma membrane, nucleus, and nucleolus. Analysis of L. japonicus sxlg2 mutants revealed transient impairment of immature nodule formation in a destructive experimental setup and inhibition of infection events in a nutrient-limited non-destructive experimental setup, with a delayed onset of established infection events and a potential impact on nodule maturation rate. Additionally, sxlg2 mutants showed a potential impairment of the root growth response in N-limited conditions. We discuss the potential utility of SXLGs in better understanding the evolution of XLGs and their possible function as transcriptional regulators, as well as the likelihood SXLGs are involved in the establishment of rhizobial and mycorrhizal symbioses through influencing membrane reorganization, such as during infection thread development.

plant biology↗

bioRxiv2023

Biosynthesis of Strained Amino Acids Through a PLP-Dependent Enzyme via Cryptic Halogenation

Amino acids (AAs) are modular and modifiable building blocks which nature uses to synthesize both macromolecules, such as proteins, and small molecule natural products, such as alkaloids and non-ribosomal peptides (NRPs). While the 20 main proteinogenic AAs display relatively limited side-chain diversity, a wide range of non-canonical amino acids (ncAAs) exist that are not used by the ribosome for protein synthesis but contain a broad array of structural features and functional groups not found in proteinogenic AAs. In this communication, we report the discovery of the biosynthetic pathway for a new ncAA, pazamine, which contains a cyclopropane ring formed in two steps. In the first step, a chlorine is added onto the C4 position of lysine by a radical halogenase PazA. The cyclopropane ring is then formed in the next step by a pyridoxal-5-phosphate-dependent enzyme, PazB, via an SN2-like attack onto C4 to eliminate chloride. Genetic studies of this pathway in the native host, Pseudomonas azotoformans, show that pazamine and its succinylated derivative, pazamide, potentially inhibit ethylene biosynthesis in growing plants based on alterations in the root phenotype of Arabidopsis thaliana seedlings. We further show that PazB can be utilized to make an alternative cyclobutane-containing AA. These discoveries may lead to advances in biocatalytic production of specialty chemicals and agricultural biotechnology.

biochemistry↗

bioRxiv2020

An iron (II) dependent oxygenase performs the last missing step of plant lysine catabolism

Due to low abundance in many staple food crops, the essential amino acid lysine must be produced industrially to meet global food supply needs. Despite intensive study, manipulation, and agricultural importance, the steps of plant lysine catabolism beyond the 2-oxoadipate (2OA) intermediate remain undescribed. Recently we described a missing step in the D-lysine catabolic pathway of the bacterium Pseudomonas putida in which 2OA is converted to D-2-hydroxyglutarate (D2HG) via hydroxyglutarate synthase (HglS), an enzyme belonging to the previously uncharacterized DUF1338 protein family. Here we solve the structure of HglS to 1.1[A] resolution in the substrate-free form and in complex with 2OA. Structural similarity to hydroxymandelate synthase suggested a successive decarboxylation and intramolecular hydroxylation mechanism forming 2HG in a Fe(II)- and O2-dependent manner, which is validated experimentally. 2OA specificity was mediated by a single arginine (R74), highly conserved across nearly all DUF1338 family proteins, including in 76% of plant enzymes. In Arabidopsis thaliana, a DUF1338 homolog is coexpressed with known lysine catabolism enzymes, and mutants show significant germination rate defects consistent with disrupted lysine catabolism. Structural and biochemical analysis of the Oryza sativa homolog FLO7 revealed identical activity to HglS despite low sequence identity. Our results suggest that nearly all DUF1338 containing enzymes likely catalyze the same biochemical reaction, exerting the same physiological function across bacteria and eukaryotes. SignificanceTo meet human demands, millions of tons of lysine are produced by bacterial fermentation annually due to its low abundance in staple crops. Here, we show the last missing step of lysine catabolism in nearly all plant endosperms is likely catalyzed by an iron-dependant DUF1338-containing enzyme homologous to the bacterial hydroxyglutarate synthase. Structural and bioninformatic analyses of DUF1338-containing enzymes showed high conservation of critical catalytic and specificity-conferring residues across multiple domains of life despite low sequence identity. These results suggest that the DUF1338 family evolved a specific physiological function within lysine catabolism across multiple domains of life.

molecular biology↗