Biology subjects

Uechi, G.-I.

Publications and source records attributed to Uechi, G.-I..

3 recordsLinked to original sources

bioRxiv2024

Engineered Oxalate decarboxylase boosts activity and stability for biological applications

Oxalate decarboxylase (OxDC) from Bacillus subtilis is a Mn-dependent hexameric enzyme which converts oxalate to carbon dioxide and formate. Recently, OxDC has attracted the interest of the scientific community, due to its biotechnological and medical applications for the treatment of hyperoxalurias, a group of pathologic conditions associated with excessive oxalate urinary excretion due to either increased endogenous production or increased exogenous absorption. The fact that OxDC displays optimum pH in the acidic range, represents a big limitation for most biotechnological applications involving processes occurring at neutral pH, where the activity and stability of the enzyme are remarkably reduced. Here, through bioinformatics-guided protein engineering, followed by combinatorial mutagenesis and analyses of activity and thermodynamic stability, we identified a double mutant of OxDC endowed with enhanced catalytic efficiency and stability under physiological conditions. The obtained engineered form of OxDC offers a potential tool for improved intestinal oxalate degradation in hyperoxaluria patients.

biochemistry↗

bioRxiv2021

Evolutionary repair reveals an unexpected role of the tRNA modification m1G37 in aminoacylation

The tRNA modification m1G37, which is introduced by the tRNA methyltransferase TrmD, is thought to be essential for growth in bacteria due to its role in suppressing translational frameshift errors at proline codons. However, because bacteria can tolerate high levels of mistranslation, it is unclear why loss of m1G37 is not tolerated. Here, we addressed this question by performing experimental evolution of trmD mutant strains of E. coli. Surprisingly, trmD mutant strains were viable even if the m1G37 modification was completely abolished, and showed rapid recovery of growth rate, mainly via tandem duplication or coding mutations in the proline-tRNA ligase gene proS. Growth assays and in vitro aminoacylation assays showed that G37-unmodified tRNAPro is aminoacylated less efficiently than m1G37-modified tRNAPro, and that growth of trmD mutant strains can be largely restored by single mutations in proS that restore aminoacylation of G37-unmodified tRNAPro. These results show that inefficient aminoacylation of tRNAPro is the main reason for growth defects observed in trmD mutant strains and that the ProRS enzyme may act as a gatekeeper of translational accuracy, preventing the use of error-prone unmodified tRNAPro in protein translation. Our work shows the utility of experimental evolution for uncovering the hidden functions of essential genes and has implications for the development of antibiotics targeting TrmD.

biochemistry↗

bioRxiv2021

Cryptic genetic variations of alanine:glyoxylate aminotransferase shape its fitness and dynamics

Genetic variations expand the conformational landscape of proteins and may underlie cryptic properties that promote environmental adaptability. However, they can also represent modifying factors for disease susceptibility, by changing frustrated regions that in turn affect protein overall intracellular fitness. In this dichotomy between conservation and innovation, understanding at structural level how genetic variations keep the balance to maintain protein fitness represents an unmet need. Herein, we took advantage of known genetic variations of human alanine:glyoxylate aminotransferase (AGT1), which is present as a common major allelic form (AGT-Ma) and a minor polymorphic form (AGT-Mi) expressed in 20% of Caucasian population. By crystallographic studies and molecular dynamics simulations we showed that the polymorphic amino acid substitutions shape the conformational flexibility of AGT1 so that three surface regions that are structured in AGT-Ma become disordered in AGT-Mi, thanks to plasticity effects propagated from the mutation site(s) to the whole structure. In-depth biochemical characterisation of variants from a library encompassing the three regions correlate this plasticity to a fitness window between AGT-Ma and AGT-Mi, and suggest the existence of cryptic functions related to protein-protein interactions. These results establish that naturally-occurring genetic variations tip the balance between stability and frustration to expand the potential innovability of the protein.

biochemistry↗