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内容記述 |
L-amino acid ligase (Lal) is a highly useful enzyme for synthesis of functional peptides, including bioactive peptides, artificial sweeteners, and antibiotics. Despite its practical utility, the detailed molecular mechanism of its catalytic reaction remains unclear.RhiM and RhiC, encoded in Bacillus subtilis ATCC6633, are homologous Lals involved in antibiotic peptide biosynthesis. While both enzymes catalyze peptide bond formation via a common ATP-dependent reaction mechanism, they have differences in substrate specificity: RhiC recognizes hydrophobic amino acids, whereas RhiM specifically recognizes L-Arg as the first substrate. Despitetheir biochemical significance, the structural basis of their substrate selectivity and catalytic mechanism remains poorly understood.To investigate their structural and functional features, crystal structures of RhiC and RhiM were determined in their ligand binding state: ATP-RhiM, AMPPNP-Leu-RhiC, ADP-intermediate analog-RhiC, and ADP-RhiC.Both RhiC and RhiM have an ATP-grasp fold composed of four domains, A-, B-, C1-, and C2-domains. Structural comparison of their respective ligand-binding states revealed that conformational transitions of the B-domain and three catalytic loops (P-loop, ω-loop, and substrate-binding loop) as the enzymatic reaction proceeds. Notably, the binding of ATP induces an inward shift of the B-domain by 5 Å, resulting in a closed conformation that stabilized the active site. This closed state was observed in the structures that bind the substrate L-Leu and intermediate-analog. In contrast, the ADP-RhiC structure, representing the post-catalytic state, exhibits a flexible loops and adopts an open conformation.Differences in substrate pocket size and surface charge between RhiC and RhiM likely contribute to their distinct substrate specificities. RhiC contains a small hydrophobic pocket, whereas RhiM features a larger, positively charged pocket that accommodates L-arginine. Structural-based mutagenesis of conserved residues involved in ligand binding and catalysis—such as glutamate residues (E258, E271) coordinating Mg2+ bound to ATP,and an arginine residue (R275) that recognizes the substrate amino acid—resulted in a significant decrease in enzymatic activity.These results highlight structural and functional features of RhiC and RhiM. Structural comparisons reveal dynamic loop rearrangements and differences in substrate pocket architecture that underlie their substrate specificities. Our findings propose a mechanistic model for L-amino acid ligase function, involving substrate-induced conformational changes, intermediate formation, and peptide bondsynthesis.gen (H) bond networks; however, difficulty in visualizing H atom positions in the protein hinders the de-tailed understanding of the protein's structure-function relationship. We here used neutron and sub-ångström resolution X-ray crystallography to directly observe H atoms in Paz. The 0.86-Å-resolution X-ray tructure shows that the peptide bond between Pro80 and the His81 Cu ligand deviates from the ideal planar structure. The 1.9-Å-resolution neutron structure confirms a long-overlooked H bond formed by the amide of His81 and the S atom of another Cu ligand Cys78. Quantum mechanics/molecular mechanics calculations show that this H bond increases the redox potential of the Cu site and explains experimental results well. Our study demonstrates the potential of neutron and sub-ångström resolution X-ray crystallography to understand the chemistry of metalloproteins at atomic and quantum levels. |
