This nMS-based workflow offers a broadly applicable framework for resolving the catalytic states and inter-site communication of oligomeric enzymes that are otherwise difficult to uncover by conventional structural methods.
Benzaldehyde is a widespread volatile compound produced by plants. Its final biosynthetic step is catalyzed by benzaldehyde synthase (BS), a peroxisomal enzyme composed of α and β subunits, both belonging to the short-chain dehydrogenase/reductase (SDR) family. Here, we report the crystal structure of Petunia hybrida BS, which reveals an α2β2 heterotetrameric arrangement. Structural and biochemical analyses show that the α subunits contain the canonical catalytic site, whereas the β subunits have lost catalytic activity but are essential for heterotetramer assembly. Notably, the C terminus of the β subunit extends into the diagonally positioned α subunit, contributing to the formation of the composite benzoyl-CoA substrate-binding pocket. Site-directed mutagenesis and subunit-mixing experiments support noncooperative, additive contributions of protomers within the heterotetramer. This work establishes BS as a rare heterotetrameric plant SDR and demonstrates how subunit specialization and intersubunit arrangement enable function, providing principles for understanding and engineering multimeric enzyme complexes.
Jason O. Matos, Jihee Lee, Ramasamy P. Kumar et al.· Science Advances· 0 citations
Bidirectional acetylation and deacetylation of small molecules by acetyltransferases (ATs) remain poorly understood. In this study, we present the structural, functional, and computational characterization of a chloramphenicol O-AT from Bacillus sp. PAMC22265 (AT65) that exhibits bidirectional catalytic activity toward the regiospecific C21-acetylation and deacetylation of steroid substrates. The crystal structure of AT65 was determined at 2.40 Å resolution (PDB ID: 24ZY), revealing a trimeric architecture in which the putative active-sites located at the interfaces between adjacent subunits. In vitro kinetic analyses of both acetylation and deacetylation reactions demonstrated substrate-dependent catalytic efficiencies, with comparable activity under the experimental conditions. Molecular docking, 100 ns molecular dynamics simulations, hydrogen-bond analysis, and MM-PBSA free-energy calculations supported stable substrate binding within the active site and suggested that His187 and Asp191 may contribute to substrate recognition and catalysis. Consistent with this observation, substitution of His187 with alanine abolished detectable enzymatic activity, highlighting its functional importance. Whole-cell biotransformation further demonstrated the selective production of C21-acetylated steroid derivatives, some of which exhibited preliminary antiproliferative activity against murine breast cancer cell lines. Collectively, these findings provide structural, biochemical, and computational insights into the bidirectional catalytic properties of AT65 and establish a foundation for further mechanistic studies and the development of selective enzymatic strategies for steroid modification.
Kamal Prasad Regmi, S. Park, Prakash Paudel et al.· International Journal of Bio...· 0 citations
Aconitate decarboxylase 1, an enzyme member of the MmgE-PrpD family of proteins, has gained significant attention in the last decade as a therapeutic target for cancer and inflammatory diseases. Its product, itaconate, is a multifunctional metabolite shown to drive several disease states. Though extensively studied in cellulo and in vivo, this protein is biochemically and mechanistically under characterized and although a family of inhibitors has been described, no ligand-bound structures have yet been determined. In this work we present a thorough structural investigation that yielded the first ligand-bound structure of this protein family, which required the generation of artifact-free apo crystals. We also developed a novel, low-consumption, robust kinetic assay and investigated active site and allosteric mutants to further elucidate structural and dynamic activity relationships of this protein.
Brent Runge, Hande Oktay, Ian J. Fucci et al.· Journal of Structural Biolog...· 0 citations
Proteolytic deubiquitinating enzymes bridge a gap in substrate recognition through complex regulatory mechanisms. A growing portion of these are accomplished through proteoforms that uniquely control association and diverse sets of cleavage capabilities that relay distinct physiological outcomes. This study describes substrate biasing governed by UCHL5 proteoforms. It demonstrates that N-terminal ubiquitination activates the enzyme towards monoubiquitin substrates, a feature that is conserved across UCHL5 homologs. Crystallographic and spectroscopic data suggest that the N-terminal ubiquitin binds intramolecularly in an allosteric binding site and inhibits branched chain substrate cleavage. Association with Rpn13/Adrm1 relieves this inhibition and reestablishes its ability to debranch, potentially controlling nonspecific debranching compared to retention of needed activity on the 26S proteasome. Collectively, this study describes the molecular basis for substrate selectivity in a deubiquitinating enzyme, an unexplored area in the enzymes that counteract ubiquitin E3 ligases.
Rishi S. Patel, Nipuni M. Pannala, Chih-Hsuan Lai et al.· bioRxiv· 0 citations
CRISPR-Cas12a is widely utilized for genome engineering and nucleic acid diagnostics, being distinguished by its indiscriminate single-stranded DNA (ssDNA) trans-cleavage activity triggered by its specific cis-target recognition. However, the precise kinetic coordination between these dual catalytic modes remains unclear because of methodological limitations, which preclude simultaneous monitoring of both activities. Here, we established a real-time, dual-wavelength fluorescence reporter system to dissect these dynamics utilizing phosphorothioate (PS) backbone modifications as chemical probes to interrogate enzyme turnover. We identified a functional decoupling and an asymmetric competitive mechanism strictly governed by “channel occupancy”. Specifically, we found that the PS modification of the cis-target abolished the trans-activity via a “product release gating” mechanism, where high-affinity product retention occluded the active site. Furthermore, we identified a critical length-dependent regulatory regime for ssDNA reporters, while short, noncleavable ligands (5-nt) acted as passive spectator molecules, and long analogues (30-nt) functioned as potent competitive inhibitors. The long ligands induced an irreversible “kinetic trap”, creating a nonproductive complex where the enzyme was permanently sequestered because of the lack of cleavage-mediated release. These findings demonstrate that the availability of the RuvC catalytic channel is determined not by induced fit binding but by the chemical cleavability of the occupant. This study establishes an integrated Cas12a-regulated kinetic model and systematically investigates the simultaneous effects of PS-modification on the cis- and trans-hydrolytic activities of Cas12a. The findings provide a theoretical framework and additional insights for developing more precise gene-editing tools and designing Cas12a-based in vitro diagnostic platforms.
The specificity and catalytic efficiency of enzymes make them attractive for applications ranging from therapeutics to chemical manufacturing. However, it remains challenging to identify specific structural and dynamic mechanisms by which enzymes achieve their catalytic rate enhancements as well as to re-engineer enzymes to improve their catalytic properties. In earlier work we reported the design and selection of KARI mutants with calculated increases in specific activity (i.e., k cat) relative to wild type (WT) for the isomerization step of one of its native substrates: 2-acetolactate (ACL), which leads to the synthesis of the amino acids valine and leucine. Eight mutants were identified with computed improvements in k cat of up to 4 orders of magnitude. In the current study, we investigate the effects of these same mutations on the isomerization of the other native substrate, 2-aceto-2-hydroxybutyrate (AHB, leading to the synthesis of isoleucine). Paralleling our previous work, we use the computational statistical mechanical method transition interface sampling (TIS) to simulate reaction kinetics and compute reaction rate constants. We find that the mutants selected for increased efficiency on ACL had varied levels of activity on AHB–some enhancing reactivity and others diminishing it–with the range in computed AHB rate constants spanning more than 7 orders of magnitude. Analysis of the simulations for WT-AHB revealed that only some of the structural mechanisms associated with mutants’ improved ACL catalysis were expected to transfer to, and thereby improve, AHB catalysis. For two mutants with significantly lower catalytic efficiency on AHB than WT, further analysis identified unique conformational changes that may explain their low activity on AHB.
Elijah Karvelis, Bruce Tidor· Journal of Physical Chemistr...· 0 citations