Monomeric Esterase: Insights into Cooperative Behavior, Hysteresis/Allokairy.

Herein, we present a novel esterase enzyme, Ade1, isolated from a metagenomic library of Amazonian dark earths soils, demonstrating its broad substrate promiscuity by hydrolyzing ester bonds linked to aliphatic groups. The three-dimensional structure of the enzyme was solved in the presence and absence of substrate (tributyrin), revealing its classification within the α/ß-hydrolase superfamily. Despite being a monomeric enzyme, enzymatic assays reveal a cooperative behavior with a sigmoidal profile (initial velocities vs substrate concentrations). Our investigation brings to light the allokairy/hysteresis behavior of Ade1, as evidenced by a transient burst profile during the hydrolysis of substrates such as p-nitrophenyl butyrate and p-nitrophenyl octanoate. Crystal structures of Ade1, coupled with molecular dynamics simulations, unveil the existence of multiple conformational structures within a single molecular state (E̅1). Notably, substrate binding induces a loop closure that traps the substrate in the catalytic site. Upon product release, the cap domain opens simultaneously with structural changes, transitioning the enzyme to a new molecular state (E̅2). This study advances our understanding of hysteresis/allokairy mechanisms, a temporal regulation that appears more pervasive than previously acknowledged and extends its presence to metabolic enzymes. These findings also hold potential implications for addressing human diseases associated with metabolic dysregulation.

The PilB-PilZ-FimX regulatory complex of the Type IV pilus from Xanthomonas citri.

Type IV pili (T4P) are thin and flexible filaments found on the surface of a wide range of Gram-negative bacteria that undergo cycles of extension and retraction and participate in a variety of important functions related to lifestyle, defense and pathogenesis. During pilus extensions, the PilB ATPase energizes the polymerization of pilin monomers from the inner membrane. In Xanthomonas citri, two cytosolic proteins, PilZ and the c-di-GMP receptor FimX, are involved in the regulation of T4P biogenesis through interactions with PilB. In vivo fluorescence microscopy studies show that PilB, PilZ and FimX all colocalize to the leading poles of X. citri cells during twitching motility and that this colocalization is dependent on the presence of all three proteins. We demonstrate that full-length PilB, PilZ and FimX can interact to form a stable complex as can PilB N-terminal, PilZ and FimX C-terminal fragments. We present the crystal structures of two binary complexes: i) that of the PilB N-terminal domain, encompassing sub-domains ND0 and ND1, bound to PilZ and ii) PilZ bound to the FimX EAL domain within a larger fragment containing both GGDEF and EAL domains. Evaluation of PilZ interactions with PilB and the FimX EAL domain in these and previously published structures, in conjunction with mutagenesis studies and functional assays, allow us to propose an internally consistent model for the PilB-PilZ-FimX complex and its interactions with the PilM-PilN complex in the context of the inner membrane platform of the X. citri Type IV pilus.

Importance of the ß5-ß6 Loop for the Structure, Catalytic Efficiency, and Stability of Carbapenem-Hydrolyzing Class D ß-Lactamase Subfamily OXA-143.

The class D ß-lactamase OXA-143 has been described as an efficient penicillinase, oxacillinase, and carbapenemase. The D224A variant, known as OXA-231, was described in 2012 as exhibiting less activity toward imipenem and increased oxacillinase activity. Additionally, the P227S mutation was reported as a case of convergent evolution for homologous enzymes. To investigate the impact of both mutations (D224A and P227S), we describe in this paper a deep investigation of the enzymatic activities of these three homologues. OXA-143(P227S) presented enhanced catalytic activity against ampicillin, oxacillins, aztreonam, and carbapenems. In addition, OXA-143(P227S) was the only member capable of hydrolyzing ceftazidime. These enhanced activities were due to a combination of a higher affinity (lower Km) and a higher turnover number (higher kcat). We also determined the crystal structure of apo OXA-231. As expected, the structure of this variant is very similar to the published OXA-143 structure, except for the two M223 conformations and the absence of electron density for three solvent-exposed loop segments. Molecular dynamics calculations showed that both mutants experience higher flexibility compared to that of the wild-type form. Therefore, our results illustrate that D224A and P227S act as deleterious and positive mutations, respectively, within the evolutionary path of the OXA-143 subfamily toward a more efficient carbapenemase.