Thermal stability and kinetic unfolding of Neisseria gonorrhoeae copper nitrite reductase.

Structural and biochemical insights into the thermostable esterase Ta0887 from Thermoplasma acidophilum

Microbial esterases are versatile and stable enzymes with a wide range of biotechnological applications. However, few esterases have been characterized from archaea, an important source of extremophilic enzymes. In this study, we report the biochemical characterization and crystal structure of Ta0887, a novel esterase from the thermoacidophilic archaeon Thermoplasma acidophilum. The protein was successfully cloned, expressed, and purified in Escherichia coli. Light scattering assays revealed that Ta0887 is a monomer in solution. Activity assays using p‐nitrophenyl (p‐NP) esters confirmed its esterase activity, showing a substrate preference for p‐NP hexanoate (C6). Furthermore, the substitution of Ser95 with alanine completely abolished enzymatic activity, thereby confirming its essential role as the nucleophilic residue of the catalytic triad. The enzyme exhibited optimal activity at 65 °C and pH 8.0. Notably, Ta0887 displayed high thermal stability, retaining 66% residual activity after incubation at 80 °C for 2 h, consistent with its thermal denaturation midpoint temperature of 80.6 °C. The crystal structure of Ta0887, resolved at 1.93 Å, revealed an α/β‐hydrolase core domain consisting of an eight‐strand β‐sheet, surrounded by seven α‐helices, and a cap domain comprising four α‐helices. Ta0887 features a large substrate‐binding pocket at the interface between the two domains that contains the conserved residues Ser95, Asp187, and His215 of the catalytic triad. Further analysis indicates that an efficiently packed hydrophobic core is a key feature for the observed thermostability. The findings from this study provide a basis for the future engineering of Ta0887 with the aim of enhancing its potential for industrial and biotechnological applications.

Analysis of the Atypical Temperature Dependence and Conformational Changes During Turnover of a Lactobacillus Chlorogenic Acid Esterase

Many bacterial chlorogenic acid esterases (ChlEs) exhibit atypical temperature behavior, featuring activities that barely change with temperature and activity maxima that fall below the thermal denaturation point. This work focuses on a ChlE fromLactobacillus helveticus (Lh–ChlE), which has a flat temperature dependence. First, it was determined that conformational changes during Lh–ChlE turnover are not rate-limiting and that the overall rate depends on the chemical step at all temperatures. Next, Lh–ChlE’s temperature dependence was investigated using a conformational equilibrium model that assumes the existence of a temperature-dependent equilibrium between an active and an inactive conformation and an activation heat capacity model that postulates a difference in heat capacity between the ground and transition states. Although the equilibrium model recapitulates the data well, it yields an unrealistically low inactivation temperature around 280 K. Circular dichroism spectroscopy suggests that Lh–ChlE does not undergo structural changes at that temperature but may undergo small structural transitions at moderately elevated temperature. The activation heat capacity model describes Lh–ChlE behavior well, yielding an activation heat capacity (ΔC p ‡) of approximately −1 kJ mol–1 K–1. Overall, the results suggest that the atypical temperature behavior of Lh–ChlE likely arises from a negative activation heat capacity. This work illustrates that contrasting thermodynamic models for atypical temperature dependence in enzymes can give rise to similar looking fits, even though they have different underlying physical meaning. Our results furthermore encourage additional analysis of the Lh–ChlE transition state structure to better understand the structural features that cause the enzyme’s nonzero activation heat capacity.

Thermostability modification and simulated application of acidic catalase

Bilirubin Reductase from Mediterraneibacter gnavus: Positional Reduction Preferences and Transient-State Analysis.

The first committed step for the excretion of bilirubin is reduction of four vinylic bonds to form urobilinogen. This reaction is catalyzed by bilirubin reductase (BilR) that uses four NADHs to accomplish the transformation. Due to the asymmetry of bilirubin, BilR must either recognize 15 different oxidant substrates or have a preferred reduction order. AlphaFold structural prediction indicates structural homology with bacterial 2,4-dienoyl-CoA reductase and 2-napthoyl-CoA reductase, both of which are single-subunit proteins with a two-domain structure that houses three cofactors: an FAD, an Fe4S4 cluster, and an FMN. In this study, anaerobic kinetic and mechanistic analysis of bilirubin reductase from Mediterraneibacter gnavus (MgBilR) is presented. Anaerobic solvent exchange studies define the stereochemistry of hydride transfer from NADH as ProS. Transient-state kinetics of the reductive half-reaction demonstrate that MgBilR can become reduced by four electrons with either NADH or NADPH, but that NADH is the preferred reductant by four orders of magnitude (based on kred/Kd values). Only the first hydride transfer from NADH occurs at a rate that is catalytically relevant. Single-turnover experiments show two dominant phases corresponding to two-electron flavin reduction and subsequent rate-limiting bilirubin reduction that occurs with flavin reoxidation. Single-turnover data correlated with steady-state assays and NMR tracking of the steady-state reaction confirm that BilR can reduce both types of vinylic bonds of bilirubin but appears to have a 4-fold preference for reduction of α-vinylic over enamine bonds that is defined almost entirely by the rate of hydride transfers to the oxidant.

inducible proteins in Geobacillus thermoleovorans B23

Insights into the enzyme kinetics, structural features, and in silico inhibition of Leishmania donovani prolidase.

Overall, this study presents the first comprehensive report on the enzyme kinetics, structural characteristics, and in silico inhibition of metal-dependent prolidase from trypanosomatid parasites.