Biology subjects

Zinovjev, K.

Publications and source records attributed to Zinovjev, K..

3 recordsLinked to original sources

bioRxiv2024

Influence of Wobbling Tryptophan and Mutations on PET Degradation Explored by QM/MM Free Energy Calculations

Plastic-degrading enzymes, particularly poly(ethylene terephthalate) (PET) hydrolases, have garnered significant attention in recent years as potential eco-friendly solutions for recycling plastic waste. However, understanding of their PET-degrading activity and influencing factors remains incomplete, impeding the development of uniform approaches for enhancing PET hydrolases for industrial applications. A key aspect of PET hydrolase engineering is optimizing the PET-hydrolysis reaction by lowering the associated free energy barrier. However, inconsistent findings have complicated these efforts. Therefore, our goal is to elucidate various aspects of enzymatic PET degradation by means of quantum mechanics / molecular mechanics (QM/MM) reaction simulations and analysis, focusing on the initial reaction step, acylation, in two thermophilic PET hydrolases: LCC and PES-H1, along with their highly active variants, LCCICCG and PES-H1FY. Our findings highlight the impact of semi-empirical QM methods on proton transfer energies, affecting the distinction between a two-step reaction involving a metastable tetrahedral intermediate and a one-step reaction. Moreover, we uncovered a concerted conformational change involving the orientation of the PET benzene ring, altering its interaction with the side-chain of the wobbling tryptophan from T-stacking to parallel {pi}-{pi} interactions, a phenomenon overlooked in prior research. Our study thus enhances the understanding of the acylation mechanism of PET hydrolases, in particular by characterizing it for the first time for the promising PES-H1FY using QM/MM simulations. It also provides insights into selecting a suitable QM method and a reaction coordinate, valuable for future studies on PET degradation processes. TOC Graphic O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=111 SRC="FIGDIR/small/591886v1_ufig1.gif" ALT="Figure 1"> View larger version (30K): org.highwire.dtl.DTLVardef@c0aa9aorg.highwire.dtl.DTLVardef@1008acdorg.highwire.dtl.DTLVardef@98b0d6org.highwire.dtl.DTLVardef@81d8e3_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2023

4-Thiaproline accelerates the slow folding phase of proteins containing cis prolines in the native state by two orders of magnitude

The cis/trans isomerization of peptidyl-prolyl peptide bonds is often the bottleneck of the refolding reaction for proteins containing cis proline residues in the native state. Proline (Pro) analogues, especially C4-substituted fluoroprolines, have been widely used in protein engineering to enhance the thermodynamic stability of peptides and proteins and to investigate folding kinetics. 4-thiaproline (Thp) has been shown to bias the ring pucker of Pro, to increase the cis population percentage of model peptides in comparison to Pro, and to diminish the activation energy barrier for the cis/trans isomerization reaction. Despite its intriguing properties, Thp has been seldom incorporated into proteins. Moreover, the impact of Thp on the folding kinetics of globular proteins has never been reported. In this study, we show that upon incorporation of Thp at cisPro76 into the thioredoxin variant Trx1P the half-life of the refolding reaction decreased from [~]2 hours to [~]35 seconds. A dramatic acceleration of the refolding rate could be observed also for the protein pseudo wild-type barstar upon replacement of cisPro48 with Thp. Quantum chemical calculations revealed that the replacement of the C{gamma}H2 group by a sulfur atom in the pyrrolidine ring, lowers the barrier for cis/trans rotation due to a weakened peptide bond. The protein variants retained their thermodynamic stability upon incorporation of Thp, while the catalytic and enzymatic activities of the modified Trx1P remained unchanged. Our results show that the Pro isostere Thp might eliminate the bottleneck of the refolding reaction of proteins containing cis proline residues in the native state, independent from the local structural environment.

biochemistry↗

bioRxiv2022

Tautomer-specific deacylation and {Omega}-loop flexibility explain carbapenem-hydrolyzing, broad-spectrum activity of the KPC-2 β-lactamase

KPC-2 (Klebsiella pneumoniae carbapenemase-2) is a globally disseminated serine-{beta}-lactamase (SBL) responsible for extensive {beta}-lactam antibiotic resistance in Gram-negative pathogens. SBLs inactivate {beta}-lactams via a mechanism involving a hydrolytically labile covalent acyl-enzyme intermediate. Carbapenems, the most potent {beta}-lactams, evade activity of many SBLs by forming long-lived inhibitory acyl-enzymes; however, carbapenemases such as KPC-2 efficiently catalyze deacylation of carbapenem-derived acyl-enzymes. We present high-resolution (1.25-1.4 [A]) crystal structures of KPC-2 acyl-enzymes with representative penicillins (ampicillin), cephalosporins (cefalothin) and carbapenems (imipenem, meropenem and ertapenem), obtained utilizing an isosteric deacylation-deficient mutant (E166Q). Mobility of the {Omega}-loop (residues 165-170) negatively correlates with antibiotic turnover rates (kcat), highlighting the role of this region in positioning catalytic residues for efficient hydrolysis of different {beta}-lactams. Carbapenem-derived acyl-enzyme structures reveal predominance of the {Delta}1-(2R) imine tautomer, except for the imipenem acyl-enzyme, which is present in dual occupancy in both {Delta}1-(2R) and (2S) configurations. Quantum mechanics/molecular mechanics (QM/MM) molecular dynamics simulations of deacylation of the KPC-2:meropenem acyl-enzyme, using an adaptive string method (ASM), show that the {Delta}1-(2R) isomer has a 7 kcal/mol higher barrier for the (rate-determining) formation of the tetrahedral deacylation intermediate than the {Delta}2 tautomer. The simulations identify tautomer-specific differences in hydrogen bonding networks involving the carbapenem C-3 carboxylate and the deacylating water, that, together with stabilization by protonated N-4 of accumulating negative charge during oxyanion formation, accelerate deacylation of the {Delta}2-enamine acyl-enzyme compared to the {Delta}1-imine. Taken together, our data show how the flexible {Omega}-loop helps confer broad spectrum activity upon KPC-2, while carbapenemase activity stems from efficient deacylation of the {Delta}2-enamine acyl-enzyme tautomer. Differentiation of the barriers associated with deacylation of these subtly different {beta}-lactam isomers further identifies ASM as a sensitive method for calculation of reaction energetics that can accurately model turnover and, potentially, predict the impact of substrate modifications or point mutations upon activity.

biochemistry↗