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The objective of this study was to establish and to mathematically describe the phenol degrading properties of a new Acinetobacter towneri CFII-87 strain, isolated from a bioreactor treating landfill leachate. For this purpose, the biokinetic parameters of phenol biodegradation at various initial phenol concentrations of the A. towneri CFII-87 strain have been experimentally measured, and four different mathematical inhibition models (Haldane, Yano, Aiba and Edwards models) have been used to simulate the substrate-inhibited phenol degradation process. The results of the batch biodegradation experiments show that the new A. towneri CFII-87 strain grows on and metabolizes phenol up to 1000 mg/L concentration, manifests significant substrate inhibition and lag time only at concentrations above 800 mg/L phenol, and has a maximum growth rate at 300 mg/L initial phenol concentration. The comparison of the model predictions with the experimental phenol and biomass data revealed that the Haldane, Aiba and Edwards models can be used with success to describe the phenol biodegradation process by A. towneri CFII-87, while the Yano model, especially at higher initial phenol concentrations, fails to describe the process. The best performing inhibition model was the Edwards model, presenting correlation coefficients of R2 > 0.98 and modelling efficiency of ME > 0.94 for the prediction of biomass and phenol concentrations on the validation datasets. The calculated biokinetic model parameters place this new strain among the bacteria with the highest tolerance towards phenol. The results suggest that the A. towneri CFII-87 strain can potentially be used in the treatment of phenolic wastewaters.

The hexavalent chromium is one of the major carcinogenic components released during the tanning process and lots of work have been carried out on the reduction of hexavalent chromium via chemical and biological routes. Different fatty oils are also employed in the tanning process and have also been released as an effluent along with chromium. However, it is difficult to find a study on the reduction of chromium in the presence of other contaminant which would help to mimic the real-time complication of treating the tannery effluent. It is the first attempt on the reduction of hexavalent chromium in the presence of synthetic fatty liquor and neatsfoot oil using Fusarium oxysporum . The maximum percentage of chromium reduction was 73.62% and 60.28% in neatsfoot oil and synthetic fatty oil, respectively, for the initial chromium concentration of 25 mg/L. The biomass productivity was better with both neatsfoot oil and synthetic fatty oil, whereas the same has decreased with the presence of chromium. The reduction of chromium was found to follow the uncompetitive substrate inhibition kinetics than the general Michaelis–Menten kinetics. The kinetic parameters were calculated using particle swarm optimization algorithm, which were compared with the already reported data. The uncompetitive substrate inhibition kinetics was represented the experimental data in both the cases and the value of substrate inhibition constant was low in the case of neatsfoot oil compared with the synthetic fatty liquor.

Mixed microbial culture collected from the wastewater treatment plant of Indian Institute of Technology Guwahati (IITG) was further grown in anaerobic condition in presence of sulfate where lactate was added as a carbon source. Sulfate addition was increased stepwise up to 1,000 mg l−1 before phenol was added at increasing concentrations from 10 mg l−1 to 300 mg l−1. Kinetics of sulfate, phenol and chemical oxygen demand reduction were studied and experimental findings were analyzed using various bio-models to estimate the bio-kinetic coefficients. This is the first detailed report on kinetics and bio-kinetic studies of sulfate reduction in presence of phenol. Experimental results showed that there was no inhibition of sulfate reduction and microbial growth up to 100 mg l−1 phenol addition. However, inhibition to different degrees was observed at higher phenol addition. The experimental data of microbial growth and substrate consumption in presence of phenol fitted well to the Edward model (R2 = 0.85, root mean square error = 0.001011) with maximum specific growth rate = 0.052 h−1, substrate inhibition constant = 88.05 mg l−1 and half saturation constant = 58.22 mg l−1. The characteristics of the cultured microbes were determined through a series of analysis and microbial tests.

Chlorophenols (CP) are listed as priority pollutants both by US EPA as well European community. In the present study, biodegradation of 4-CP was investigated using a cells immobilized in calcium alginate beads system. The batch shake flask study of the cells entrapped in calcium alginate beads system followed substrate inhibition kinetics. The bio-kinetic constants that are necessary to design and simulation of the bioreactor system were estimated from the best fitted Edward model. Further, performance of the cells immobilized in calcium alginate beads system for the degradation of 4-CP was evaluated in an upflow packed bed reactor (PBR) by varying its influent concentration from 100 to 250 mg/L and hydraulic retention time (HRT) between 8 h and 2 h. Almost complete removal of 4-CP and 98.6% effluent toxicity removal were obtained in the reactor at a 4-CP loading rate of 1275 mg/L/d or lesser. However, at higher 4-CP loading rates, the performance of the packed bed reactor (PBR) was found to be deteriorated due to the temporary accumulation of 4-chlorocatehol. The study reveals the potential of cells entrapped in calcium alginate beads system for biodegradation of 4-CP from contaminated wastewater.

Abstract Metformin is a first-line therapy for the treatment of type 2 diabetes, due to its robust glucose-lowering effects, well-established safety profile, and relatively low cost. While metformin has been shown to have pleotropic effects on glucose metabolism, there is a general consensus that the major glucose-lowering effect in patients with type 2 diabetes is mostly mediated through inhibition of hepatic gluconeogenesis. However, despite decades of research, the mechanism by which metformin inhibits this process is still highly debated. A key reason for these discrepant effects is likely due to the inconsistency in dosage of metformin across studies. Widely studied mechanisms of action, such as complex I inhibition leading to AMPK activation, have only been observed in the context of supra-pharmacological (>1 mM) metformin concentrations, which do not occur in the clinical setting. Thus, these mechanisms have been challenged in recent years and new mechanisms have been proposed. Based on the observation that metformin alters cellular redox balance, a redox-dependent mechanism of action has been described by several groups. Recent studies have shown that clinically relevant (50-100 μM) concentrations of metformin inhibit hepatic gluconeogenesis in a substrate-selective manner both in vitro and in vivo, supporting a redox-dependent mechanism of metformin action. Here, we review the current literature regarding metformin’s cellular and molecular mechanisms of action. Graphical Abstract Graphical Abstract

Substrate inhibition of enzymes can be a major obstacle to the production of valuable chemicals in engineered microorganisms. Here, we show substrate inhibition of lycopene cyclase as the main limitation in carotenoid biosynthesis in Yarrowia lipolytica . To overcome this bottleneck, we exploit two independent approaches. Structure-guided protein engineering yields a variant, Y27R, characterized by complete loss of substrate inhibition without reduction of enzymatic activity. Alternatively, establishing a geranylgeranyl pyrophosphate synthase-mediated flux flow restrictor also prevents the onset of substrate inhibition by diverting metabolic flux away from the inhibitory metabolite while maintaining sufficient flux towards product formation. Both approaches result in high levels of near-exclusive β-carotene production. Ultimately, we construct strains capable of producing 39.5 g/L β-carotene at a productivity of 0.165 g/L/h in bioreactor fermentations (a 1441-fold improvement over the initial strain). Our findings provide effective approaches for removing substrate inhibition in engineering pathways for efficient synthesis of natural products. Substrate inhibition has not been widely studied in the context of synthetic biology and metabolic engineering. Here, the authors report removal of lycopene substrate inhibition by two different strategies and enable high carotenoid productivity in Yarrowia lipolytica .

Molecular-motor coordinationThe proteasome is a cytosolic molecular machine that recognizes and degrades unneeded or damaged proteins that have been tagged with ubiquitin. A heterohexameric adenosine triphosphatase motor pulls the substrate into the proteolytic chamber, while at the same time, a protein located at the entrance of this motor removes the ubiquitin. De la Peña et al. trapped the substrate inside the motor by inhibiting removal of ubiquitin. This allowed them to determine cryo–electron microscopy structures in the presence of substrate and adenosine triphosphate (ATP). The findings distinguish three sequential conformational states that show how ATP binding, hydrolysis, and phosphate release are coordinated between the six subunits of the motor to cause the conformational changes that translocate the substrate through the proteasome.Science, this issue p. eaav0725INTRODUCTIONAs the major protease in eukaryotic cells and the final component of the ubiquitin-proteasome system, the 26S proteasome is responsible for protein homeostasis and the regulation of numerous vital processes. Misfolded, damaged, or obsolete regulatory proteins are marked for degradation by the attachment of polyubiquitin chains, which bind to ubiquitin receptors of the proteasome. A heterohexameric ring of AAA+ (ATPases associated with diverse cellular activities) subunits then uses conserved pore loops to engage, mechanically unfold, and translocate protein substrates into a proteolytic core for cleavage while the deubiquitinase Rpn11 removes substrate-attached ubiquitin chains.RATIONALEDespite numerous structural and functional studies, the mechanisms by which adenosine triphosphate (ATP) hydrolysis drives the conformational changes responsible for protein degradation remained elusive. Structures of related homohexameric AAA+ motors, in which bound substrates were stabilized with ATP analogs or hydrolysis-eliminating mutations, revealed snapshots of ATPase subunits in different nucleotide states and spiral-staircase arrangements of pore loops around the substrate. These structures gave rise to “hand-over-hand” translocation models by inferring how individual subunits may progress through various substrate-binding conformations. However, the coordination of ATP-hydrolysis steps and their mechanochemical coupling to propelling substrate were unknown.RESULTSWe present the cryo–electron microscopy (cryo-EM) structures of the actively ATP-hydrolyzing, substrate-engaged 26S proteasome with four distinct motor conformations. Stalling substrate translocation at a defined position by inhibiting deubiquitination led to trapped states in which the substrate-attached ubiquitin remains functionally bound to the Rpn11 deubiquitinase, and the scissile isopeptide bond of ubiquitin is aligned with the substrate-translocation trajectory through the AAA+ motor. Our structures suggest a ubiquitin capture mechanism, in which mechanical pulling on the substrate by the AAA+ motor delivers ubiquitin modifications directly into the Rpn11 catalytic groove and accelerates isopeptide cleavage for efficient, cotranslocational deubiquitination.These structures also show how the substrate polypeptide traverses from the Rpn11 deubiquitinase, through the AAA+ motor, and into the core peptidase. The proteasomal motor thereby adopts staircase arrangements with five substrate-engaged subunits and one disengaged subunit. Four of the substrate-engaged subunits are ATP bound, whereas the subunit at the bottom of the staircase and the disengaged subunit are bound to adenosine diphosphate (ADP).CONCLUSIONOf the four distinct motor states we observed, three apparently represent sequential stages of ATP binding, hydrolysis, and substrate translocation and hence reveal the coordination of individual steps in the ATPase cycle and their mechanochemical coupling with translocation. ATP hydrolysis occurs in the fourth substrate-engaged subunit from the top, concomitantly with exchange of ADP for ATP in the disengaged subunit. The subsequent transition, which is likely triggered by phosphate release from the fourth, posthydrolysis subunit of the staircase, then involves major conformational changes of the entire ATPase hexamer. The bottom ADP-bound subunit is displaced and the previously disengaged subunit binds the substrate at the top of the staircase, while the four engaged subunits move downward as a rigid body and translocate substrate toward the peptidase. Our likely consecutive proteasome conformations, together with previously determined substrate-free structures, suggest a sequential progression of ATPase subunits through the ATP-hydrolysis cycle. We hypothesize that, in general, hexameric AAA+ translocases function by this sequential mechanism.The 26S proteasome is the primary eukaryotic degradation machine and thus is critically involved in numerous cellular processes. The heterohexameric adenosine triphosphatase (ATPase) motor of the proteasome unfolds and translocates targeted protein substrates into the open gate of a proteolytic core while a proteasomal deubiquitinase concomitantly removes substrate-attached ubiquitin chains. However, the mechanisms by which ATP hydrolysis drives the conformational changes responsible for these processes have remained elusive. Here we present the cryo–electron microscopy structures of four distinct conformational states of the actively ATP-hydrolyzing, substrate-engaged 26S proteasome. These structures reveal how mechanical substrate translocation accelerates deubiquitination and how ATP-binding, -hydrolysis, and phosphate-release events are coordinated within the AAA+ (ATPases associated with diverse cellular activities) motor to induce conformational changes and propel the substrate through the central pore.

β-Amyloid peptides, which are derived from amyloid precursor protein (APP), form the plaques in the brain that are characteristic of Alzheimer's disease. Zhou et al. report a high-resolution structure of a transmembrane segment of APP bound to human γ-secretase, the transmembrane protease that cleaves APP to give β-amyloid peptides (see the Perspective by Lichtenthaler and Güner). Disease-associated mutations within presenilin-1, the catalytic subunit of APP, likely affect how the substrate is bound and thus which peptides are generated, with some being more amyloidogenic. It may now be possible to exploit the features of substrate binding to design inhibitors. Science , this issue p. eaaw0930 ; see also p. 690 A view of how the substrate is recognized facilitates understanding of Alzheimer’s disease–associated mutations in γ-secretase. Cleavage of amyloid precursor protein (APP) by the intramembrane protease γ-secretase is linked to Alzheimer’s disease (AD). We report an atomic structure of human γ-secretase in complex with a transmembrane (TM) APP fragment at 2.6-angstrom resolution. The TM helix of APP closely interacts with five surrounding TMs of PS1 (the catalytic subunit of γ-secretase). A hybrid β sheet, which is formed by a β strand from APP and two β strands from PS1, guides γ-secretase to the scissile peptide bond of APP between its TM and β strand. Residues at the interface between PS1 and APP are heavily targeted by recurring mutations from AD patients. This structure, together with that of γ-secretase bound to Notch, reveal contrasting features of substrate binding, which may be applied toward the design of substrate-specific inhibitors.

ABCB1, also known as P-glycoprotein, actively extrudes xenobiotic compounds across the plasma membrane of diverse cells, which contributes to cellular drug resistance and interferes with therapeutic drug delivery. We determined the 3.5-angstrom cryo–electron microscopy structure of substrate-bound human ABCB1 reconstituted in lipidic nanodiscs, revealing a single molecule of the chemotherapeutic compound paclitaxel (Taxol) bound in a central, occluded pocket. A second structure of inhibited, human-mouse chimeric ABCB1 revealed two molecules of zosuquidar occupying the same drug-binding pocket. Minor structural differences between substrate- and inhibitor-bound ABCB1 sites are amplified toward the nucleotide-binding domains (NBDs), revealing how the plasticity of the drug-binding site controls the dynamics of the adenosine triphosphate–hydrolyzing NBDs. Ordered cholesterol and phospholipid molecules suggest how the membrane modulates the conformational changes associated with drug binding and transport.

The 26S proteasome is involved in degrading and regulating the majority of proteins in eukaryotic cells, which requires a sophisticated balance of specificity and promiscuity. In this Review, we discuss the principles that underly substrate recognition and ATP-dependent degradation by the proteasome. We focus on recent insights into the mechanisms of conventional ubiquitin-dependent and ubiquitin-independent protein turnover, and discuss the plethora of modulators for proteasome function, including substrate-delivering cofactors, ubiquitin ligases and deubiquitinases that enable the targeting of a highly diverse substrate pool. Furthermore, we summarize recent progress in our understanding of substrate processing upstream of the 26S proteasome by the p97 protein unfoldase. The advances in our knowledge of proteasome structure, function and regulation also inform new strategies for specific inhibition or harnessing the degradation capabilities of the proteasome for the treatment of human diseases, for instance, by using proteolysis targeting chimera molecules or molecular glues. Most proteins are degraded and regulated by the 26S proteasome. A balance of specificity and promiscuity underlies substrate recognition and ATP-dependent degradation, which can occur in a ubiquitin-dependent or ubiquitin-independent manner. Recent insights into the mechanisms and regulation of substrate delivery, processing and degradation will inform new strategies for the treatment of human diseases.