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Cellobiohydrolases directly convert crystalline cellulose into cellobiose and are of biotechnological interest to achieve efficient biomass utilization. As a result, much research in the field has focused on identifying cellobiohydrolases that are very fast. Cellobiohydrolase A from the bacterium Cellulomonas fimi (CfCel6B) and cellobiohydrolase II from the fungus Trichoderma reesei (TrCel6A) have similar catalytic domains (CDs) and show similar hydrolytic activity. However, TrCel6A and CfCel6B have different cellulose-binding domains (CBDs) and linkers: TrCel6A has a glycosylated peptide linker, whereas CfCel6B's linker consists of three fibronectin type 3 domains. We previously found that TrCel6A's linker plays an important role in increasing the binding rate constant to crystalline cellulose. However, it was not clear whether CfCel6B's linker has similar function. Here we analyze kinetic parameters of CfCel6B using single-molecule fluorescence imaging to compare CfCel6B and TrCel6A. We find that CBD is important for initial binding of CfCel6B, but the contribution of the linker to the binding rate constant or to the dissociation rate constant is minor. The crystal structure of the CfCel6B CD showed longer loops at the entrance and exit of the substrate-binding tunnel compared with TrCel6A CD, which results in higher processivity. Furthermore, CfCel6B CD showed not only fast surface diffusion but also slow processive movement, which is not observed in TrCel6A CD. Combined with the results of a phylogenetic tree analysis, we propose that bacterial cellobiohydrolases are designed to degrade crystalline cellulose using high-affinity CBD and high-processivity CD., Competing Interests: Conflict of interest—The authors declare that they have no conflicts of interest with the contents of this article., (© 2020 Nakamura et al.)

Chitin degradation is important for biomass conversion and has potential applications for agriculture, biotechnology, and the pharmaceutical industry. Chitinase A from the Gram-negative bacterium Serratia marcescens ( Sm ChiA) is a processive enzyme that hydrolyzes crystalline chitin as it moves linearly along the substrate surface. In a previous study, the catalytic activity of Sm ChiA against crystalline chitin was found to increase after the tryptophan substitution of two phenylalanine residues (F232W and F396W), located at the entrance and exit of the substrate binding cleft of the catalytic domain, respectively. However, the mechanism underlying this high catalytic activity remains elusive. In this study, single-molecule fluorescence imaging and high-speed atomic force microscopy were applied to understand the mechanism of this high-catalytic-activity mutant. A reaction scheme including processive catalysis was used to reproduce the properties of Sm ChiA WT and F232W/F396W, in which all of the kinetic parameters were experimentally determined. High activity of F232W/F396W mutant was caused by a high processivity and a low dissociation rate constant after productive binding. The turnover numbers for both WT and F232W/F396W, determined by the biochemical analysis, were well-replicated using the kinetic parameters obtained from single-molecule imaging analysis, indicating the validity of the reaction scheme. Furthermore, alignment of amino acid sequences of 258 Sm ChiA-like proteins revealed that tryptophan, not phenylalanine, is the predominant amino acid at the corresponding positions (Phe-232 and Phe-396 for Sm ChiA). Our study will be helpful for understanding the kinetic mechanisms and further improvement of crystalline chitin hydrolytic activity of Sm ChiA mutants., (© 2020 Visootsat et al.)

V 1 -ATPase (V 1 ), the catalytic domain of an ion-pumping V-ATPase, is a molecular motor that converts ATP hydrolysis-derived chemical energy into rotation. Here, using a gold nanoparticle probe, we directly observed rotation of V 1 from the pathogen Enterococcus hirae (EhV 1 ). We found that 120° steps in each ATP hydrolysis event are divided into 40 and 80° substeps. In the main pause before the 40° substep and at low ATP concentration ([ATP]), the time constant was inversely proportional to [ATP], indicating that ATP binds during the main pause with a rate constant of 1.0 × 10 7 m -1 s -1 At high [ATP], we observed two [ATP]-independent time constants (0.5 and 0.7 ms). One of two time constants was prolonged (144 ms) in a rotation driven by slowly hydrolyzable ATPγS, indicating that ATP is cleaved during the main pause. In another subpause before the 80° substep, we noted an [ATP]-independent time constant (2.5 ms). Furthermore, in an ATP-driven rotation of an arginine-finger mutant in the presence of ADP, -80 and -40° backward steps were observed. The time constants of the pauses before -80° backward and +40° recovery steps were inversely proportional to [ADP] and [ATP], respectively, indicating that ADP- and ATP-binding events trigger these steps. Assuming that backward steps are reverse reactions, we conclude that 40 and 80° substeps are triggered by ATP binding and ADP release, respectively, and that the remaining time constant in the main pause represents phosphate release. We propose a chemo-mechanical coupling scheme of EhV 1 , including substeps largely different from those of F 1 -ATPases., (© 2019 Iida et al.)

Trichoderma reesei Cel6A (TrCel6A) is a cellobiohydrolase that hydrolyzes crystalline cellulose into cellobiose. Here we directly observed the reaction cycle (binding, surface movement, and dissociation) of single-molecule intact TrCel6A, isolated catalytic domain (CD), cellulose-binding module (CBM), and CBM and linker (CBM-linker) on crystalline cellulose I α The CBM-linker showed a binding rate constant almost half that of intact TrCel6A, whereas those of the CD and CBM were only one-tenth of intact TrCel6A. These results indicate that the glycosylated linker region largely contributes to initial binding on crystalline cellulose. After binding, all samples showed slow and fast dissociations, likely caused by the two different bound states due to the heterogeneity of cellulose surface. The CBM showed much higher specificity to the high affinity site than to the low affinity site, whereas the CD did not, suggesting that the CBM leads the CD to the hydrophobic surface of crystalline cellulose. On the cellulose surface, intact molecules showed slow processive movements (8.8 ± 5.5 nm/s) and fast diffusional movements (30-40 nm/s), whereas the CBM-Linker, CD, and a catalytically inactive full-length mutant showed only fast diffusional movements. These results suggest that both direct binding and surface diffusion contribute to searching of the hydrolysable point of cellulose chains. The duration time constant for the processive movement was 7.7 s, and processivity was estimated as 68 ± 42. Our results reveal the role of each domain in the elementary steps of the reaction cycle and provide the first direct evidence of the processive movement of TrCel6A on crystalline cellulose., (© 2016 by The American Society for Biochemistry and Molecular Biology, Inc.)

V-ATPase (V(o)V1) converts the chemical free energy of ATP into an ion-motive force across the cell membrane via mechanical rotation. This energy conversion requires proper interactions between the rotor and stator in V(o)V1 for tight coupling among chemical reaction, torque generation, and ion transport. We developed an Escherichia coli expression system for Enterococcus hirae V(o)V1 (EhV(o)V1) and established a single-molecule rotation assay to measure the torque generated. Recombinant and native EhV(o)V1 exhibited almost identical dependence of ATP hydrolysis activity on sodium ion and ATP concentrations, indicating their functional equivalence. In a single-molecule rotation assay with a low load probe at high ATP concentration, EhV(o)V1 only showed the "clear" state without apparent backward steps, whereas EhV1 showed two states, "clear" and "unclear." Furthermore, EhV(o)V1 showed slower rotation than EhV1 without the three distinct pauses separated by 120° that were observed in EhV1. When using a large probe, EhV(o)V1 showed faster rotation than EhV1, and the torque of EhV(o)V1 estimated from the continuous rotation was nearly double that of EhV1. On the other hand, stepping torque of EhV1 in the clear state was comparable with that of EhV(o)V1. These results indicate that rotor-stator interactions of the V(o) moiety and/or sodium ion transport limit the rotation driven by the V1 moiety, and the rotor-stator interactions in EhV(o)V1 are stabilized by two peripheral stalks to generate a larger torque than that of isolated EhV1. However, the torque value was substantially lower than that of other rotary ATPases, implying the low energy conversion efficiency of EhV(o)V1., (© 2014 by The American Society for Biochemistry and Molecular Biology, Inc.)

Trichoderma reesei cellobiohydrolase I (TrCel7A) is a molecular motor that directly hydrolyzes crystalline celluloses into water-soluble cellobioses. It has recently drawn attention as a tool that could be used to convert cellulosic materials into biofuel. However, detailed mechanisms of action, including elementary reaction steps such as binding, processive hydrolysis, and dissociation, have not been thoroughly explored because of the inherent challenges associated with monitoring reactions occurring at the solid/liquid interface. The crystalline cellulose Iα and IIII were previously reported as substrates with different crystalline forms and different susceptibilities to hydrolysis by TrCel7A. In this study, we observed that different susceptibilities of cellulose Iα and IIII are highly dependent on enzyme concentration, and at nanomolar enzyme concentration, TrCel7A shows similar rates of hydrolysis against cellulose Iα and IIII. Using single-molecule fluorescence microscopy and high speed atomic force microscopy, we also determined kinetic constants of the elementary reaction steps for TrCel7A against cellulose Iα and IIII. These measurements were performed at picomolar enzyme concentration in which density of TrCel7A on crystalline cellulose was very low. Under this condition, TrCel7A displayed similar binding and dissociation rate constants for cellulose Iα and IIII and similar fractions of productive binding on cellulose Iα and IIII. Furthermore, once productively bound, TrCel7A processively hydrolyzes and moves along cellulose Iα and IIII with similar translational rates. With structural models of cellulose Iα and IIII, we propose that different susceptibilities at high TrCel7A concentration arise from surface properties of substrate, including ratio of hydrophobic surface and number of available lanes., (© 2014 by The American Society for Biochemistry and Molecular Biology, Inc.)

V-ATPases are rotary molecular motors that generally function as proton pumps. We recently solved the crystal structures of the V1 moiety of Enterococcus hirae V-ATPase (EhV1) and proposed a model for its rotation mechanism. Here, we characterized the rotary dynamics of EhV1 using single-molecule analysis employing a load-free probe. EhV1 rotated in a counterclockwise direction, exhibiting two distinct rotational states, namely clear and unclear, suggesting unstable interactions between the rotor and stator. The clear state was analyzed in detail to obtain kinetic parameters. The rotation rates obeyed Michaelis-Menten kinetics with a maximal rotation rate (Vmax) of 107 revolutions/s and a Michaelis constant (Km) of 154 μM at 26 °C. At all ATP concentrations tested, EhV1 showed only three pauses separated by 120°/turn, and no substeps were resolved, as was the case with Thermus thermophilus V1-ATPase (TtV1). At 10 μM ATP (<>Km), the distribution of the durations of the catalytic pause was reproduced by a consecutive reaction with two time constants of 2.6 and 0.5 ms. These kinetic parameters were similar to those of TtV1. Our results identify the common properties of rotary catalysis of V1-ATPases that are distinct from those of F1-ATPases and will further our understanding of the general mechanisms of rotary molecular motors.

F(1)-ATPase (F(1)) is an ATP-driven rotary motor wherein the γ subunit rotates against the surrounding α(3)β(3) stator ring. The 3 catalytic sites of F(1) reside on the interface of the α and β subunits of the α(3)β(3) ring. While the catalytic residues predominantly reside on the β subunit, the α subunit has 1 catalytically critical arginine, termed the arginine finger, with stereogeometric similarities with the arginine finger of G-protein-activating proteins. However, the principal role of the arginine finger of F(1) remains controversial. We studied the role of the arginine finger by analyzing the rotation of a mutant F(1) with a lysine substitution of the arginine finger. The mutant showed a 350-fold longer catalytic pause than the wild-type; this pause was further lengthened by the slowly hydrolyzed ATP analog ATPγS. On the other hand, the mutant F(1) showed highly unidirectional rotation with a coupling ratio of 3 ATPs/turn, the same as wild-type, suggesting that cooperative torque generation by the 3 β subunits was not impaired. The hybrid F(1) carrying a single copy of the α mutant revealed that the reaction step slowed by the mutation occurs at +200° from the binding angle of the mutant subunit. Thus, the principal role of the arginine finger is not to mediate cooperativity among the catalytic sites, but to enhance the rate of the ATP cleavage by stabilizing the transition state of ATP hydrolysis. Lysine substitution also caused frequent pauses because of severe ADP inhibition, and a slight decrease in ATP-binding rate.

F(1)-ATPase (F(1)), a soluble portion of F(o)F(1)-ATP synthase (F(o)F(1)), is an ATP-driven motor in which gammaepsilon subunits rotate in the alpha(3)beta(3) cylinder. Activity of F(1) and F(o)F(1) from Bacillus PS3 is attenuated by the epsilon subunit in an inhibitory extended form. In this study we observed ATP-dependent transition of epsilon in single F(1) molecules from extended form to hairpin form by fluorescence resonance energy transfer. The results justify the previous bulk experiments and ensure that fraction of F(1) with hairpin epsilon directly determines the fraction of active F(1) at any ATP concentration. Next, mechanical activation and stiffness of epsilon-inhibited F(1) were examined by the forced rotation of magnetic beads attached to gamma. Compared with ADP inhibition, which is another manner of inhibition, rotation by a larger angle was required for the activation from epsilon inhibition when the beads were forced to rotate to ATP hydrolysis direction, and more torque was required to reach the same rotation angle when beads were forced to rotate to ATP synthesis direction. The results imply that if F(o)F(1) is resting in the epsilon-inhibited state, F(o) motor must transmit to gamma a torque larger than expected from thermodynamic equilibrium to initiate ATP synthesis.

F(1)-ATPase is a rotary molecular motor in which the gamma-subunit rotates against the alpha(3)beta(3) cylinder. The unitary gamma-rotation is a 120 degrees step comprising 80 and 40 degrees substeps, each of these initiated by ATP binding and ADP release and by ATP hydrolysis and inorganic phosphate release, respectively. In our previous study on gamma-rotation at low temperatures, a highly temperature-sensitive (TS) reaction step of F(1)-ATPase from thermophilic Bacillus PS3 was found below 9 degrees C as an intervening pause before the 80 degrees substep at the same angle for ATP binding and ADP release. However, it remains unclear as to which reaction step the TS reaction corresponds. In this study, we found that the mutant F(1)(beta E190D) from thermophilic Bacillus PS3 showed a clear pause of the TS reaction below 18 degrees C. In an attempt to identify the catalytic state of the TS reaction, the rotation of the hybrid F(1), carrying a single copy of beta E190D, was observed at 18 degrees C. The hybrid F(1) showed a pause of the TS reaction at the same angle as for the ATP binding of the incorporated beta E190D, although kinetic analysis revealed that the TS reaction is not the ATP binding step. These findings suggest that the TS reaction is a structural rearrangement of beta before or after ATP binding.

The epsilon subunit of bacterial FoF1-ATP synthase (FoF1), a rotary motor protein, is known to inhibit the ATP hydrolysis reaction of this enzyme. The inhibitory effect is modulated by the conformation of the C-terminal alpha-helices of epsilon, and the "extended" but not "hairpin-folded" state is responsible for inhibition. Although the inhibition of ATP hydrolysis by the C-terminal domain of epsilon has been extensively studied, the effect on ATP synthesis is not fully understood. In this study, we generated an Escherichia coli FoF1 (EFoF1) mutant in which the epsilon subunit lacked the C-terminal domain (FoF1epsilonDeltaC), and ATP synthesis driven by acid-base transition (DeltapH) and the K+-valinomycin diffusion potential (DeltaPsi) was compared in detail with that of the wild-type enzyme (FoF1epsilonWT). The turnover numbers (kcat) of FoF1epsilonWT were severalfold lower than those of FoF1epsilonDeltaC. FoF1epsilonWT showed higher Michaelis constants (Km). The dependence of the activities of FoF1epsilonWT and FoF1epsilonDeltaC on various combinations of DeltapH and DeltaPsi was similar, suggesting that the rate-limiting step in ATP synthesis was unaltered by the C-terminal domain of epsilon. Solubilized FoF1epsilonWT also showed lower kcat and higher Km values for ATP hydrolysis than the corresponding values of FoF1epsilonDeltaC. These results suggest that the C-terminal domain of the epsilon subunit of EFoF1 slows multiple elementary steps in both the ATP synthesis/hydrolysis reactions by restricting the rotation of the gamma subunit.

It has been proposed that C-terminal two alpha-helices of the epsilon subunit of F1-ATPase can undergo conformational transition between retracted folded-hairpin form and extended form. Here, using F(1) from thermophilic Bacillus PS3, we monitored this transition in real time by fluorescence resonance energy transfer (FRET) between a donor dye and an acceptor dye attached to N terminus of the gamma subunit and C terminus of the epsilon subunit, respectively. High FRET (extended form) of F1 turned to low FRET (retracted form) by ATP, which then reverted as ATP was hydrolyzed to ADP. 5'-Adenyl-beta,gamma-imidodiphosphate, ADP + AlF4-, ADP + NaN3, and GTP also caused the retracted form, indicating that ATP binding to the catalytic beta subunits induces the transition. The ATP-induced transition from high FRET to low FRET occurred in a similar time scale to the ATP-induced activation of ATPase from inhibition by the epsilon subunit, although detailed kinetics were not the same. The transition became faster as temperature increased, but the extrapolated rate at 65 degrees C (physiological temperature of Bacillus PS3) was still too slow to assign the transition as an obligate step in the catalytic turnover. Furthermore, binding affinity of ATP to the isolated epsilon subunit was weakened as temperature increased, and the dissociation constant extrapolated to 65 degrees C reached to 0.67 mm, a consistent value to assume that the epsilon subunit acts as a sensor of ATP concentration in the cell.

The epsilon subunit in F0F1-ATPase/synthase undergoes drastic conformational rearrangement, which involves the transition of two C-terminal helices between a hairpin "down"-state and an extended "up"-state, and the enzyme with the up-fixed epsilon cannot catalyze ATP hydrolysis but can catalyze ATP synthesis (Tsunoda, S. P., Rodgers, A. J. W., Aggeler, R., Wilce, M. C. J., Yoshida, M., and Capaldi, R. A. (2001) Proc. Natl. Acad. Sci. U. S. A. 98, 6560-6564). Here, using cross-linking between introduced cysteine residues as a probe, we have investigated the causes of the transition. Our findings are as follows. (i) In the up-state, the two helices of epsilon are fully extended to insert the C terminus into a deeper position in the central cavity of F1 than was thought previously. (ii) Without a nucleotide, epsilon is in the up-state. ATP induces the transition to the down-state, and ADP counteracts the action of ATP. (iii) Conversely, the enzyme with the down-state epsilon can bind an ATP analogue, 2',3'-O-(2,4,6-trinitrophenyl)-ATP, much faster than the enzyme with the up-state epsilon. (iv) Proton motive force stabilizes the up-state. Thus, responding to the increase of proton motive force and ADP, F0F1-ATPase/synthase would transform the epsilon subunit into the up-state conformation and change gear to the mode for ATP synthesis.