Identification of a Long-range Protein Network That Modulates Active Site Dynamics in Extremophilic Alcohol Dehydrogenases

Nagel, Zachary D.; Cun, Shujian; Klinman, Judith P.

doi:10.1074/jbc.m113.453951

Cited by 44 publications

(78 citation statements)

References 49 publications

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“…Studies of alcohol dehydrogenases have shown that longrange conformation selection is important in catalysis (111). The work presented here suggests that long-range conformational interactions may also play an important role in the PSII reaction center.…”

Section: Discussionmentioning

confidence: 82%

An Intrinsically Disordered Photosystem II Subunit, PsbO, Provides a Structural Template and a Sensor of the Hydrogen-bonding Network in Photosynthetic Water Oxidation

Offenbacher

Polander

Barry

2013

Journal of Biological Chemistry

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Background:PsbO is an intrinsically disordered subunit of photosystem II. Results: Temperature-sensitive PsbO dynamics are identified by reaction-induced Fourier transform infrared spectroscopy and the removal and reconstitution of PsbO. Conclusion: PsbO serves as an organizational template and undergoes flash-induced hydrogen-bonding changes, coupled with the catalytic cycle of water oxidation. Significance: PsbO samples a rough conformational landscape when bound to its target, the photosystem II reaction center.

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Section: Discussionmentioning

confidence: 82%

An Intrinsically Disordered Photosystem II Subunit, PsbO, Provides a Structural Template and a Sensor of the Hydrogen-bonding Network in Photosynthetic Water Oxidation

Offenbacher

Polander

Barry

2013

Journal of Biological Chemistry

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“…As indicated in Figure 9, an Arrhenius plot of the transformed data clearly shows the non-linearity of the rate constant with respect to temperature. Specifically, two transition temperatures are observed, indicating a strong coupling between conformational dynamics and activation energy [48-54]. There are two transition points along the Arrhenius plot, with one around 28.3 °C and another around 34.8 °C.…”

Section: Resultsmentioning

confidence: 99%

“…A convex Arrhenius plot indicates a decreasing activation energy with increasing temperature [51], as is observed for the Mtu RecA mini -intein in the 30.5 °C to 35 °C temperature range. In many cases, the lowest-energy states in enzyme catalysis can be conformationally flexible and are favorable at higher temperatures, leading to observed biphasic rate constants with changes in temperature [48-54]. For example, wild-type tetrameric thermophilic alcohol dehydrogenase from Bacillus stearothermophilus (ht-ADH) has small kink in its Arrhenius plot [54], indicating an obvious yet weak change in activation energy.…”

Section: Discussionmentioning

confidence: 99%

Dynamics differentiate between active and inactive inteins

Cronin

Coolbaugh

Nellis

et al. 2015

European Journal of Medicinal Chemistry

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The balance between stability and dynamics for active enzymes can be somewhat quantified by studies of intein splicing and cleaving reactions. Inteins catalyze the ligation of flanking host exteins while excising themselves. The potential for applications led to engineering of a mini-intein splicing domain, where the homing endonuclease domain of the Mycobacterium tuberculosis RecA (Mtu recA) intein was removed. The remaining domains were linked by several short peptides, but splicing activity in all was substantially lower than the full-length intein. Native splicing activity was restored in some cases by a V67L mutation. Using computations and experiments, we examine the impact of this mutation on the stability and conformational dynamics of the mini-intein splicing domain. Molecular dynamics simulations were used to delineate the factors that determine the active state, including the V67L mini-intein mutant, and peptide linker. We found that (1) the V67L mutation lowers the global fluctuations in all modeled mini-inteins, stabilizing the mini-intein constructs; (2) the connecting linker length affects intein dynamics; and (3) the flexibilities of the linker and intein core are higher in the active structure. We have observed that the interaction of the linker region and a turn region around residues 35-41 provides the pathway for the allostery interaction. Our experiments reveal that intein catalysis is characterized by non-linear Arrhenius plot, confirming the significant contribution of protein conformational dynamics to intein function. We conclude that while the V67L mutation stabilizes the global structure, cooperative dynamics of all intein regions appear more important for intein function than high stability. Our studies suggest that effectively quenching the conformational dynamics of an intein through engineered allosteric interactions could deactivate intein splicing or cleaving.

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“…Previously, the use of a bulky substrate benzyl alcohol in primary H/D substrate kinetic isotope effect (KIE) measurements revealed a “breakpoint” for the temperature dependence, where the KIE was largely temperature independent above 30 °C but increased sharply at lower temperatures 7. Site‐directed mutagenesis and hydrogen–deuterium exchange studies showed that the flexibility of a group of residues greatly affects the temperature dependence of the substrate KIE 6b, 7a,7b,7d. It has been suggested that these residues are responsible for modifying the distance between hydride donor and acceptor 8.…”

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confidence: 99%

Isotope Substitution of Promiscuous Alcohol Dehydrogenase Reveals the Origin of Substrate Preference in the Transition State

et al. 2018

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The origin of substrate preference in promiscuous enzymes was investigated by enzyme isotope labelling of the alcohol dehydrogenase from Geobacillus stearothermophilus (BsADH). At physiological temperature, protein dynamic coupling to the reaction coordinate was insignificant. However, the extent of dynamic coupling was highly substrate‐dependent at lower temperatures. For benzyl alcohol, an enzyme isotope effect larger than unity was observed, whereas the enzyme isotope effect was close to unity for isopropanol. Frequency motion analysis on the transition states revealed that residues surrounding the active site undergo substantial displacement during catalysis for sterically bulky alcohols. BsADH prefers smaller substrates, which cause less protein friction along the reaction coordinate and reduced frequencies of dynamic recrossing. This hypothesis allows a prediction of the trend of enzyme isotope effects for a wide variety of substrates.

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Identification of a Long-range Protein Network That Modulates Active Site Dynamics in Extremophilic Alcohol Dehydrogenases

Cited by 44 publications

References 49 publications

An Intrinsically Disordered Photosystem II Subunit, PsbO, Provides a Structural Template and a Sensor of the Hydrogen-bonding Network in Photosynthetic Water Oxidation

An Intrinsically Disordered Photosystem II Subunit, PsbO, Provides a Structural Template and a Sensor of the Hydrogen-bonding Network in Photosynthetic Water Oxidation

Dynamics differentiate between active and inactive inteins

Isotope Substitution of Promiscuous Alcohol Dehydrogenase Reveals the Origin of Substrate Preference in the Transition State

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