Analysis of proton wires in the enzyme active site suggests a mechanism of c‐di‐GMP hydrolysis by the EAL domain phosphodiesterases

Grigorenko, Bella L.; Knyazeva, M. A.; Nemukhin, Alexander V.

doi:10.1002/prot.25108

Cited by 8 publications

(11 citation statements)

References 49 publications

(138 reference statements)

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“…The DgcC amino acid sequence includes a catalytic center motif (the “A-site” or GGDEF ) and an intact “I-site” motif ( R SG D ; conferring potential product inhibition) as characteristic for DGCs [ 34 ] and also the EAL domain of PdeK contains the amino acids crucially involved in PDE activity [ [35] , [36] , [37] , [38] ]. Nevertheless, we wanted to show their enzymatic activities directly in vitro , which is not trivial since both proteins are membrane-integrated.…”

Section: Resultsmentioning

confidence: 99%

Local c-di-GMP Signaling in the Control of Synthesis of the E. coli Biofilm Exopolysaccharide pEtN-Cellulose

Richter

Possling

Malysheva

et al. 2020

Journal of Molecular Biology

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In many bacteria, the biofilm-promoting second messenger c-di-GMP is produced and degraded by multiple diguanylate cyclases (DGC) and phosphodiesterases (PDE), respectively. High target specificity of some of these enzymes has led to theoretical concepts of “local” c-di-GMP signaling. In Escherichia coli K-12, which has 12 DGCs and 13 PDEs, a single DGC, DgcC, is specifically required for the biosynthesis of the biofilm exopolysaccharide pEtN-cellulose without affecting the cellular c-di-GMP pool, but the mechanistic basis of this target specificity has remained obscure. DGC activity of membrane-associated DgcC, which is demonstrated in vitro in nanodiscs, is shown to be necessary and sufficient to specifically activate cellulose biosynthesis in vivo . DgcC and a particular PDE, PdeK (encoded right next to the cellulose operon), directly interact with cellulose synthase subunit BcsB and with each other, thus establishing physical proximity between cellulose synthase and a local source and sink of c-di-GMP. This arrangement provides a localized, yet open source of c-di-GMP right next to cellulose synthase subunit BcsA, which needs allosteric activation by c-di-GMP. Through mathematical modeling and simulation, we demonstrate that BcsA binding from the low cytosolic c-di-GMP pool in E. coli is negligible, whereas a single c-di-GMP molecule that is produced and released in direct proximity to cellulose synthase increases the probability of c-di-GMP binding to BcsA several hundred-fold. This local c-di-GMP signaling could provide a blueprint for target-specific second messenger signaling also in other bacteria where multiple second messenger producing and degrading enzymes exist.

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Section: Resultsmentioning

confidence: 99%

Local c-di-GMP Signaling in the Control of Synthesis of the E. coli Biofilm Exopolysaccharide pEtN-Cellulose

Richter

Possling

Malysheva

et al. 2020

Journal of Molecular Biology

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“…The importance of proton wires in protein systems is documented in previous works. [27][28][29][30][31][32][33][34] Simulations described here extend the experience with proton wires in the two-metal catalysis.…”

Section: Summary and Additional Commentsmentioning

confidence: 54%

Mechanisms of ATP to cAMP Conversion Catalyzed by the Mammalian Adenylyl Cyclase: A Role of Magnesium Coordination Shells and Proton Wires

Grigorenko¹,

Polyakov²,

Nemukhin

2019

Preprint

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We report a mechanism of adenosine triphosphate (ATP) to cyclic adenosine monophosphate (cAMP) conversion by the mammalian type V adenylyl cyclase revealed in molecular dynamics (MD) and quantum mechanics/molecular mechanics (QM/MM) simulations. We characterize a set of computationally derived enzyme-substrate (ES) structures showing an important role of coordination shells of magnesium ions in the solvent accessible active site. Several stable six-fold coordination shells of Mg A 2+ are observed in MD simulations of ES complexes. In the lowest energy ES conformation, the coordination shell of Mg A 2+ does not include the O δ1 atom of the conserved Asp440 residue. Starting from this conformation, a one-step reaction mechanism is characterized which includes proton transfer from the ribose O 3' H 3' group in ATP to Asp440 via a shuttling water molecule and P A -O 3A bond cleavage and O 3' -P A bond formation. The energy profile of this route is consistent with the observed reaction kinetics. In a higher energy ES conformation, Mg A 2+ is bound to the O δ1 (Asp440) atom as suggested in the relevant crystal structure of the protein with a substrate analog. The computed energy profile initiated by this ES is characterized by higher energy expenses to complete the reaction. Consistently with experimental data, we show that the Asp440Ala mutant of the enzyme should exhibit a reduced but retained activity. All considered reaction pathways include proton wires from the O 3' H 3' group via shuttling water molecules.

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“…Such bridges (water wires) can conduct protons and effectively support proton‐coupled electron transfer mechanism. Because of this effect, water wires were recognized to be often essential for enzyme efficiency . In fact, it has been shown that water molecules are as crucial for proton transport and biological functions of proteins as are amino acids …”

Section: Resultsmentioning

confidence: 99%

“…Because of this effect, water wires were recognized to be often essential for enzyme efficiency. 27,28 In fact, it has been shown that water molecules are as crucial for proton transport and biological functions of proteins as are amino acids. 5 Our previous studies showed that electromagnetic radiation in the infrared range had the ability to enhance alignment of water molecules near protein interfaces, by increasing strength and cooperativeness of H-bonds, 7 which further affects protein-protein and protein-surface interactions.…”

Section: Resultsmentioning

confidence: 99%

Beyond esterase‐like activity of serum albumin. Histidine‐(nitro)phenol radical formation in conversion cascade of p‐nitrophenyl acetate and the role of infrared light

Kowacz

Warszyński

2019

J of Molecular Recognition

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Serum albumin, recognized mainly for its capacity to act as a carrier protein for many compounds, can also actively transform some organic molecules. As a starting point in this study, we consider esterase‐like activity of bovine serum albumin (BSA) toward p‐nitrophenyl acetate (p‐NPA). Our results reveal that the reaction goes beyond ester hydrolysis step. In fact, the transformation product, p‐nitrophenol (p‐NP), becomes a substrate for further reaction with BSA in which its nitro group in subtracted and released in the form of HNO2. Spectral data indicate that this cascade of events proceeds through formation of phenoxyl radical via proton‐coupled electron transport (PCET) between OH group of p‐NP and imidazole ring of histidine from the protein. Furthermore, the effect of application of electromagnetic radiation in the infrared range suggests that this remote physical trigger can support interactions based on PCET mechanism by acting on polarization and mutual alignment of water dipoles serving as effective water wires.

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Analysis of proton wires in the enzyme active site suggests a mechanism of c‐di‐GMP hydrolysis by the EAL domain phosphodiesterases

Cited by 8 publications

References 49 publications

Local c-di-GMP Signaling in the Control of Synthesis of the E. coli Biofilm Exopolysaccharide pEtN-Cellulose

Local c-di-GMP Signaling in the Control of Synthesis of the E. coli Biofilm Exopolysaccharide pEtN-Cellulose

Mechanisms of ATP to cAMP Conversion Catalyzed by the Mammalian Adenylyl Cyclase: A Role of Magnesium Coordination Shells and Proton Wires

Beyond esterase‐like activity of serum albumin. Histidine‐(nitro)phenol radical formation in conversion cascade of p‐nitrophenyl acetate and the role of infrared light

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