scholarly journals A Bacteroidetes locus dedicated to fungal 1,6-β-glucan degradation: Unique substrate conformation drives specificity of the key endo-1,6-β-glucanase

2017 ◽  
Vol 292 (25) ◽  
pp. 10639-10650 ◽  
Author(s):  
Max J. Temple ◽  
Fiona Cuskin ◽  
Arnaud Baslé ◽  
Niall Hickey ◽  
Gaetano Speciale ◽  
...  
Keyword(s):  
Substrate Conformation

A Dynamic Substrate is Required for MhuD-catalyzed Degradation of Heme to Mycobilin

2021 ◽  
Author(s):  
Biswash Thakuri ◽  
Bruce O'Rourke ◽  
Amanda Graves ◽  
Matthew Liptak
Keyword(s):  
Dynamic Equilibrium ◽  
Ionization Mass ◽  
Esi Ms ◽  
Enzymatic Mechanism ◽  
Single Turnover ◽  
Rate Limiting Step ◽  
Planar Substrate ◽  
Out Of Plane ◽  
Substrate Conformation ◽  
Rate Limiting

The non-canoncial heme oxygenase MhuD from <i>Mycobacterium tuberculosis</i> binds a heme substrate that adopts a dynamic equilibrium between planar and out-of-plane ruffled conformations. MhuD degrades this substrate to an unusual mycobilin product via successive monooxygenation and dioxygenation reactions. This article establishes a causal relationship between heme substrate dynamics and MhuD-catalyzed heme degradation resulting in a refined enzymatic mechanism. UV/Vis absorption (Abs) and electrospray ionization mass spectrometry (ESI-MS) data demonstrated that a second-sphere substitution favoring population of the ruffled heme conformation changed the rate-limiting step of the reaction resulting in a measurable build-up of the monooxygenated meso-hydroxyheme intermediate. In addition, UV/Vis Abs and ESI-MS data for a second-sphere variant that favored the planar substrate conformation showed that this change altered the enzymatic mechanism resulting in an alpha-biliverdin product. Single-turnover kinetic analyses for three MhuD variants revealed that the rate of heme monooxygenation depends upon the population of the ruffled substrate conformation. These kinetic analyses also revealed that the rate of meso-hydroxyheme dioxygenation by MhuD depends upon the population of the planar substrate conformation. Thus, the ruffled haem conformation supports rapid heme monooxygenation by MhuD, but further oxygenation to the mycobilin product is inhibited. In contrast, the planar substrate conformation exhibits altered heme monooxygenation regiospecificity followed by rapid oxygenation of meso-hydroxyheme. Altogether, these data yielded a refined enzymatic mechanism for MhuD where access to both substrate conformations is needed for rapid incorporation of three oxygen atoms into heme yielding mycobilin.<br>

scholarly journals Substrate Conformation Correlates with the Outcome of Hyoscyamine 6β-Hydroxylase Catalyzed Oxidation Reactions

2018 ◽  
Vol 140 (24) ◽  
pp. 7433-7436 ◽  
Author(s):  
Richiro Ushimaru ◽  
Mark W. Ruszczycky ◽  
Wei-chen Chang ◽  
Feng Yan ◽  
Yung-nan Liu ◽  
...  
Keyword(s):  
Oxidation Reactions ◽  
Substrate Conformation ◽  
Catalyzed Oxidation

scholarly journals Role of the Substrate Conformation and of the S1 Protein in the Cleavage Efficiency of the T4 Endoribonuclease RegB

2000 ◽  
Vol 276 (16) ◽  
pp. 13264-13272 ◽  
Author(s):  
Isabelle Lebars ◽  
Rouh-Mei Hu ◽  
Jean-Yves Lallemand ◽  
Marc Uzan ◽  
François Bontems
Keyword(s):  
Cleavage Efficiency ◽  
Substrate Conformation

A Dynamic Substrate is Required for MhuD-catalyzed Degradation of Heme to Mycobilin

2020 ◽  
Author(s):  
Biswash Thakuri ◽  
Bruce O'Rourke ◽  
Amanda Graves ◽  
Matthew Liptak
Keyword(s):  
Dynamic Equilibrium ◽  
Ionization Mass ◽  
Esi Ms ◽  
Enzymatic Mechanism ◽  
Single Turnover ◽  
Rate Limiting Step ◽  
Planar Substrate ◽  
Out Of Plane ◽  
Substrate Conformation ◽  
Rate Limiting

The non-canoncial heme oxygenase MhuD from <i>Mycobacterium tuberculosis</i> binds a heme substrate that adopts a dynamic equilibrium between planar and out-of-plane ruffled conformations. MhuD degrades this substrate to an unusual mycobilin product via successive monooxygenation and dioxygenation reactions. This article establishes a causal relationship between heme substrate dynamics and MhuD-catalyzed heme degradation resulting in a revised enzymatic mechanism. UV/Vis absorption (Abs) and electrospray ionization mass spectrometry (ESI-MS) data demonstrated that a second-sphere substitution favoring population of the ruffled heme conformation changed the rate-limiting step of the reaction resulting in a measurable build-up of the monooxygenated meso-hydroxyheme intermediate. In addition, UV/Vis Abs and ESI-MS data for a second-sphere variant that favored the planar substrate conformation showed that this change altered the enzymatic mechanism resulting in an alpha-biliverdin product. Single-turnover kinetic analyses for three MhuD variants revealed that the rate of heme monooxygenation depends upon the population of the ruffled substrate conformation. These kinetic analyses also revealed that the rate of meso-hydroxyheme dioxygenation by MhuD depends upon the population of the planar substrate conformation. Thus, the ruffled haem conformation supports rapid heme monooxygenation by MhuD, but further oxygenation to the mycobilin product is inhibited. In contrast, the planar substrate conformation exhibits altered heme monooxygenation regiospecificity followed by rapid oxygenation of meso-hydroxyheme. Altogether, these data yielded a revised enzymatic mechanism for MhuD where access to both substrate conformations is needed for rapid incorporation of three oxygen atoms into heme yielding mycobilin.<br>

Nanostructured substrate conformation can decrease osteoblast-like cell dysfunction in simulated microgravity conditions

2012 ◽  
Vol 8 (12) ◽  
pp. 978-988 ◽  
Author(s):  
Ljupcho Prodanov ◽  
Jack J. W. A. van Loon ◽  
Joost te Riet ◽  
John A. Jansen ◽  
X. Frank Walboomers
Keyword(s):  
Simulated Microgravity ◽  
Cell Dysfunction ◽  
Microgravity Conditions ◽  
Substrate Conformation

scholarly journals Structural and functional analysis of Oceanobacillus iheyensis macrodomain reveals a network of waters involved in substrate binding and catalysis

Open Biology ◽  
2017 ◽  
Vol 7 (4) ◽  
pp. 160327 ◽  
Author(s):  
Rubén Zapata-Pérez ◽  
Fernando Gil-Ortiz ◽  
Ana Belén Martínez-Moñino ◽  
Antonio Ginés García-Saura ◽  
Jordi Juanhuix ◽  
...  
Keyword(s):  
Functional Analysis ◽  
Enzyme Activity ◽  
Substrate Binding ◽  
Therapeutic Agents ◽  
Water Molecules ◽  
Substrate Conformation ◽  
Coordinated Water ◽  
Alkaliphilic Bacterium ◽  
Hydrolysis Of

Macrodomains are ubiquitous conserved domains that bind or transform ADP-ribose (ADPr) metabolites. In humans, they are involved in transcription, X-chromosome inactivation, neurodegeneration and modulating PARP1 signalling, making them potential targets for therapeutic agents. Unfortunately, some aspects related to the substrate binding and catalysis of MacroD-like macrodomains still remain unclear, since mutation of the proposed catalytic aspartate does not completely abolish enzyme activity. Here, we present a functional and structural characterization of a macrodomain from the extremely halotolerant and alkaliphilic bacterium Oceanobacillus iheyensis (OiMacroD), related to hMacroD1/hMacroD2, shedding light on substrate binding and catalysis. The crystal structures of D40A, N30A and G37V mutants, and those with MES, ADPr and ADP bound, allowed us to identify five fixed water molecules that play a significant role in substrate binding. Closure of the β6–α4 loop is revealed as essential not only for pyrophosphate recognition, but also for distal ribose orientation. In addition, a novel structural role for residue D40 is identified. Furthermore, it is revealed that OiMacroD not only catalyses the hydrolysis of O -acetyl-ADP-ribose but also reverses protein mono-ADP-ribosylation. Finally, mutant G37V supports the participation of a substrate-coordinated water molecule in catalysis that helps to select the proper substrate conformation.

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