Dynamism and Cooperativity Essential for Efficient Catalysis in Aspergillus Niger Monoamine Oxidase

10.26434/chemrxiv.7707857 ◽

2019 ◽

Author(s):

Christian Curado-Carballada ◽

Ferran Feixas ◽

Sílvia Osuna

Keyword(s):

Aspergillus Niger ◽

Monoamine Oxidase ◽

Active Site ◽

Conformational Dynamics ◽

Catalytic Efficiency ◽

Building Blocks ◽

Chiral Amine ◽

Evolutionary Pathway ◽

Accelerated Molecular Dynamics ◽

Open States

Aspergillus niger Monoamine Oxidase (MAO-N) is a homodimeric enzyme responsible for the oxidation of amines into the corresponding imine. Laboratory evolved variants of MAO-N in combination with a non-selective chemical reductant represents a powerful strategy for the deracemisation of chiral amine mixtures and, thus, is of interest for obtaining chiral amine building blocks. MAO-N presents a rich conformational dynamics with a flexible ß-hairpin region that can adopt closed, partially closed and open states. Despite the ß-hairpin conformational dynamics is altered along the laboratory evolutionary pathway of MAO-N, the connection between the ß-hairpin conformational dynamics and active site catalysis still remains unclear. In this work, we use accelerated molecular dynamics to elucidate the potential interplay between the ß-hairpin conformational dynamics and catalytic activity in MAO-N wild type and its evolved D5 variant. Our study reveals a delicate communication between both MAO-N subunits that impacts the active site architecture, and thus its catalytic efficiency. In both MAO-N WT and the laboratory evolved D5 variant, the ß-hairpin conformation in one of the monomers affects the productive binding of the substrate in the active site of the other subunit. However, both MAO-N WT and D5 variants show a quite different behaviour due to the distal mutations introduced experimentally with Directed Evolution.

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Hidden Conformations in Aspergillus niger Monoamine Oxidase are Key for Catalytic Efficiency

Angewandte Chemie International Edition ◽

10.1002/anie.201812532 ◽

2019 ◽

Vol 58 (10) ◽

pp. 3097-3101 ◽

Cited By ~ 5

Author(s):

Christian Curado‐Carballada ◽

Ferran Feixas ◽

Javier Iglesias‐Fernández ◽

Sílvia Osuna

Keyword(s):

Aspergillus Niger ◽

Monoamine Oxidase ◽

Catalytic Efficiency

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Conserved Conformational Hierarchy across Functionally Divergent Glycosyltransferases of the GT-B Structural Superfamily as Determined from Microsecond Molecular Dynamics

International Journal of Molecular Sciences ◽

10.3390/ijms22094619 ◽

2021 ◽

Vol 22 (9) ◽

pp. 4619

Author(s):

Carlos A. Ramirez-Mondragon ◽

Megin E. Nguyen ◽

Jozafina Milicaj ◽

Bakar A. Hassan ◽

Frank J. Tucci ◽

...

Keyword(s):

Molecular Dynamics ◽

Communication Networks ◽

Active Site ◽

Conformational Dynamics ◽

Catalytic Efficiency ◽

Potential Effect ◽

Structural Class ◽

Terminal Domain ◽

Cross Correlation Analysis ◽

Dynamics Simulations

It has long been understood that some proteins undergo conformational transitions en route to the Michaelis Complex to allow chemistry. Examination of crystal structures of glycosyltransferase enzymes in the GT-B structural class reveals that the presence of ligand in the active site triggers an open-to-closed conformation transition, necessary for their catalytic functions. Herein, we describe microsecond molecular dynamics simulations of two distantly related glycosyltransferases that are part of the GT-B structural superfamily, HepI and GtfA. Simulations were performed using the open and closed conformations of these unbound proteins, respectively, and we sought to identify the major dynamical modes and communication networks that interconnect the open and closed structures. We provide the first reported evidence within the scope of our simulation parameters that the interconversion between open and closed conformations is a hierarchical multistep process which can be a conserved feature of enzymes of the same structural superfamily. Each of these motions involves of a collection of smaller molecular reorientations distributed across both domains, highlighting the complexities of protein dynamic involved in the interconversion process. Additionally, dynamic cross-correlation analysis was employed to explore the potential effect of distal residues on the catalytic efficiency of HepI. Multiple distal nonionizable residues of the C-terminal domain exhibit motions anticorrelated to positively charged residues in the active site in the N-terminal domain involved in substrate binding. Mutations of these residues resulted in a reduction in negatively correlated motions and an altered enzymatic efficiency that is dominated by lower Km values with kcat effectively unchanged. The findings suggest that residues with opposing conformational motions involved in the opening and closing of the bidomain HepI protein can allosterically alter the population and conformation of the “closed” state, essential to the formation of the Michaelis complex. The stabilization effects of these mutations likely equally influence the energetics of both the ground state and the transition state of the catalytic reaction, leading to the unaltered kcat. Our study provides new insights into the role of conformational dynamics in glycosyltransferase’s function and new modality to modulate enzymatic efficiency.

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Simulating Multiple Substrate Binding Events by γ-Glutamyltransferase using Accelerated Molecular Dynamics

10.1101/2020.04.20.050500 ◽

2020 ◽

Author(s):

Francesco Oliva ◽

Jose C. Flores-Canales ◽

Stefano Pieraccini ◽

Carlo F. Morelli ◽

Maurizio Sironi ◽

...

Keyword(s):

Molecular Dynamics ◽

Electrostatic Interactions ◽

Substrate Binding ◽

Conformational Dynamics ◽

Binding Pocket ◽

Thermal Fluctuations ◽

Accelerated Molecular Dynamics ◽

Binding Cavity ◽

Multiple Substrate ◽

Open States

Abstractγ-glutamyltransferase (GGT) is an enzyme that uses γ-glutamyl compounds as substrate and catalyzes their transfer into a water molecule or an acceptor substrate with varied physiological-function in bacteria, plants and animals. Crystal structures of GGT are known for different species and in different states of the chemical reaction; however, structural dynamics of the substrate binding to the catalytic site of GGT is unknown. Here, we modeled Escherichia Coli GGT’s glutamine binding by using a swarm of accelerated molecular dynamics (aMD) simulations. Characterization of multiple binding events identified three structural binding motifs composed of polar residues in the binding pocket that govern glutamine binding into the active site. Simulated open and closed conformations of a lid-loop protecting the binding cavity suggests its role as a gating element by allowing or blocking substrates entry into the binding pocket. Partially open states of the lid-loop are accessible within thermal fluctuations, while the estimated free energy cost of a complete open state is 2.4 kcal/mol. Our results suggest that both specific electrostatic interactions and GGT conformational dynamics dictate the molecular recognition of substrate-GGT complexes.

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Mix-and-inject XFEL crystallography reveals gated conformational dynamics during enzyme catalysis

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1901864116 ◽

2019 ◽

Vol 116 (51) ◽

pp. 25634-25640 ◽

Cited By ~ 10

Author(s):

Medhanjali Dasgupta ◽

Dominik Budday ◽

Saulo H. P. de Oliveira ◽

Peter Madzelan ◽

Darya Marchany-Rivera ◽

...

Keyword(s):

Active Site ◽

Enzyme Catalysis ◽

Conformational Dynamics ◽

Catalytic Efficiency ◽

Enzyme Mechanism ◽

Time Resolved ◽

X Ray ◽

Protein Motions ◽

X Ray Crystallography ◽

Active Site Cysteine

How changes in enzyme structure and dynamics facilitate passage along the reaction coordinate is a fundamental unanswered question. Here, we use time-resolved mix-and-inject serial crystallography (MISC) at an X-ray free electron laser (XFEL), ambient-temperature X-ray crystallography, computer simulations, and enzyme kinetics to characterize how covalent catalysis modulates isocyanide hydratase (ICH) conformational dynamics throughout its catalytic cycle. We visualize this previously hypothetical reaction mechanism, directly observing formation of a thioimidate covalent intermediate in ICH microcrystals during catalysis. ICH exhibits a concerted helical displacement upon active-site cysteine modification that is gated by changes in hydrogen bond strength between the cysteine thiolate and the backbone amide of the highly strained Ile152 residue. These catalysis-activated motions permit water entry into the ICH active site for intermediate hydrolysis. Mutations at a Gly residue (Gly150) that modulate helical mobility reduce ICH catalytic turnover and alter its pre-steady-state kinetic behavior, establishing that helical mobility is important for ICH catalytic efficiency. These results demonstrate that MISC can capture otherwise elusive aspects of enzyme mechanism and dynamics in microcrystalline samples, resolving long-standing questions about the connection between nonequilibrium protein motions and enzyme catalysis.

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Hidden Conformations in Aspergillus niger Monoamine Oxidase are Key for Catalytic Efficiency

Angewandte Chemie ◽

10.1002/ange.201812532 ◽

2019 ◽

Vol 131 (10) ◽

pp. 3129-3133

Author(s):

Christian Curado‐Carballada ◽

Ferran Feixas ◽

Javier Iglesias‐Fernández ◽

Sílvia Osuna

Keyword(s):

Aspergillus Niger ◽

Monoamine Oxidase ◽

Catalytic Efficiency

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Molecular Dynamics Simulations on Aspergillus niger Monoamine Oxidase: Conformational Dynamics and Inter‐monomer Communication Essential for Its Efficient Catalysis

Advanced Synthesis & Catalysis ◽

10.1002/adsc.201900158 ◽

2019 ◽

Author(s):

Christian Curado‐Carballada ◽

Ferran Feixas ◽

Sílvia Osuna

Keyword(s):

Molecular Dynamics ◽

Aspergillus Niger ◽

Monoamine Oxidase ◽

Molecular Dynamics Simulations ◽

Conformational Dynamics ◽

Dynamics Simulations

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Correlation of membrane protein conformational and functional dynamics

Nature Communications ◽

10.1038/s41467-021-24660-1 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Raghavendar Reddy Sanganna Gari ◽

Joel José Montalvo‐Acosta ◽

George R. Heath ◽

Yining Jiang ◽

Xiaolong Gao ◽

...

Keyword(s):

Membrane Protein ◽

Conformational Changes ◽

Single Channel ◽

Conformational Dynamics ◽

Md Simulations ◽

High Temporal Resolution ◽

Dependent Manner ◽

Functional Dynamics ◽

Ph Dependent ◽

Open States

AbstractConformational changes in ion channels lead to gating of an ion-conductive pore. Ion flux has been measured with high temporal resolution by single-channel electrophysiology for decades. However, correlation between functional and conformational dynamics remained difficult, lacking experimental techniques to monitor sub-millisecond conformational changes. Here, we use the outer membrane protein G (OmpG) as a model system where loop-6 opens and closes the β-barrel pore like a lid in a pH-dependent manner. Functionally, single-channel electrophysiology shows that while closed states are favored at acidic pH and open states are favored at physiological pH, both states coexist and rapidly interchange in all conditions. Using HS-AFM height spectroscopy (HS-AFM-HS), we monitor sub-millisecond loop-6 conformational dynamics, and compare them to the functional dynamics from single-channel recordings, while MD simulations provide atomistic details and energy landscapes of the pH-dependent loop-6 fluctuations. HS-AFM-HS offers new opportunities to analyze conformational dynamics at timescales of domain and loop fluctuations.

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Crystal Structure of Escherichia coli Agmatinase: Catalytic Mechanism and Residues Relevant for Substrate Specificity

International Journal of Molecular Sciences ◽

10.3390/ijms22094769 ◽

2021 ◽

Vol 22 (9) ◽

pp. 4769

Author(s):

Pablo Maturana ◽

María S. Orellana ◽

Sixto M. Herrera ◽

Ignacio Martínez ◽

Maximiliano Figueroa ◽

...

Keyword(s):

Escherichia Coli ◽

Active Site ◽

Catalytic Mechanism ◽

Conformational Dynamics ◽

High Specificity ◽

Arginine Decarboxylase ◽

Space Groups ◽

X Ray Crystallography ◽

High Dynamics ◽

Dynamics Simulations

Agmatine is the product of the decarboxylation of L-arginine by the enzyme arginine decarboxylase. This amine has been attributed to neurotransmitter functions, anticonvulsant, anti-neurotoxic, and antidepressant in mammals and is a potential therapeutic agent for diseases such as Alzheimer’s, Parkinson’s, and cancer. Agmatinase enzyme hydrolyze agmatine into urea and putrescine, which belong to one of the pathways producing polyamines, essential for cell proliferation. Agmatinase from Escherichia coli (EcAGM) has been widely studied and kinetically characterized, described as highly specific for agmatine. In this study, we analyze the amino acids involved in the high specificity of EcAGM, performing a series of mutations in two loops critical to the active-site entrance. Two structures in different space groups were solved by X-ray crystallography, one at low resolution (3.2 Å), including a guanidine group; and other at high resolution (1.8 Å) which presents urea and agmatine in the active site. These structures made it possible to understand the interface interactions between subunits that allow the hexameric state and postulate a catalytic mechanism according to the Mn2+ and urea/guanidine binding site. Molecular dynamics simulations evaluated the conformational dynamics of EcAGM and residues participating in non-binding interactions. Simulations showed the high dynamics of loops of the active site entrance and evidenced the relevance of Trp68, located in the adjacent subunit, to stabilize the amino group of agmatine by cation-pi interaction. These results allow to have a structural view of the best-kinetic characterized agmatinase in literature up to now.

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