Modeling catalytic promiscuity in the alkaline phosphatase superfamily

Duarte, Fernanda; Amrein, Beat Anton; Kamerlin, Shina Caroline Lynn

doi:10.1039/c3cp51179k

Cited by 47 publications

(39 citation statements)

References 245 publications

(476 reference statements)

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“…The question of whether the large mobility of the metals in this kind of enzymes is an artifact of the computing methods or reveals the plasticity of the metals in the active site requires further validation to provide a definitive answer, as recently proposed in a perspective paper by Kamerlin and co-workers. 57 In particular, there are no experimental X-ray structures of the Michaelis complex of mono-nuclear metallo-beta-lactamases with different substrates to confirm the observations obtained in the present paper, where the QM/MM MD simulations have been performed using the semiempirical PM3 hamiltonian.…”

Section: Discussionsupporting

confidence: 66%

Theoretical studies of the hydrolysis of antibiotics catalyzed by a metallo-β-lactamase

Meliá

Ferrer

Moliner

et al. 2015

Archives of Biochemistry and Biophysics

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In this paper, hybrid QM/MM molecular dynamics (MD) simulations have been performed to explore the mechanisms of hydrolysis of two antibiotics, Imipenen (IMI), an antibiotic belonging to the subgroup of carbapenems, and the Cefotaxime (CEF), a third-generation cephalosporin antibiotic, in the active site of a mono-nuclear β-lactamase, CphA from Aeromonas hydrophila. According to our results, significant different transition state structures are obtained for the hydrolysis of both antibiotics:while the TS of the CEF is a ionic species with negative charge on nitrogen, the IMI TS presents a tetrahedral-like character with negative charge on oxygen atom of the carbonyl group of the lactam ring. Thus, dramatic conformational changes can take place in the cavity of CphA to accommodate different substrates, which would be the origin of its substrate promiscuity. This feature of the β-lactamase would be in turn, associated to the different mechanisms that the protein employs to hydrolyze the different antibiotics; i.e. the catalytic promiscuity. Since CphA shows only activity against carbapenem antibiotic, this study will be used to shed some light into the origin of the selectivity of the different MbL and, as a consequence, into the discovery of specific and potent MβL inhibitors against a broad spectrum of bacterial pathogens.

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Section: Discussionsupporting

confidence: 66%

Theoretical studies of the hydrolysis of antibiotics catalyzed by a metallo-β-lactamase

Meliá

Ferrer

Moliner

et al. 2015

Archives of Biochemistry and Biophysics

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“…Characterised AP enzymes vary in the nature and number of metal ions required for activity and, consequently, active-site architecture differs substantially between those of known structure25. Our structural comparisons identify the ligands to the MCR-1 Zn1 site, Asp465, His466, Glu246 and Thr285, to be well conserved between MCR-1, LptA and EptC and between MCR-1 and other AP family members, although the identity of the nucleophile differs between enzymes.…”

Section: Discussionmentioning

confidence: 86%

Insights into the Mechanistic Basis of Plasmid-Mediated Colistin Resistance from Crystal Structures of the Catalytic Domain of MCR-1

Hinchliffe

Qin

Portal

et al. 2017

Sci Rep

126

189

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The polymixin colistin is a “last line” antibiotic against extensively-resistant Gram-negative bacteria. Recently, the mcr-1 gene was identified as a plasmid-mediated resistance mechanism in human and animal Enterobacteriaceae, with a wide geographical distribution and many producer strains resistant to multiple other antibiotics. mcr-1 encodes a membrane-bound enzyme catalysing phosphoethanolamine transfer onto bacterial lipid A. Here we present crystal structures revealing the MCR-1 periplasmic, catalytic domain to be a zinc metalloprotein with an alkaline phosphatase/sulphatase fold containing three disulphide bonds. One structure captures a phosphorylated form representing the first intermediate in the transfer reaction. Mutation of residues implicated in zinc or phosphoethanolamine binding, or catalytic activity, restores colistin susceptibility of recombinant E. coli. Zinc deprivation reduces colistin MICs in MCR-1-producing laboratory, environmental, animal and human E. coli. Conversely, over-expression of the disulphide isomerase DsbA increases the colistin MIC of laboratory E. coli. Preliminary density functional theory calculations on cluster models suggest a single zinc ion may be sufficient to support phosphoethanolamine transfer. These data demonstrate the importance of zinc and disulphide bonds to MCR-1 activity, suggest that assays under zinc-limiting conditions represent a route to phenotypic identification of MCR-1 producing E. coli, and identify key features of the likely catalytic mechanism.

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“…Promiscuity is thought to play an essential role in the evolution of new functions as evinced by modern enzymes (34), which are highly efficient specialists and primordial enzymes, which were likely promiscuous generalists. Modern-day proteins can be thought of as evolved from generalists into specialists (35)(36)(37)(38)(39)(40). Remarkably, there are only a few structural differences-in particular at the active-site regions-between the resurrected ancestral enzymes and penicillin-specialist modern b-lactamase.…”

Section: Introductionmentioning

confidence: 99%

The Role of Conformational Dynamics and Allostery in the Disease Development of Human Ferritin

Kumar

Glembo

Ozkan

2015

Biophysical Journal

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Determining the three-dimensional structure of myoglobin, the first solved structure of a protein, fundamentally changed the way protein function was understood. Even more revolutionary was the information that came afterward: protein dynamics play a critical role in biological functions. Therefore, understanding conformational dynamics is crucial to obtaining a more complete picture of protein evolution. We recently analyzed the evolution of different protein families including green fluorescent proteins (GFPs), β-lactamase inhibitors, and nuclear receptors, and we observed that the alteration of conformational dynamics through allosteric regulation leads to functional changes. Moreover, proteome-wide conformational dynamics analysis of more than 100 human proteins showed that mutations occurring at rigid residue positions are more susceptible to disease than flexible residue positions. These studies suggest that disease-associated mutations may impair dynamic allosteric regulations, leading to loss of function. Thus, in this study, we analyzed the conformational dynamics of the wild-type light chain subunit of human ferritin protein along with the neutral and disease forms. We first performed replica exchange molecular dynamics simulations of wild-type and mutants to obtain equilibrated dynamics and then used perturbation response scanning (PRS), where we introduced a random Brownian kick to a position and computed the fluctuation response of the chain using linear response theory. Using this approach, we computed the dynamic flexibility index (DFI) for each position in the chain for the wild-type and the mutants. DFI quantifies the resilience of a position to a perturbation and provides a flexibility/rigidity measurement for a given position in the chain. The DFI analysis reveals that neutral variants and the wild-type exhibit similar flexibility profiles in which experimentally determined functionally critical sites act as hinges in controlling the overall motion. However, disease mutations alter the conformational dynamic profile, making hinges more loose (i.e., softening the hinges), thus impairing the allosterically regulated dynamics.

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Modeling catalytic promiscuity in the alkaline phosphatase superfamily

Abstract: This Perspective discusses advances in computational approaches to study catalytic promiscuity, and, by extension, functional evolution of proteins.

Cited by 47 publications

References 245 publications

Theoretical studies of the hydrolysis of antibiotics catalyzed by a metallo-β-lactamase

Theoretical studies of the hydrolysis of antibiotics catalyzed by a metallo-β-lactamase

Insights into the Mechanistic Basis of Plasmid-Mediated Colistin Resistance from Crystal Structures of the Catalytic Domain of MCR-1

The Role of Conformational Dynamics and Allostery in the Disease Development of Human Ferritin

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