Catalytic Metal Ion Rearrangements Underline Promiscuity and Evolvability of a Metalloenzyme

Ben‐David, Moshe; Wieczorek, Grzegorz; Elias, Mikael; Silman, Israel; Sussman, Joel L.; Tawfik, Dan S.

doi:10.1016/j.jmb.2013.01.009

Cited by 60 publications

(100 citation statements)

References 46 publications

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“…The likelihood of transitions between mononuclear and binuclear metal binding sites in the evolutionary trajectory of the AHS has already been hypothesized (73). MolA and PuhB may represent a transition from a binuclear carboxypeptidase ancestor, where loss of the second metal binding site enables the efficient hydrolysis of molinate and diuron.…”

Section: Figmentioning

confidence: 98%

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Evolutionary Expansion of the Amidohydrolase Superfamily in Bacteria in Response to the Synthetic Compounds Molinate and Diuron

Sugrue

Fraser

Hopkins

et al. 2015

Appl Environ Microbiol

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The amidohydrolase superfamily has remarkable functional diversity, with considerable structural and functional annotation of known sequences. In microbes, the recent evolution of several members of this family to catalyze the breakdown of environmental xenobiotics is not well understood. An evolutionary transition from binuclear to mononuclear metal ion coordination at the active sites of these enzymes could produce large functional changes such as those observed in nature, but there are few clear examples available to support this hypothesis. To investigate the role of binuclear-mononuclear active-site transitions in the evolution of new function in this superfamily, we have characterized two recently evolved enzymes that catalyze the hydrolysis of the synthetic herbicides molinate (MolA) and phenylurea (PuhB). In this work, the crystal structures, mutagenesis, metal ion analysis, and enzyme kinetics of both MolA and PuhB establish that these enzymes utilize a mononuclear active site. However, bioinformatics and structural comparisons reveal that the closest putative ancestor of these enzymes had a binuclear active site, indicating that a binuclear-mononuclear transition has occurred. These proteins may represent examples of evolution modifying the characteristics of existing catalysts to satisfy new requirements, specifically, metal ion rearrangement leading to large leaps in activity that would not otherwise be possible.T he amidohydrolase superfamily (AHS) has been extensively studied, with Ͼ36,000 attributed sequences since its first classification in 1998, most of which are from bacteria (1, 2). All AHS members have a (␤/␣) 8 -barrel structural fold and catalyze metaldependent hydrolysis reactions (3). The scissile bond cleaved varies between AHS enzymes, with C-O, P-O, P-S, C-N, C-S, and C-Cl bonds all having been reported to be hydrolyzed (4-8). In accordance with the metal-dependent mechanism, a mononuclear or binuclear metal binding site is observed in all AHS enzymes (9, 10). The role of the metal ion(s) in catalysis varies, depending on the substrate being hydrolyzed and the enzyme involved (10, 11). Generally, one or two metals are enlisted to lower the pK a of a catalytic water molecule, favoring the formation of a nucleophilic hydroxide (12). A second metal may polarize the substrate directly, often at a carbonyl or phosphoryl bond, or stabilize a negative charge in the transition state (13,14).Enzymes within the AHS can be separated into subtypes on the basis of the specific metal binding ligands at the conserved mononuclear or binuclear metal binding sites. Since the comprehensive review published by Seibert and Raushel in 2005 (9), in which seven subtypes were defined, two additional subtypes have been identified (Table 1) (6,15). A typical binuclear metal binding site (subtypes I, II, and VI) is characterized by a bridging ligand (generally a glutamate or carboxylated lysine residue) that coordinates both (M ␣ and M ␤ ) metal ions (16-18). Mononuclear metal binding ligands are more variable, ...

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Section: Figmentioning

confidence: 98%

“…alternative binding mode of a catalytic calcium ion could have been the initiator of divergence in enzymatic activity (73). The likelihood of transitions between mononuclear and binuclear metal binding sites in the evolutionary trajectory of the AHS has already been hypothesized (73).…”

Section: Figmentioning

confidence: 99%

Evolutionary Expansion of the Amidohydrolase Superfamily in Bacteria in Response to the Synthetic Compounds Molinate and Diuron

Sugrue

Fraser

Hopkins

et al. 2015

Appl Environ Microbiol

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“…[7,[98][99][100] The flexibility of metal ion coordination in a B1 MbL (Bacillus cereus), through the variable positioning of metal ion-coordinating ligands on flexible loop regions and the use of coordination state-flexible zinc ions, is thought to be crucial for its catalysis of diverse substrates and antibiotics. [100] Rapid-freeze-quench doubleelectron electron resonance spectroscopy was used to analyse loop movements in the New Delhi metallo-b-lactamase (NDM, Klebsiella pneumoniae).…”

Section: Active Site Changes and Activity Divergencementioning

confidence: 99%

“…[99] However, a secondary low-metal-ionoccupancy binding mode is also possible, where the asparagine coordinating ligands coordinate to a water molecule absent in the most common binding mode, and exhibit different orientations. [99] When PON1 was subjected to directed evolution to enhance its promiscuous paraoxonase activity, the variants had higher metal ion occupancy in the alternative metal binding site. [99,108] This alternative binding site, 1.8 Å away from the native site, is postulated to be the initiator of divergence in activity between lactonase and paraoxonase enzymes.…”

Section: Active Site Changes and Activity Divergencementioning

confidence: 99%

“…[99] When PON1 was subjected to directed evolution to enhance its promiscuous paraoxonase activity, the variants had higher metal ion occupancy in the alternative metal binding site. [99,108] This alternative binding site, 1.8 Å away from the native site, is postulated to be the initiator of divergence in activity between lactonase and paraoxonase enzymes. The mutation of a histidine residue in the directed evolution of PON1 is thought to change the calcium ion binding position, optimising the ligating residues for paraoxon hydrolysis.…”

Section: Active Site Changes and Activity Divergencementioning

confidence: 99%

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The Evolution of New Catalytic Mechanisms for Xenobiotic Hydrolysis in Bacterial Metalloenzymes

et al. 2016

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An increasing number of bacterial metalloenzymes have been shown to catalyse the breakdown of xenobiotics in the environment, while others exhibit a variety of promiscuous xenobiotic-degrading activities. Several different evolutionary processes have allowed these enzymes to gain or enhance xenobiotic-degrading activity. In this review, we have surveyed the range of xenobiotic-degrading metalloenzymes, and discuss the molecular and catalytic basis for the development of new activities. We also highlight how our increased understanding of the natural evolution of xenobiotic-degrading metalloenzymes can be been applied to laboratory enzyme design.

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Molecular Dynamics

Wieczorek¹,

Niedziałek²

2020

Encyclopedia of Life Sciences

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Molecular dynamics (MD) is a handy computer simulation method which unveils principles of a large variety of fundamental processes by mimicking the real life perpetual motion of atoms triggered by interatomic forces. In MD, these forces are expressed through analytical functions and associated parameters, which are commonly referred to as the force field. The forces' effect on the movement of atoms is determined by Newton's second law. Most importantly, MD simulations are able to retrace the exact trajectories of interacting atoms by following their spatial evolution in time from a defined initial configuration. The resulting integrated trajectory of the system can be visualised and analysed with rigorous mathematical expressions of statistical mechanics, which relate averaged microscopic states to macroscopic properties relevant to the problem at hand. Key Concepts Molecular dynamics is a computation tool successfully used to unveil principles of a large variety of fundamental processes down to the atomic resolution. Thanks to recent advances in computer simulations, atomistic MD can reach millisecond timescales, thus emerging as a powerful alternative method that successfully complements experimental measurements in biology. Computational efficiency of molecular dynamics algorithms continues to pose challenges. The collective efforts of many groups of researchers have enabled a great deal of force fields applied to many problems in biology. Biomolecular simulations require the presence of solvent; hence, a reliable representation of water is crucial for achieving realistic results. Several specialised methods stemming from MD allow to investigate various processes of biomolecules: REMD, SMD and TMD.

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Catalytic Metal Ion Rearrangements Underline Promiscuity and Evolvability of a Metalloenzyme

Cited by 60 publications

References 46 publications

Evolutionary Expansion of the Amidohydrolase Superfamily in Bacteria in Response to the Synthetic Compounds Molinate and Diuron

Evolutionary Expansion of the Amidohydrolase Superfamily in Bacteria in Response to the Synthetic Compounds Molinate and Diuron

The Evolution of New Catalytic Mechanisms for Xenobiotic Hydrolysis in Bacterial Metalloenzymes

Molecular Dynamics

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