Conformational gating in ammonia lyases

https://doi.org/10.1016/j.bbagen.2020.129605Get rights and content

Highlights

  • 3-methylaspartase ammonia lyase (MAL) is a dimer in solution with asymmetric dynamics

  • The β5-α2 loop and the hhl subdomain of MAL populates ‘open’ and ‘occluded’ states to modulate the accessibility of the catalytic site

  • The hhl subdomain acts as a gate for rearrangements of the β5-α2 loop

  • A sequential mechanism of couple conformational changes of the two structural elements is in play

Abstract

Background

Ammonia lyases are enzymes of industrial and biomedical interest. Knowledge of structure-dynamics-function relationship in ammonia lyases is instrumental for exploiting the potential of these enzymes in industrial or biomedical applications.

Methods

We investigated the conformational changes in the proximity of the catalytic pocket of a 3-methylaspartate ammonia lyase (MAL) as a model system. At this scope, we used microsecond all-atom molecular dynamics simulations, analyzed with dimensionality reduction techniques, as well as in terms of contact networks and correlated motions.

Results

We identify two regulatory elements in the MAL structure, i.e., the β5-α2 loop and the helix-hairpin-loop subdomain. These regulatory elements undergo conformational changes switching from ‘occluded’ to ‘open’ states. The rearrangements are coupled to changes in the accessibility of the active site. The β5-α2 loop and the helix-hairpin-loop subdomain modulate the formation of tunnels from the protein surface to the catalytic site, making the active site more accessible to the substrate when they are in an open state.

Conclusions

Our work pinpoints a sequential mechanism, in which the helix-hairpin-loop subdomain of MAL needs to break a subset of intramolecular interactions first to favor the displacement of the β5-α2 loop. The coupled conformational changes of these two elements contribute to modulate the accessibility of the catalytic site.

General significance

Similar molecular mechanisms can have broad relevance in other ammonia lyases with similar regulatory loops. Our results also imply that it is important to account for protein dynamics in the design of variants of ammonia lyases for industrial and biomedical applications.

Introduction

Ammonia lyases (ALs) are a broad class of enzymes that catalyze different transformations based on α- and β-amino acid scaffolds. For example, ALs are involved in the deamination and isomerization of natural amino acids through the reversible cleavage or the shifting of a C-N bond. ALs are highly heterogeneous in their structures and mechanisms of action, as attested by the fact that they cover 31 Enzyme Commission (EC) sub-classes, and they have high stereoselectivity for their substrates [1].

AL enzymes are commercially appealing for their industrial [2,3] and biomedical applications [1,2]. For example, they have been suggested as potential cancer biotherapeutics [1] due to the fact that ALs could prevent the supply of the tumor cells with essential metabolites. One example is the histidine ammonia lyase, which influences the growth of ovarian and prostate cancer cells by histidine deamination [1]. The deamination reaction produces urocanic acid and ammonia, eliminating histidine as building block and contributes to prevent protein synthesis, which is essential for the growth of cancer cells.

The catalytic mechanism of ALs is well known [[3], [4], [5], [6], [7], [8], [9]]. Among them, 3-methylaspartate ammonia lyases, or methylaspartases (MALs) [3] catalyze the reversible deamination of 3-methylaspartate to mesaconate. MALs belong to the enolase superfamily and they catalyze a broader range of reactions than other ammonia lyases [1]. They are versatile and promising targets to design new variants with different substrate specificity or improved activity and stability. For example, the MAL variant isolated from Citrobacter amalonaticus (CaMAL) has been engineered for enantioselective synthesis of N-substituted aspartic acids, which are essential building blocks for pharmaceutical, artificial sweeteners, synthetic enzymes and peptidomimetics [10,11].

MALs are dimeric enzymes. Only a few crystallographic three-dimensional (3D) structures of the dimeric form of MALs are available, deposited in the Protein Data Bank (PDB), as the entries 1KKO, 1KKR [12], 1KD0, 1KCZ [13], along with two mutated variants with PDB entries 3ZVH, 3ZVI [11]. CaMAL is a homodimeric enzyme where each monomer (here referred to as A and B) is composed by 413 amino acids and can be divided into two domains: a N-terminal (residues 1–160) and a C-terminal (residues 170–413) domain connected by β-sheet regions (Fig. 1A and B). The C-terminal domain folds into a triosephosphate-isomerase (TIM) barrel structure, consisting of eight α-helices and eight parallel β-strands, while the N-terminal domain is composed of a three antiparallel β-strands and four α-helices (Fig. 1B). The catalytic mechanism of CaMAL has been inferred from the analysis of its 3D structure, the comparison with other members of the enolase family, and validated by experimental mutagenesis [14,15]. The mutagenesis studies reveal the importance of specific residues on the structural integrity, activity, and regio- and diastereoselectivity of CaMAL (Fig. 1C). The reaction catalyzed by CaMAL requires magnesium (Mg2+) in the catalytic pocket, and the residues K331, H194 and Q329 as a catalytic triad (Fig. 1C). K331 acts as the base catalyst, while the Mg2+ metal ion and the residues H194 and Q329 are responsible for the stabilization of the enolate anion and the binding to the 4-carboxylate group of the substrate [14]. Additionally, Q73, F170, Q172, Y356, T360, C361, and L384 interact with different functional groups of the substrate or in the pocket (Fig. 1C). The N-terminal domain of CaMAL bears, in the proximity of the catalytic pocket, two structural elements, that we refer to as β5-α2 loop (residues 70–85) and helix-hairpin-loop subdomain (hhl, residues 12–51) (Fig. 1C). The β5-α2 loop connects the strand β5 with the α2 helix and includes Q73, which form water-mediated hydrogen bonds with the substrate and alters CaMAL kcat when mutated [15]. The hhl subdomain contains a short α-helix (residues 19–25), a small β-hairpin (β2-β3 strands, residues 29–34) and the loop between the β3 and β4 strands. The hhl subdomain is involved in intermolecular interactions at the interface between the monomers [12].

To the best of our knowledge, there is no information available on the conformational changes of MAL enzymes that could be related to their function. A detailed investigation of the dynamics of MALs could be essential to understand how to engineer them for applicative purposes since dynamics and function in enzymes are tightly related [[16], [17], [18], [19], [20]]. It is a common feature of several enzymes that disordered or partially structured regions of a folded protein undergo conformational changes that modulate the access of the substrate to the active site or allow for its formation [8,[21], [22], [23], [24], [25], [26], [27], [28], [29]]. In this context, biomolecular simulations are useful to describe protein functional dynamics, from local to global motions. This includes local changes in the proximity of the catalytic site [[30], [31], [32], [33], [34], [35], [36]] up to conformational changes of large amplitude associated with the opening and closing of gating loops or domains that can modulate the access to the catalytic site of an enzyme [21,26,37]. Here, we focus on CaMAL, presenting the first all-atom Molecular Dynamics (MD) investigation of this family of enzymes and its implications for activity.

Section snippets

Data and code availability

All the software used are freely available. The R and bash scripts, input and outputs files generated during this study are freely available in a GitHub repository associated to our publication (https://github.com/ELELAB/MAL_MD). The MD trajectories are available at OSF (https://osf.io/c5mhj/).

CaMAL expression and purification

We retrieved the gene sequence encoding for MAL from Citrobacter amalonaticus (CaMAL) (NCBI accession numbers AB005294) from the National Center for Biotechnology Information database and codon optimized

CaMAL is a dimer in solution

We investigated the preferred quaternary structures of CaMAL in solution to select the form to study with MD simulations. In particular, we performed Size-Exclusion Chromatography (SEC) to estimate CaMAL molecular weight (MW). We used proteins with a known MW as calibration standards (see Materials and Methods). The resulting partition coefficients (Kav) plotted against the logarithm of MW were fitted with a linear function (correlation coefficient ≈ 0.99) (Fig. 2A). We identify one single

Conclusions

An essential step toward the application of enzymes to industrial processes, and biomedical treatments, is to understand their structure, dynamics, and unveil the link with enzyme function and activity. An example is the study of ALs, which have been proposed as anticancer molecules. Low stability and rapid clearance hindered their applications [1]. We contributed to structural studies of ALs using molecular simulations. Our study highlights two important regulatory elements for CaMAL, i.e.,

Acknowledgments

The study was funded by The Swedish Research Council for Environment, Agricultural Sciences and Spatial Planning (FORMAS) (project number 942-2015-1628), the NovoNordisk Foundation under the program for Biotechnology-based Synthesis and Production Research (reference number NNF-17OC0027588) to EP and LO groups and a European Biophysical Society Association (EBSA) bursary granted to VSJ to visit EP group in 2017. EP group is part of the Center of Excellence in Autophagy, Recycling and Disease

References (72)

  • M. Kokkinidis et al.

    Protein flexibility and enzymatic catalysis

    Adv. Protein Chem. Struct. Biol.

    (2012)
  • F. del Caño-Ochoa et al.

    Characterization of the catalytic flexible loop in the dihydroorotase domain of the human multi-enzymatic protein CAD

    J. Biol. Chem.

    (2018)
  • E. Papaleo et al.

    Flexibility and enzymatic cold-adaptation: a comparative molecular dynamics investigation of the elastase family

    Biochim. Biophys. Acta

    (2006)
  • E. Papaleo et al.

    Coupled motions during dynamics reveal a tunnel toward the active site regulated by the N-terminal α-helix in an acylaminoacyl peptidase

    J. Mol. Graph. Model.

    (2012)
  • F.W. Studier

    Protein production by auto-induction in high-density shaking cultures

    Protein Expr. Purif.

    (2005)
  • M.J. Abraham et al.

    GROMACS: high performance molecular simulations through multi-level parallelism from laptops to supercomputers

    SoftwareX.

    (2015)
  • E. Papaleo et al.

    Free-energy landscape, principal component analysis, and structural clustering to identify representative conformations from molecular dynamics simulations: the myoglobin case

    J. Mol. Graph. Model.

    (2009)
  • K.R. Óskarsson et al.

    A single mutation Gln142Lys doubles the catalytic activity of VPR, a cold adapted subtilisin-like serine proteinase

    Biochim. Biophys. Acta

    (2016)
  • M. Manak et al.

    Hybrid Voronoi diagrams, their computation and reduction for applications in computational biochemistry

    J. Mol. Graph. Model.

    (2017)
  • D. Mercadante et al.

    CONAN: a tool to decode dynamical information from molecular interaction maps

    Biophys. J.

    (2018)
  • E. Papaleo et al.

    Dynamics fingerprint and inherent asymmetric flexibility of a cold-adapted homodimeric enzyme. A case study of the Vibrio alkaline phosphatase

    Biochim. Biophys. Acta

    (2013)
  • J.M. Flynn et al.

    Mechanistic asymmetry in Hsp90 dimers

    J. Mol. Biol.

    (2015)
  • F. Parmeggiani et al.

    Synthetic and therapeutic applications of ammonia-lyases and aminomutases

    Chem. Rev.

    (2017)
  • M. De Villiers et al.

    Catalytic mechanisms and biocatalytic applications of aspartate and methylaspartate ammonia lyases

    ACS Chem. Biol.

    (2012)
  • A. Heine et al.

    High resolution crystal structure of Clostridium propionicum β-alanyl-CoA: Ammonia lyase, a new member of the “hot dog fold” protein superfamily

    Proteins.

    (2014)
  • A.L. Seff et al.

    Computational investigation of the histidine ammonia-lyase reaction: a modified loop conformation and the role of the zinc(II) ion

    J. Mol. Model.

    (2011)
  • C. Levy et al.

    Structure and function of amino acid ammonia-lyases

    Biocatal. Biotransform.

    (2004)
  • W.J. Quax et al.

    Enantioselective synthesis of N-substituted aspartic acids using an angineered variant of Methylaspartate ammonia lyase

    ChemCatChem.

    (2013)
  • H. Raj et al.

    Engineering methylaspartate ammonia lyase for the asymmetric synthesis of unnatural amino acids

    Nat. Chem.

    (2012)
  • H. Raj et al.

    Alteration of the diastereoselectivity of 3-methlaspartate ammonia lyase by using structure-based mutagenesis

    ChemBioChem.

    (2009)
  • D. Tobi et al.

    Structural changes involved in protein binding correlate with intrinsic motions of proteins in the unbound state

    Proc. Natl. Acad. Sci. U. S. A.

    (2005)
  • Z. Kurkcuoglu et al.

    Coupling between catalytic loop motions and enzyme global dynamics

    PLoS Comput. Biol.

    (2012)
  • R.O. Dror et al.

    Biomolecular simulation: a computational microscope for molecular biology

    Annu. Rev. Biophys.

    (2012)
  • E. Papaleo et al.

    An acidic loop and cognate phosphorylation sites define a molecular switch that modulates ubiquitin charging activity in Cdc34-like enzymes

    PLoS Comput. Biol.

    (2011)
  • E. Papaleo et al.

    Loop 7 of E2 enzymes : an ancestral conserved functional motif involved in the E2-mediated steps of the ubiquitination Cascade

    PLoS One

    (2012)
  • S. Pilbák et al.

    The essential tyrosine-containing loop conformation and the role of the C-terminal multi-helix region in eukaryotic phenylalanine ammonia-lyases

    FEBS J.

    (2006)
  • View full text