New tricks for the glycyl radical enzyme family

Abstract

Glycyl radical enzymes (GREs) are important biological catalysts in both strict and facultative anaerobes, playing key roles both in the human microbiota and in the environment. GREs contain a backbone glycyl radical that is post-translationally installed, enabling radical-based mechanisms. GREs function in several metabolic pathways including mixed acid fermentation, ribonucleotide reduction and the anaerobic breakdown of the nutrient choline and the pollutant toluene. By generating a substrate-based radical species within the active site, GREs enable C-C, C-O and C-N bond breaking and formation steps that are otherwise challenging for nonradical enzymes. Identification of previously unknown family members from genomic data and the determination of structures of well-characterized GREs have expanded the scope of GRE-catalyzed reactions as well as defined key features that enable radical catalysis. Here, we review the structures and mechanisms of characterized GREs, classifying members into five categories. We consider the open questions about each of the five GRE classes and evaluate the tools available to interrogate uncharacterized GREs.

Keywords: Glycyl radical enzymes; anaerobic metabolism; benzylsuccinate synthase; choline trimethylamine-lyase; class III ribonucleotide reductase; pyruvate formate-lyase; radical chemistry; radical decarboxylases.

Figure 1. Classification of characterized GREs

Five classes of GREs include: GRE formate-lyases (pyruvate formate-lyases, PFL, and ketobutyrate formate-lyases, TdcE); GRE 1,2-eliminases (choline trimethylamine-lyase, CutC, glycerol dehydratase, GDH, and propane-1,2-diol dehydratase, PDH); GRE ribonucleotide reductases (class III RNRs); and GRE decarboxylases (4-hydroxyphenylacetate decarboxylase, HPAD); and X-succinate synthases (XSSs where X= toluene in the case of benzylsuccinate synthase, BSS).

Figure 2. General overview of GRE activation and catalysis

A. Ten β-strands surrounding the active site and two catalytic loops, the Gly (yellow) and Cys (green) loops, in the GRE PFL (PDB ID: 1H18). The α-helices and connecting loops are not shown. B. Active site of GRE activase PFL-AE (teal), showing the [4Fe-4S] cluster (orange and yellow), AdoMet (carbons in grey), and a 7-mer peptide substrate (yellow carbons) (PDB ID: 3CB8). Dashed line represents the 3.9-Å distance between the 5′ position of AdoMet and glycine residue of the 7-residue peptide, which has the same sequence as the Gly radical loop of PFL. C. A [4Fe-4S]¹⁺ cluster reductively cleaves AdoMet to generate L-methionine and a 5′-dAdo radical species. The 5′-dAdo radical directly abstracts the pro-S hydrogen atom from the GRE glycine. Hydrogen atom transfer is thought to occur between the Gly radical and a neighboring Cys in the Cys loop to reversibly form a thiyl radical. This thiyl radical initiates catalysis. D. GRE-AE (teal) installs a Gly radical on the GRE in a reaction that is thought to require a conformational change of a region of the GRE known as the Gly radical domain (yellow). A color version of this figure is available online.

Figure 3. Structure and mechanism of PFL

A. Ribbon drawing of the PFL homodimer with Gly radical as spheres and Gly radical domain in yellow (PDB ID: 2PFL) B. Structure of PFL with pyruvate and CoA (PDB ID: 1H16). Active site is set up for the first half reaction with Gly734 close to Cys419 and the Sγ of Cys418 close to C2 of pyruvate (2.6 Å). CoA is not in a catalytic position. It is found in the rare syn conformation, disengaged from the active site, with the CoA pyrophosphate group approximately 20 Å and the CoA thiyl group approximately 30 Å away from C2 of pyruvate. In order for the thiyl group of CoA to reach the active site to be acetylated, a dramatic conformational change must occur (Becker and Kabsch, 2002). C. Putative PFL mechanism (Becker and Kabsch, 2002). See text for discussion. A color version of this figure is available online.

Figure 4. Structure and mechanism of GRE 1,2-eliminases

A. Dimeric structure of CutC (pink, PDB ID: 5FAU) with Gly radical as spheres and Gly radical domain in yellow. B. Active sites of CutC with choline (pink) and AdoCbl-dependent ethanolamine ammonia-lyase with ethanolamine (EAL, teal) (PDB: 3ABO) showing that CutC has no residue that is equivalent to Glu287 or Met392 in EAL. C. Active sites of CutC, GDH, and PDH with hydrogen bonds shown as black dashed lines and CH-O interactions in CutC as gray dashed lines. Substrate positioning was determined by crystallography for CutC and GDH and is modeled for PDH. D. A proposed 1,2-migration mechanism for EAL (Wetmore et al., 2002). E. A proposed 1,2-elimination mechanism for CutC (Bodea et al., 2016). GDH and PDH are also likely to proceed through 1,2-elimination reactions. A color version of this figure is available online.

Figure 5. Structure and mechanism(s) of class III RNRs (NrdDs)

A. Two NrdD structures with helices comprising the dimeric interface shown in brown. Location of an allosteric site at the dimeric interface is highlighted in yellow. Zoomed out views of the active sites are shown highlighted against a blue background. Both structures contain a zinc (grey sphere) site in the Gly radical domain (shown in yellow), but in the T. maritima structure (PDB 4U3E), the Cys loop (pink) is missing, replaced by a different loop (Ile359 loop in green). B. Active site models that are based on the structures of a formate-utilizing NrdD1 (T4 phage NrdD) and a disulfide-utilizing NrdD2 (T. maritima NrdD). Key residues for catalysis are marked in bold. Distances are given in ångstroms. CTP in the T4NrdD structure is modeled from TmNrdD (PDB ID 4COJ). The Cys loop in TmNrdD was generated manually in PyMol through in silico mutagenesis and torsion angle adjustment to relieve clashes. C. Mechanistic proposals for NrdD1 and NrdD2 enzymes (Wei et al., 2014a, Wei et al., 2014b). See text for description. A color version of this figure is available online.

Figure 6. Structure and mechanism of HPAD

A. HPAD heterotetramer displaying large catalytic β-subunits (teal, with glycyl radical domain in yellow) and small γ-subunits (magenta) each binding two [4Fe-4S] clusters (PDB code: 2YAJ). B. 4-Hydroxyphenylacetate (4HPA, purple) bound in the active site of HPAD. Active site residues are shown as sticks and labeled. Notably, 4HPA binds with the carboxylate group proximal to the catalytic Cys503, suggesting radical transfer to the carboxylate group. C. Proposed decarboxylation mechanism for decarboxylation of 4HPA (Martins et al., 2011). A color version of this figure is available online.

Figure 7. Structure and mechanism of BSS

A. The BSS heterohexamer is formed by a central BSSα dimer (green with Gly radical domain in yellow) and associated BSSβ (salmon) and BSSγ (light blue) monomers. BSSβ contains a [4Fe-4S] cluster (spheres) whereas BSSγ is partially disordered in the crystal structure and is missing its cluster. B. BSSβ (salmon) contacts the Gly radical domain (yellow) and juts into the putative substrate access channel (green). In the structure without BSSβ (inset), the access channel is more open and the Gly radical domain is shifted away from the Cys loop (closed state shown as faded model, open state as solid). C. The active site of BSS positions the substrates toluene and fumarate such that the expected radical transfer distances (red dashes) are minimized. Hydrogen bonds (black dashes) and van der Waals contacts secure fumarate into place above the Cys loop. D. Van der Waals interactions from the “hydrophobic wall” secure toluene positioning. E. Proposed structure-based mechanism for BSS (Funk et al., 2015). A color version of this figure is available online.

Figure 8. Comparison of CutC structure with GDH-based CutC homology model

A. Homology model of CutC (Bodea et al., 2016) created in Modeller using GDH as the starting model; choline (magenta) was docked using Schrodinger Suite 2012. Residues that differ substantially from the observed positions in the crystal structure are colored gray, with most signification differences observed for Tyr298, Thr502, and Ser503. B. The crystal structure of CutC bound to choline (magenta, PDB ID: 5FAU) in the same orientation as in A. A color version of this figure is available online.