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. 2018:606:269-318.
doi: 10.1016/bs.mie.2018.04.013. Epub 2018 Jul 7.

Mechanistic Studies of Radical SAM Enzymes: Pyruvate Formate-Lyase Activating Enzyme and Lysine 2,3-Aminomutase Case Studies

Amanda S Byer  1 , Elizabeth C McDaniel  1 , Stella Impano  1 , William E Broderick  1 , Joan B Broderick  2
Affiliations

Mechanistic Studies of Radical SAM Enzymes: Pyruvate Formate-Lyase Activating Enzyme and Lysine 2,3-Aminomutase Case Studies

Amanda S Byer et al. Methods Enzymol. 2018.

Abstract

The radical SAM enzyme superfamily is large and diverse, with ever-increasing numbers of examples of characterized reactions. This chapter focuses on the methodology we have developed over the last 25 years for working with these enzymes, with the specific examples discussed being the pyruvate formate-lyase activating enzyme (PFL-AE) and lysine 2,3-aminomutase (LAM). Both enzymes are purified from overexpressing Escherichia coli, but differ in that PFL-AE is expressed without an affinity tag and does not require iron-sulfur cluster reconstitution, while LAM purification is carried out through use of a His6 affinity tag and the enzyme benefits from cluster reconstitution. Because of radical SAM enzymes' catalytic need for a [4Fe-4S] cluster, we present methods for characterization and incorporation of a full [4Fe-4S] cluster in addition to enzyme activity assay protocols. Synthesis of SAM (S-adenosyl-l-methionine) and its analogs have played an important role in our mechanistic studies of radical SAM enzymes, and their synthetic methods are also presented in detail.

Keywords: Glycyl radical enzyme; Lysine 2,3-aminomutase; Purification; Pyruvate formate-lyase; Radical SAM; Reconstitution; S-adenosylmethionine.

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Figures

Figure 1.
Figure 1.. Generation of the dAdo• Radical by Radical SAM Enzymes.
The resting enzyme with a [4Fe-4S]2+ (left; sulfur, yellow spheres; iron, rust-color spheres) is reduced by one electron to generate the catalytically active state containing a [4Fe-4S]1+ cluster (center). An electron is thought to undergo inner-sphere electron transfer to the sulfonium of SAM, promoting S-C(5’) bond cleavage and generation of a 5’-dAdo• radical intermediate and methionine (right).
Figure 2.
Figure 2.. The Organometallic Intermediate Ω Observed in PFL-AE.
This Ω intermediate was captured through rapid-freeze quench preparations of spectroscopy samples; electron paramagnetic resonance and electron-nuclear double resonance spectroscopy characterization illustrated a direct bond between the 5’-C of the dAdo moiety and the unique iron of the cluster. (sulfur, yellow spheres; iron, rust-color spheres)
Figure 3.
Figure 3.. Radical SAM Enzyme Catalysis Scheme.
This scheme represents a simplification of the current understanding of how radical SAM enzymes use SAM as a cofactor (green pathway) or a cosubstrate (orange pathway). Prior to catalysis, radical SAM enzymes house a [4Fe-4S] cluster in the 2+ state (A; sulfur, yellow spheres; iron, rust-color spheres). One electron reduction of this cluster generates the catalytically-active [4Fe-4S]+ state (See Figure 1); in this cluster state, an electron can be transferred to the sulfonium of SAM to promote homolytic bond cleavage of the S-C(5’) bond to generate a 5’-dAdo• radical intermediate (B). This dAdo• radical abstracts a hydrogen from substrate to generate a substrate based radical and dAdoH (C). From here, the scheme branches depending on whether SAM is used as a cofactor (green arrows) or as a co-substrate (orange arrows). If SAM is used as a co-factor, a product radical abstracts a hydrogen from dAdoH to re-generate the dAdo radical (C→B) and the product leaves the active-site. If SAM is used as a co-substrate, the substrate-based radical reacts to form the product, while methionine and dAdoH leave the active-site (C→D) allowing a new SAM molecule to enter the enzyme active-site (D→A) and catalysis to begin again.
Figure 4.
Figure 4.. Radical SAM Enzymes Full or Partial TIM Barrels.
In all structures, the alpha helices of the TIM barrel are shown in grey and the beta strands are displayed in black and the enzyme structure remainder is shown in light grey; in the center of the TIM barrel, essential components of radical SAM enzymes are displayed: the [4Fe-4S] (sulfur, light grey spheres; iron, dark grey spheres), SAM (light grey sticks), and substrate (orange sticks, when present). A) The full TIM barrel structure (ßα)8 is [FeFe]-hydrogenase maturase, HydE; no substrate is present in this structure (PDB: 3IIZ). B) In LAM’s partial TIM barrel (ßα)6, the substrate (lysine, orange sticks) and LAM’s second cofactor PLP (pink sticks) are visible above the SAM-bound [4Fe-4S] cluster; a zinc ion (blue-grey sphere) resides at the subunit interface, however the additional subunits are not displayed (PDB: 2A5H). C) PFL-AE’s partial TIM barrel (ßα)6 houses the cluster, SAM, and a peptide (orange sticks) representing the arm of PFL that sits in the PFL-AE active-site; additionally in this active site, a monovalent cation (Na+, magenta) is positioned in close proximity to SAM and the cluster (PDB: 3CB8).
Figure 5.
Figure 5.. Radical SAM Enzyme Active-site.
Both structures of LAM (A) and PFL-AE (B) illustrate similar approximate positioning (from bottom to top) of the [4Fe-4S] cluster (sulfur, yellow sphere; iron, rust color sphere), SAM (green sticks), and substrate (orange sticks). A) The LAM X-ray crystal structure (PDB: 2A5H) shows an additional cofactor pyridoxyl 5’-phosphate (pink sticks). B) PFL-AE’s active-site (PDB: 3CB8) depicts a monovalent cation (Na+, magenta sphere) and only part of the PFL substrate (orange sticks; for clarity only three residues are shown, S733-G734-Y735, with the G734 highlighted in a darker orange).
Figure 6.
Figure 6.. PFL Reaction Scheme.
Through this proposed ping-pong mechanism pathway, PFL converts pyruvate into formate, and CoA into acetyl-CoA. Activated PFL (PFLa) has a glycyl radical at residue G734 which abstracts a hydrogen from residue C418, generating a cysteinyl radical which initiates a nucleophilic attack on the 2’C of pyruvate to generate an oxygen-centered pyruvate-based radical. Homolytic cleavage between the C1 and C2 position on pyruvate produces a carbonyl radical, which quickly reacts to form a different cysteinyl radical, this time on C419, while formate is released from the active-site. This C419-based radical initiates a nucleophilic attack on the incoming CoA, generating a CoA sulfur-based radical. Finally, this CoA-based radical initiates a nucleophilic attack on the C418-bound carbonyl to form acetyl-CoA and reestablish the C418 sulfur-based radical so the catalytic cycle can repeat.
Figure 7.
Figure 7.. PFL-AE and PFL Over-expression.
A) SDS PAGE of PFL-AE over-expression shows cellular protein content before induction (−IPTG) and after (+IPTG, 2x dilute) induction. B) SDS PAGE of PFL over-expression showing cellular protein content before induction before induction (−IPTG) and after (+IPTG, 2x dilute) induction.. All samples are run against a BioRad Broad Range Molecular Marker with molecular weights indicated as appropriate; in panel B, the gel fragments are juxtaposed and aligned appropriately, but remain separated to illustrate gel lanes were not adjacent.
Figure 7.
Figure 7.. PFL-AE and PFL Over-expression.
A) SDS PAGE of PFL-AE over-expression shows cellular protein content before induction (−IPTG) and after (+IPTG, 2x dilute) induction. B) SDS PAGE of PFL over-expression showing cellular protein content before induction before induction (−IPTG) and after (+IPTG, 2x dilute) induction.. All samples are run against a BioRad Broad Range Molecular Marker with molecular weights indicated as appropriate; in panel B, the gel fragments are juxtaposed and aligned appropriately, but remain separated to illustrate gel lanes were not adjacent.
Figure 8.
Figure 8.. PFL-AE Purifications.
PFL-AE is purified twice on a 1 L (5 cm x 55 cm) Superdex 75 prep-grade gel filtration column, while UV Vis traces at 280 nm and 426 nm monitor protein and iron content, respectively. A) First PFL-AE Purification. The elution chromatogram shows two peaks, wherein the first peak is an impurity and the second is the PFL-AE protein; in this chromatogram, the 0 – 1600 mAU scale applies only to the 426 nm trace, as the 280 nm trace is on a scale of approximately 0 – 4500 mAU. A representative fraction (inset) illustrates the brown color of the PFL-AE solution isolated from a first purification; select fractions (shaded) are run over the column a second time. B) Second PFL-AE Purification. This second purification chromatogram shows PFL-AE eluting at the same volume as the first run, but without the impurity; both UV-Vis traces are on the same scale. The brown color of PFL-AE isolated from this second purification (inset) is often darker than that of the first purification fractions. To select enzyme with the best cluster content, fractions are partitioned into A (highlighted in blue) and B (highlighted in green) sets, based on A426nm:A280nm ratios.
Figure 8.
Figure 8.. PFL-AE Purifications.
PFL-AE is purified twice on a 1 L (5 cm x 55 cm) Superdex 75 prep-grade gel filtration column, while UV Vis traces at 280 nm and 426 nm monitor protein and iron content, respectively. A) First PFL-AE Purification. The elution chromatogram shows two peaks, wherein the first peak is an impurity and the second is the PFL-AE protein; in this chromatogram, the 0 – 1600 mAU scale applies only to the 426 nm trace, as the 280 nm trace is on a scale of approximately 0 – 4500 mAU. A representative fraction (inset) illustrates the brown color of the PFL-AE solution isolated from a first purification; select fractions (shaded) are run over the column a second time. B) Second PFL-AE Purification. This second purification chromatogram shows PFL-AE eluting at the same volume as the first run, but without the impurity; both UV-Vis traces are on the same scale. The brown color of PFL-AE isolated from this second purification (inset) is often darker than that of the first purification fractions. To select enzyme with the best cluster content, fractions are partitioned into A (highlighted in blue) and B (highlighted in green) sets, based on A426nm:A280nm ratios.
Figure 9.
Figure 9.. PFL Protein Purifications.
A) PFL elution off the Accell Plus™ quarternary methylamine anion exchange column will show a trailing peak beginning roughly halfway through the linear gradient. The SDS PAGE (inset and boxed) illustrates only those fractions without the lower molecular weight impurity are kept for buffer exchange and further purification. B) PFL elution off of the HR 16/10 Highload Phenyl Sepharose High Performance column commences around 70 mL, halfway through the linear gradient into the no-salt buffer. The collected fractions (inset and boxed) are predominantly PFL protein.
Figure 9.
Figure 9.. PFL Protein Purifications.
A) PFL elution off the Accell Plus™ quarternary methylamine anion exchange column will show a trailing peak beginning roughly halfway through the linear gradient. The SDS PAGE (inset and boxed) illustrates only those fractions without the lower molecular weight impurity are kept for buffer exchange and further purification. B) PFL elution off of the HR 16/10 Highload Phenyl Sepharose High Performance column commences around 70 mL, halfway through the linear gradient into the no-salt buffer. The collected fractions (inset and boxed) are predominantly PFL protein.
Figure 10.
Figure 10.. PFL-AE Coupling Assay Scheme.
Inactive PFL (PFLi) is activated by PFL-AE and SAM to generate active PFL (PFLa) and enable conversion of pyruvate and CoA to formate and acetyl-CoA. Catalyzed by citrate synthase, acetyl-CoA reacts with oxaloacetate (replenished by the reversible reaction catalyzed by malic dehydrogenase) to produce CoA and citrate. Monitored by UV-Vis absorbance (A340nm), PFL-AE activity is measured by malic dehydrogenase catalyzed NADH (purple) production. As the assay requires mixing of two solutions, this scheme illustrates select components of the coupling solution (blue) and activation solution (red).
Figure 11.
Figure 11.. UV-Vis Spectroscopy of Pre- and Post-Reconstitution of a Radical SAM Enzyme.
A) Radical SAM enzyme solutions pre- and post-reconstitution demonstrate iron-sulfur cluster content changes can be observed with the human eye. B)The UV-Vis absorbance spectrum of an as-purified radical SAM enzyme (dashed line) illustrates iron-sulfur cluster content; however, after reconstitution, this same radical SAM enzyme has significantly increased iron-sulfur cluster content (solid line) as demonstrated by an increase in intensity of the LMCT bands between 400 – 500 nm.
Figure 11.
Figure 11.. UV-Vis Spectroscopy of Pre- and Post-Reconstitution of a Radical SAM Enzyme.
A) Radical SAM enzyme solutions pre- and post-reconstitution demonstrate iron-sulfur cluster content changes can be observed with the human eye. B)The UV-Vis absorbance spectrum of an as-purified radical SAM enzyme (dashed line) illustrates iron-sulfur cluster content; however, after reconstitution, this same radical SAM enzyme has significantly increased iron-sulfur cluster content (solid line) as demonstrated by an increase in intensity of the LMCT bands between 400 – 500 nm.
Figure 12.
Figure 12.. UV-Vis Spectroscopy of FeS Clusters Pre- and Post-Reduction.
The UV-Vis absorbance spectrum of a reconstituted radical SAM enzyme (dashed line) displays iron sulfur cluster content that can be associated with a [4Fe-4S]2+ cluster state; after partial reduction, the LMCT bands diminish, illustrating the oxidation state change in clusters to the [4Fe-4S]+ state (solid line).
Figure 13.
Figure 13.. EPR Spectroscopy of FeS Clusters in Radical SAM Enzymes.
The top spectrum (black) is of an as-purified enzyme and illustrates an isotropic [2Fe-2S]+ cluster signal shown at 40 K, Topt. The middle spectrum (blue) is of a reduced enzyme and displays an axial [4Fe-4S]+ cluster signal. The lower spectrum (red) is of a reduced enzyme with exogenous SAM added and displays two overlapping axial [4Fe-4S]+ cluster signals, suggesting cluster content with and without SAM bound. The lower two spectra are shown at 10 K, as this temperature is the Topt for these [4Fe-4S]+ cluster signals.
Figure 14.
Figure 14.. SAM Purification Monitors.
A) TLC can illustrate formation of SAM, using a solvent system of butanol : acetic acid : water : 2 M formic acid (8:2:2:1) on a silica solid phase. B) While the SAM elution profile is typically consistent, monitoring the UV-Vis absorbance during the described purification can be incredibly useful for SAM analogues that can have a slightly altered elution profile.
Figure 14.
Figure 14.. SAM Purification Monitors.
A) TLC can illustrate formation of SAM, using a solvent system of butanol : acetic acid : water : 2 M formic acid (8:2:2:1) on a silica solid phase. B) While the SAM elution profile is typically consistent, monitoring the UV-Vis absorbance during the described purification can be incredibly useful for SAM analogues that can have a slightly altered elution profile.
Figure 15.
Figure 15.. SAM Analogue - anSAM.
The structure of anSAM is depicted on the left illustrating the double bond in the between the 3’C and 4’C of the ribose; upon a 5’C-S bond cleavage during radical SAM enzyme catalysis, an allylic radical is generated and stabilized across the 3’, 4’, and 5’ carbons (as depicted in red) allowing for spectroscopic observation.
Figure 16.
Figure 16.. AdoK Affinity Purification.
A) AdoK elution profile off the HisTrap™ column has a double peak; all fractions in the red highlighted section contain useful AdoK protein and are collected. B) In the SDS PAGE of fractions collected, the red highlighted fractions show presence of AdoK and appear relatively pure, despite the need for further purification; the BioRad Broad Range Molecular Marker shows weights of 200 kDa, 116.5 kDa, 97.4 kDa, 66.2 kDa, 45 kDa, 31 kDa, 21.5 kDa, and 14.4 kDa, (top to bottom) with AdoK protein correlating to ~45 kDa.
Figure 16.
Figure 16.. AdoK Affinity Purification.
A) AdoK elution profile off the HisTrap™ column has a double peak; all fractions in the red highlighted section contain useful AdoK protein and are collected. B) In the SDS PAGE of fractions collected, the red highlighted fractions show presence of AdoK and appear relatively pure, despite the need for further purification; the BioRad Broad Range Molecular Marker shows weights of 200 kDa, 116.5 kDa, 97.4 kDa, 66.2 kDa, 45 kDa, 31 kDa, 21.5 kDa, and 14.4 kDa, (top to bottom) with AdoK protein correlating to ~45 kDa.
Figure 17.
Figure 17.. DEAE Sephacel AdoK Purification.
A) AdoK elution profile with AdoK fractions highlighted, B) SDS PAGE illustration of fraction purity, wherein ‘Ldr’ refers to the BioRad Broad Range Molecular Marker, ‘FT’ is the flow through during loading of the column, ‘0%’ is the wash with 0% Buffer B, and ‘F1’-’F5’ are fractions. Fractions F2 and F3 both have active AdoK.
Figure 17.
Figure 17.. DEAE Sephacel AdoK Purification.
A) AdoK elution profile with AdoK fractions highlighted, B) SDS PAGE illustration of fraction purity, wherein ‘Ldr’ refers to the BioRad Broad Range Molecular Marker, ‘FT’ is the flow through during loading of the column, ‘0%’ is the wash with 0% Buffer B, and ‘F1’-’F5’ are fractions. Fractions F2 and F3 both have active AdoK.
Figure 18.
Figure 18.. HPLC and TLC Distinguish Nucleotides During anATP Synthesis.
A) HPLC standards elution profile illustrates clear baseline separation between nucleotides and adenosine; the peak at ~31.5 minutes is a column impurity. B) Details of the HPLC method necessary to achieve this separation using the appropriate C18 reverse phase column and buffer system. C) TLC plate drawing demonstrates distinction between adenosine, AMP, and ADP using a solvent system of formic acid (0.15 M): EtOH (2:3) on a silica solid phase. D) TLC plate drawing illustrates separation between ADP and ATP using a solvent system of dioxane: isopropanol: NH4OH: H2O (4:2:3:4) on a silica solid phase.
Figure 18.
Figure 18.. HPLC and TLC Distinguish Nucleotides During anATP Synthesis.
A) HPLC standards elution profile illustrates clear baseline separation between nucleotides and adenosine; the peak at ~31.5 minutes is a column impurity. B) Details of the HPLC method necessary to achieve this separation using the appropriate C18 reverse phase column and buffer system. C) TLC plate drawing demonstrates distinction between adenosine, AMP, and ADP using a solvent system of formic acid (0.15 M): EtOH (2:3) on a silica solid phase. D) TLC plate drawing illustrates separation between ADP and ATP using a solvent system of dioxane: isopropanol: NH4OH: H2O (4:2:3:4) on a silica solid phase.
Figure 18.
Figure 18.. HPLC and TLC Distinguish Nucleotides During anATP Synthesis.
A) HPLC standards elution profile illustrates clear baseline separation between nucleotides and adenosine; the peak at ~31.5 minutes is a column impurity. B) Details of the HPLC method necessary to achieve this separation using the appropriate C18 reverse phase column and buffer system. C) TLC plate drawing demonstrates distinction between adenosine, AMP, and ADP using a solvent system of formic acid (0.15 M): EtOH (2:3) on a silica solid phase. D) TLC plate drawing illustrates separation between ADP and ATP using a solvent system of dioxane: isopropanol: NH4OH: H2O (4:2:3:4) on a silica solid phase.
Figure 18.
Figure 18.. HPLC and TLC Distinguish Nucleotides During anATP Synthesis.
A) HPLC standards elution profile illustrates clear baseline separation between nucleotides and adenosine; the peak at ~31.5 minutes is a column impurity. B) Details of the HPLC method necessary to achieve this separation using the appropriate C18 reverse phase column and buffer system. C) TLC plate drawing demonstrates distinction between adenosine, AMP, and ADP using a solvent system of formic acid (0.15 M): EtOH (2:3) on a silica solid phase. D) TLC plate drawing illustrates separation between ADP and ATP using a solvent system of dioxane: isopropanol: NH4OH: H2O (4:2:3:4) on a silica solid phase.
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