Discovery, structure and mechanism of a tetraether lipid synthase.

Archaea synthesize isoprenoid-based ether-linked membrane lipids, which enable them to withstand extreme environmental conditions, such as high temperatures, high salinity, and low or high pH values1-5. In some archaea, such as Methanocaldococcus jannaschii, these lipids are further modified by forming carbon-carbon bonds between the termini of two lipid tails within one glycerophospholipid to generate the macrocyclic archaeol or forming two carbon-carbon bonds between the termini of two lipid tails from two glycerophospholipids to generate the macrocycle glycerol dibiphytanyl glycerol tetraether (GDGT)1,2. GDGT contains two 40-carbon lipid chains (biphytanyl chains) that span both leaflets of the membrane, providing enhanced stability to extreme conditions. How these specialized lipids are formed has puzzled scientists for decades. The reaction necessitates the coupling of two completely inert sp3-hybridized carbon centres, which, to our knowledge, has not been observed in nature. Here we show that the gene product of mj0619 from M. jannaschii, which encodes a radical S-adenosylmethionine enzyme, is responsible for biphytanyl chain formation during synthesis of both the macrocyclic archaeol and GDGT membrane lipids6. Structures of the enzyme show the presence of four metallocofactors: three [Fe4S4] clusters and one mononuclear rubredoxin-like iron ion. In vitro mechanistic studies show that Csp3-Csp3 bond formation takes place on fully saturated archaeal lipid substrates and involves an intermediate bond between the substrate carbon and a sulfur of one of the [Fe4S4] clusters. Our results not only establish the biosynthetic route for tetraether formation but also improve the use of GDGT in GDGT-based paleoclimatology indices7-10.

Design of a Superpositively Charged Enzyme: Human Carbonic Anhydrase II Variant with Ferritin Encapsulation and Immobilization.

Supercharged proteins exhibit high solubility and other desirable properties, but no engineered superpositively charged enzymes have previously been made. Superpositively charged variants of proteins such as green fluorescent protein have been efficiently encapsulated within Archaeoglobus fulgidus thermophilic ferritin (AfFtn). Encapsulation by supramolecular ferritin can yield systems with a variety of sequestered cargo. To advance applications in enzymology and green chemistry, we sought a general method for supercharging an enzyme that retains activity and is compatible with AfFtn encapsulation. The zinc metalloenzyme human carbonic anhydrase II (hCAII) is an attractive encapsulation target based on its hydrolytic activity and physiologic conversion of carbon dioxide to bicarbonate. A computationally designed variant of hCAII contains positively charged residues substituted at 19 sites on the protein's surface, resulting in a shift of the putative net charge from -1 to +21. This designed hCAII(+21) exhibits encapsulation within AfFtn without the need for fusion partners or additional reagents. The hCAII(+21) variant retains esterase activity comparable to the wild type and spontaneously templates the assembly of AfFtn 24mers around itself. The AfFtn-hCAII(+21) host-guest complex exhibits both greater activity and thermal stability when compared to hCAII(+21). Upon immobilization on a solid support, AfFtn-hCAII(+21) retains enzymatic activity and exhibits an enhancement of activity at elevated temperatures.

Expression, purification, and characterization of a membrane-associated cyclic oligo-adenylate degrader from Sulfolobus islandicus.

Type III CRISPR-cas systems initiate cyclic oligo-adenylate (cOA) signaling to initiate immune response. Previously, we identified that a membrane-associated DHH-DHHA1 family protein from Sulfolobus islandicus efficiently degrades cOA. Here, we provide detailed protocols for expression and purification of the protein from its native host and a cOA degradation assay with the purified enzyme. The methodology should be of interest for researchers studying Sulfolobus, membrane-associated proteins, or type III CRISPR-cas systems. For complete details on the use and execution of this protocol, please refer to Zhao et al. (2020).

Optimal secretion of thermostable Beta-glucosidase in Bacillus subtilis by signal peptide optimization.

Commercial applications of ß-glucosidase (BGL) demands its purity and availability on a large scale. In the present study, we aim to optimize the expression and secretion of a thermostable BGL from Pyrococcus furiosus (PfuBGL) in B. subtilis strain RIK1285. Initial studies with base strain BV002 harboring aprE signal peptide (aprESP) showed PfuBGL yield of 0.743 ± 0.19 pNP U/ml only. A library of 173 different homologous SPs from B. subtilis 168 genome was fused with target PfuBGL gene (PF0073) in pBE-S vector and extracellularly expressed in RIK1285 strain to identify optimal SP for PfuBGL secretion. High-throughput screening of the resulting SP library for BGL activity with a synthetic substrate followed by systematic scaling of the clones yielded a gene construct with CitHSP reporting a sixteen fold enhancement of PfuBGL secretion in comparison to base strain. Batch fermentation (7.5 L scale) PfuBGL yield of the BV003 strain with CitHSP-PF0073 fusion was observed to be 12.08 ± 0.21 pNP U/ml with specific activity of 35.52 ± 0.53 U/mg. Thus, the study represents report on the secretory expression of thermostable PfuBGL using B. subtilis as a host organism and demonstrating its high potential for industrial production of any protein/enzyme.

Characterization of a novel type III alcohol dehydrogenase from Thermococcus barophilus Ch5.

The genome of the hyperthermophilic and piezophilic euryarchaeaon Thermococcus barophilus Ch5 encodes three putative alcohol dehydrogenases (Tba ADHs). Herein, we characterized Tba ADH547 biochemically and probed its catalytic mechanism by mutational studies. Our data demonstrate that Tba ADH547 can oxidize ethanol and reduce acetaldehyde at high temperature with the same optimal temperature (75 °C) and exhibit similar thermostability for oxidization and reduction reactions. However, Tba ADH547 has different optimal pH for oxidation and reduction: 8.5 for oxidation and 7.0 for reduction. Tba ADH547 is dependent on a divalent ion for its oxidation activity, among which Mn2+ is optimal. However, Tba ADH547 displays about 20% reduction activity without a divalent ion, and the maximal activity with Fe2+. Furthermore, Tba ADH547 showcases a strong substrate preference for 1-butanol and 1-hexanol over ethanol and other alcohols. Similarly, Tba ADH547 prefers butylaldehyde to acetaldehyde as its reduction substrate. Mutational studies showed that the mutations of residues D195, H199, H262 and H274 to Ala result in the significant activity loss of Tba ADH547, suggesting that residues D195, H199, H262 and H274 are responsible for catalysis. Overall, Tba ADH547 is a thermoactive ADH with novel biochemical characteristics, thereby allowing this enzyme to be a potential biocatalyst.

A Membrane-Associated DHH-DHHA1 Nuclease Degrades Type III CRISPR Second Messenger.

Type III CRISPR-Cas systems initiate an intracellular signaling pathway to confer immunity. The signaling pathway includes synthesis of cyclic oligo-adenylate (cOA) and activation of the RNase activity of type III accessory ribonuclease Csm6/Csx1 by cOA. After the immune response, cOA should be cleared on time to avoid constant cellular RNA degradation. In this study, we find a metal-dependent cOA degradation activity in Sulfolobus islandicus. The activity is associated with the cell membrane and able to accelerate cOA clearance at a high cOA level. Further, we show that a metal-dependent and membrane-associated DHH-DHHA1 family nuclease (MAD) rapidly cleaves cOA and deactivates Csx1 ribonuclease. The cOA degradation efficiency of MAD is much higher than the cellular ring nuclease. However, the subcellular organization may prevent it from degrading nascent cOA. Together, the data suggest that MAD acts as the second cOA degrader after the ring nuclease to remove diffused redundant cOA.

Gene cloning, expression enhancement in Escherichia coli and biochemical characterization of a highly thermostable amylomaltase from Pyrobaculum calidifontis.

Pcal_0768 gene encoding an amylomaltase, a 4-α-glucanatransferase belonging to family 77 of glycosyl hydrolases, from Pyrobaculum calidifontis was cloned and expressed in Escherichia coli. The recombinant protein was produced in E. coli in soluble and active form. However, the expression level was not very high. Analysis of the mRNA of initial seven codons at the 5'-end of the gene revealed the presence of a hair pin like secondary structure. This secondary structure was removed by site directed mutagenesis, without altering the amino acids, which resulted in enhanced expression of the cloned gene. Recombinant Pcal_0768 exhibited optimal amylomaltase activity at 80 °C and pH 6.9. Under these conditions, the specific activity was 690 U/ mg. Recombinant Pcal_0768 was highly thermostable with a half-life of 6 h at 100 °C. It exhibited the highest kcat value among the characterized glucanotransferases. No metal ions were required for activity or stability of the enzyme. Recombinant Pcal_0768 was successfully employed in the synthesis of modified starch for producing thermoreversible gel. To the best of our knowledge, till now this is the most thermostable enzyme among the characterized amylomaltases. High thermostability and starch modification potential make it a novel and distinct amylomaltase.

MALDI-TOF analysis of a novel extremophile peptide purified from Halarchaeum acidiphilum ASDL78 with antiarchaeal and antibacterial activities.

In hypersaline environments, halophilic archaea synthesize antimicrobial substances called halocins. There is a promise to make new drugs for antibiotic-resistant strains. Here, we report the antibacterial activity of a new haloarchaea selected from Lut Desert, Iran. A total of 38 isolated halophilic bacteria and archaea were screened for the antagonistic activity test of each strain against other bacterial and archaeal strains. Finally, a strain, recognized as Halarchaeum acidiphilum, with a fast grown strain and high antagonistic potential against different strains was identified by morphological, physiological, and molecular characteristics. The halocin was produced in a semisolid submerge medium and partially purified by heat treatments and molecular weight ultrafiltration cutoff (3, 50, and 10 kDa). It was a cell-free, heat-resistant (85°C for 2 h) protein with a molecular mass near to 20 kDa produced at the endpoint of logarithmic growth. The molecular weight of halocin was 17 kDa, and indicated no apparent homology with known halocins, suggesting that this might be a new halocin. Therefore, a new strain belonging to Halarchaeum genus was isolated and characterized here that produced an antimicrobial and anti-haloarchaea halocin.

Double Electron-Electron Resonance Shows That the Substrate but Not the Inhibitors Causes Disorder in the F/G Loop of CYP119 in Solution.

CYP119, a bacterial thermophilic protein from the cytochrome P450 superfamily, has previously been observed in three different conformations with different inhibitors bound using X-ray crystallography. The significance of these states in solution and in the function of the enzyme is not well-known. Double electron-electron resonance (DEER) was used to measure distances and distance distributions between spin-labels for populated conformational states in solution. DEER spectroscopy and molecular dynamics for the ligand-free enzyme suggest that the G helix is in a slightly different conformation than seen previously by crystallography, with the F/G loop in a slightly open conformation. Inhibitor-bound samples showed that this conformation remains as the predominant form, but partial conversion is indicated to a more closed conformation of the F/G loop. However, when the enzyme binds to lauric acid, the proposed substrate, it induces the conversion to a state that is characterized by increased disorder. We propose that similar to recent results with soluble CYP3A4, binding of the inhibitor to CYP119 is accompanied by only small changes in the enzyme structure, but substrate binding results in greater heterogeneity in the structure of the F/G loop region.

Complete sequential assignment and secondary structure prediction of the cannulae forming protein CanA from the hyperthermophilic archaeon Pyrodictium abyssi.

CanA from Pyrodictium abyssi forms a heat-resistant organic hollow-fiber network together with CanB and CanC. An N-terminally truncated construct of CanA (K1-CanA) gave NMR spectra of good quality that could be assigned by three-dimensional NMR methods on 15N and 13C-15N enriched protein. We assigned the chemical shifts of 96% of all backbone 1HN atoms, 98% of all backbone 15N atoms, 100% of all 13Cα atoms, 100% of all 1Hα atoms, 90% of all 13C' atoms, and 100% of the 13Cß atoms. Two short helices and 10 ß-strands are estimated from an analysis of the chemical shifts leading to a secondary structure content of K1-CanA of 6% helices, 44% ß-pleated sheets, and 50% coils.

Characterization of Extracellular Protease from the Haloarcheon Halococcus sp. Strain GUGFAWS-3 (MF425611).

Halococcus agarilyticus GUGFAWS-3 (MF425611) was isolated from a marine white sponge of Haliclona sp., inhabiting the rocks in the intertidal region of Anjuna, Goa, India. Uniquely, the microbe simultaneously produces two halo-extremozymes in 25% NaCl, namely protease and lipase at 49.5 ± 0.4 and 3.67 ± 0.02 (U mL-1), respectively. The protease is constitutively produced in starch mineral salts medium with consistent 4 ± 1.0 mm zone of enzyme production, regardless of the non-availability of protein as substrate. The ethanol precipitated enzyme on dialysis and Sephadex G-200 gel filtration chromatography was partially purified to 12.26-fold and was active between 20 and 80 °C, 0-5 M NaCl, and pH 3-13. Optimum activity, however, was at 70 °C, 3 M NaCl, and pH 7. The enzyme was thermo stable at 70 °C with 50.26 ± 2.40% of relative enzyme activity at 75 min. Furthermore, it was stable in the presence of polar and non-polar organic solvents, detergents, and hydrocarbons. Several metal cations enhanced its activity in the order of Ca2+ > Ni2+ > Fe3+ > Co2+ > Mg2+ > Cu2+ > Mn2+. Dependence of enzyme on cysteine; serine, and metal ions was confirmed by ß-mercaptoethanol; PMSF and EDTA, respectively which induced its partial inhibition. Additionally, protease inhibited in vitro biofilm formation in Staphylococcus aureus. Conclusively, the production of a neutral halo-thermophilic protease is reported for the first time in the genus Halococcus.

Characterization of an extremely thermo-active archaeal ß-glucosidase and its activity towards glucan and mannan in concert with an endoglucanase.

A metagenome from an enrichment culture of a hydrothermal vent sample taken at Vulcano Island (Italy) was sequenced and an endoglucanase-encoding gene (vul_cel5A) was identified in a previous work. Vul_Cel5A with maximal activity at 115 °C was characterized as the most heat-active endoglucanase to date. Based on metagenome sequences, genomes were binned and bin4 included vul_cel5A as well as a putative GH1 ß-glycosidase-encoding gene (vul_bgl1A) with highest identities to sequences from the archaeal genus Thermococcus. The recombinant ß-glucosidase Vul_Bgl1A produced in E. coli BL21 pQE-80L exhibited highest activity at 105 °C and pH 7.0 (76.12 ± 5.4 U/mg, 100%) using 4NP ß-D-glucopyranoside as substrate and 61% relative activity at 120 °C. Accordingly, Vul_Bgl1A represents one of the most heat-active ß-glucosidases to date. The enzyme has a broad substrate specificity with 155% activity towards 4NP ß-D-mannopyranoside in comparison with 4NP ß-D-glucopyranoside. Moreover, nearly complete hydrolysis of cellobiose was demonstrated. The enzyme exhibited a high glucose tolerance with 26% residual activity in presence of 2 M glucose and was furthermore activated at glucose concentrations of up to 0.5 M. When the endoglucanase Vul_Cel5A and the ß-glucosidase Vul_Bgl1A were applied simultaneously at 99 °C, 158% activity towards barley ß-glucan and 215% towards mannan were achieved compared with the activity of Vul_Cel5A alone (100%). Consequently, a significant increase in glucose formation was observed when both enzymes were incubated with ß-glucan and mannan suggesting a synergistic effect. Hence, the two archaeal extremozymes are ideal candidates for complete glucan and mannan saccharification at temperatures above the boiling point of water.

Purification and characterization of a novel thermophilic ß-galactosidase from Picrophilus torridus of potential industrial application.

Intracellular ß-galactosidase (E.C 3.2.1.23) produced by the thermoacidophilic archeon Picrophilus torridus DSM 9790 was purified to homogeneity using a combination of DEAE Sepharose, gel filtration, hydroxyapatite and chromatofocusing chromatographies. LC-MS/MS analysis was used to confirm the identity of the purified protein. The enzyme was found to be a homotrimer, with a molecular mass of 157.0 kDa and an isoelectric point of 5.7. To our knowledge, this enzyme has the lowest pH optimum of any intracellular ß-galactosidase characterized to date. Maximal activity was exhibited at acidic pH values of 5.0-5.5 and at 70 °C. The enzyme retained > 95% activity after heating to 70 °C for 1 h, or after incubation at pH 5.5 for 1 h. The enzyme may be of interest for high-temperature bioprocessing, such as in the production of lactulose. This investigation suggests that the ß-galactosidase activity produced by P. torridus is potentially more useful than several enzymes already characterized for such an application.

Microbial diversity analysis and screening for novel xylanase enzymes from the sediment of the Lobios Hot Spring in Spain.

Here, we describe the metagenome composition of a microbial community in a hot spring sediment as well as a sequence-based and function-based screening of the metagenome for identification of novel xylanases. The sediment was collected from the Lobios Hot Spring located in the province of Ourense (Spain). Environmental DNA was extracted and sequenced using Illumina technology, and a total of 3.6 Gbp of clean paired reads was produced. A taxonomic classification that was obtained by comparison to the NCBI protein nr database revealed a dominance of Bacteria (93%), followed by Archaea (6%). The most abundant bacterial phylum was Acidobacteria (25%), while Thaumarchaeota (5%) was the main archaeal phylum. Reads were assembled into contigs. Open reading frames (ORFs) predicted on these contigs were searched by BLAST against the CAZy database to retrieve xylanase encoding ORFs. A metagenomic fosmid library of approximately 150,000 clones was constructed to identify functional genes encoding thermostable xylanase enzymes. Function-based screening revealed a novel xylanase-encoding gene (XynA3), which was successfully expressed in E. coli BL21. The resulting protein (41 kDa), a member of glycoside hydrolase family 11 was purified and biochemically characterized. The highest activity was measured at 80 °C and pH 6.5. The protein was extremely thermostable and showed 94% remaining activity after incubation at 60 °C for 24 h and over 70% remaining activity after incubation at 70 °C for 24 h. Xylanolytic activity of the XynA3 enzyme was stimulated in the presence of ß-mercaptoethanol, dithiothreitol and Fe3+ ions. HPLC analysis showed that XynA3 hydrolyzes xylan forming xylobiose with lower proportion of xylotriose and xylose. Specific activity of the enzyme was 9080 U/mg for oat arabinoxylan and 5080 U/mg for beechwood xylan, respectively, without cellulase activity.

FruBPase II and ADP-PFK1 are involved in the modulation of carbon flow in the metabolism of carbohydrates in Methanosarcina acetivorans.

To enhance our understanding of the control of archaeal carbon central metabolism, a detailed analysis of the regulation mechanisms of both fructose1,6-bisphosphatase (FruBPase) and ADP-phosphofructokinase-1 (ADP-PFK1) was carried out in the methanogen Methanosarcina acetivorans. No correlations were found among the transcript levels of the MA_1152 and MA_3563 (frubpase type II and pfk1) genes, the FruBPase and ADP-PFK1 activities, and their protein contents. The kinetics of the recombinant FruBPase II and ADP-PFK1 were hyperbolic and showed simple mixed-type inhibition by AMP and ATP, respectively. Under physiological metabolite concentrations, the FruBPase II and ADP-PFK1 activities were strongly modulated by their inhibitors. To assess whether these enzymes were also regulated by a phosphorylation/dephosphorylation process, the recombinant enzymes and cytosolic-enriched fractions were incubated in the presence of commercial protein phosphatase or protein kinase. De-phosphorylation of ADP-PFK1 slightly decreased its activity (i.e. Vmax) and did not change its kinetic parameters and oligomeric state. Thus, the data indicated a predominant metabolic regulation of both FruBPase and ADP-PFK1 activities by adenine nucleotides and suggested high degrees of control on the respective pathway fluxes.

Recombinant expression, purification and characterization of acetylated LysargiNase from Escherichia coli with high activity and stability.

RATIONALE: LysargiNase is a novel characterized metalloprotease that can cleave the N-terminii of lysine or arginine residues. The peptides generated by LysargiNase are just mirrors to those generated by trypsin. These characteristics of LysargiNase provide a powerful tool for mass spectrometry (MS)-based proteomics research. A highly active and stable LysargiNase produced by an easy and inexpensive method could greatly benefit proteomics research. Here, we report the soluble recombinant expression, purification and acetyl modification of LysargiNase in Escherichia coli. METHODS: The coding sequence of LysargiNase with an enterokinase cleavage site at the N-terminus was inserted into plasmid pGEX-4 T-2 and transformed into E. coli BL21 (DE3). The strain was cultured in a 14-L fermenter with a working volume of 5 L. The protein expression was induced by adding isopropyl-ß-D-thiogalactoside (IPTG) to a final concentration of 1 mM. The recombinant LysargiNase was loaded onto a GSTrap and an on-column digestion was performed to remove the GST tag and was subsequently purified by chromatographic purification. In vitro acetylation of LysargiNase was performed by using acetic anhydride. The digestion efficiency and specificity of recombinant LysargiNase and acetylated LysargiNase were compared with simple protein substrate, human serum albumin (HSA), and a complex proteomic sample, yeast lysate, by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and liquid chromatography/tandem mass spectrometry (LC/MS/MS). RESULTS: Highly soluble expression of recombinant LysargiNase was achieved by plasmid pGEX-4 T-2 in E. coli BL21 (DE3). In addition, acetylation of purified LysargiNase significantly increased its resistance to autolysis, which resulted in a more complete digestion of proteomics samples and more identified peptides and proteins by LC/MS/MS. CONCLUSIONS: In this study, we constructed a highly soluble expression system for producing recombinant LysargiNase in E. coli, which gave tremendous advantages in the downstream purification process. We also confirmed that acetyl modification can increase the stability and activity of recombinant LysargiNase. The study provided a superior way to produce this powerful tool for proteomics research.

Antifungal and anti-biofilm activity of the first cryptic antimicrobial peptide from an archaeal protein against Candida spp. clinical isolates.

Candida species cause cutaneous and systemic infections with a high mortality rate, especially in immunocompromised patients. The emergence of resistance to the most common antifungal drugs, also due to biofilm formation, requires the development of alternative antifungal agents. The antimicrobial peptide VLL-28, isolated from an archaeal transcription factor, shows comparable antifungal activity against 10 clinical isolates of Candida spp. Using a fluoresceinated derivative of this peptide, we found that VLL-28 binds to the surface of planktonic cells. This observation suggested that it could exert its antifungal activity by damaging the cell wall. In addition, analyses performed on biofilms via confocal microscopy revealed that VLL-28 is differentially active on all the strains tested, with C. albicans and C. parapsilosis being the most sensitive ones. Notably, VLL-28 is the first example of an archaeal antimicrobial peptide that is active towards Candida spp. Thus, this points to archaeal microorganisms as a possible reservoir of novel antifungal agents.

TYW1: A Radical SAM Enzyme Involved in the Biosynthesis of Wybutosine Bases.

Transfer RNA is extensively modified by the actions of a variety of enzymes. The radical S-adenosyl-l-methionine enzyme TYW1 modifies tRNAPhe forming the characteristic tricyclic ring via the condensation of carbons 2 and 3 of pyruvate. This chapter details methods that are required for studies of TYW1.

Radical S-Adenosyl-l-Methionine (SAM) Enzyme Involved in the Maturation of the Nitrogenase Cluster.

Nitrogenase is the only known enzymatic system that converts atmospheric dinitrogen (N2) into bioavailable ammonia (NH3). The active-site cofactor responsible for this reactivity is a [(R-homocitrate)MoFe7S9C] cluster that is designated as the M-cluster. This important cofactor is assembled stepwise from a pair of [Fe4S4] clusters that become fused into a [Fe8S9C] core before additional refinements take place to complete the biosynthesis. NifB, a member of the radical S-adenosyl-l-methionine (SAM) superfamily, facilitates the conversion of the [Fe4S4] clusters (called the K-cluster) to the [Fe8S9C] core (called the L-cluster). This transformation includes a SAM-dependent carbide insertion with concomitant incorporation of an additional sulfur. While difficulties with the purification of NifB have historically prevented detailed biochemical analyses, we have developed a heterologous expression system in Escherichia coli that yields stable NifB proteins from various N2-fixing methanogenic organisms that can be used for studies. This chapter details the procedures necessary to prepare an active NifB protein. The methods used for the biochemical characterization of the SAM-dependent carbide insertion reactions are also described.

Methods for Studying the Radical SAM Enzymes in Diphthamide Biosynthesis.

Diphthamide is a unique posttranslational modification on translation elongation factor 2 (EF2) in archaea and eukaryotes. Biosynthesis of diphthamide was proposed to involve four steps. The first step is a CC bond forming reaction catalyzed by unique radical S-adenosylmethionine (SAM) enzymes. Classical radical SAM enzymes use SAM and [4Fe-4S] clusters to generate a 5'-deoxyadenynal radical and catalyze numerous reactions. Radical SAM enzymes in diphthamide biosynthesis cleave a different CS bond in SAM to generate a 3-amino-3-carboxypropyl radical and modify a histidine residue of substrate protein EF2. Here, we describe our investigations on these unique radical SAM enzymes, including the preparation, characterization, and activity assays we have developed.