Mutational and structural studies of (ßα)8-barrel fold methylene-tetrahydropterin reductases utilizing a common catalytic mechanism.

Methylene-tetrahydropterin reductases catalyze the reduction of a methylene to a methyl group bound to a reduced pterin as C1 carrier in various one-carbon (C1) metabolisms. F420-dependent methylene-tetrahydromethanopterin (methylene-H4MPT) reductase (Mer) and the flavin-independent methylene-tetrahydrofolate (methylene-H4F) reductase (Mfr) use a ternary complex mechanism for the direct transfer of a hydride from F420H2 and NAD(P)H to the respective methylene group, whereas FAD-dependent methylene-H4F reductase (MTHFR) uses FAD as prosthetic group and a ping-pong mechanism to catalyze the reduction of methylene-H4F. A ternary complex structure and a thereof derived catalytic mechanism of MTHFR is available, while no ternary complex structures of Mfr or Mer are reported. Here, Mer from Methanocaldococcus jannaschii (jMer) was heterologously produced and the crystal structures of the enzyme with and without F420 were determined. A ternary complex of jMer was modeled on the basis of the jMer-F420 structure and the ternary complex structure of MTHFR by superimposing the polypeptide after fixing hydride-transferring atoms of the flavins on each other, and by the subsequent transfer of the methyl-tetrahydropterin from MTHFR to jMer. Mutational analysis of four functional amino acids, which are similarly positioned in the three reductase structures, indicated despite the insignificant sequence identity, a common catalytic mechanism with a 5-iminium cation of methylene-tetrahydropterin as intermediate protonated by a shared glutamate. According to structural, mutational and phylogenetic analysis, the evolution of the three reductases most likely proceeds via a convergent development although a divergent scenario requiring drastic structural changes of the common ancestor cannot be completely ruled out.

Isolation of an H2 -dependent electron-bifurcating CO2 -reducing megacomplex with MvhB polyferredoxin from Methanothermobacter marburgensis.

In the hydrogenotrophic methanogenic pathway, formylmethanofuran dehydrogenase (Fmd) catalyzes the formation of formylmethanofuran through reducing CO2 . Heterodisulfide reductase (Hdr) provides two low potential electrons for the Fmd reaction using a flavin-based electron-bifurcating mechanism. [NiFe]-hydrogenase (Mvh) or formate dehydrogenase (Fdh) complexes with Hdr and provides electrons to Hdr from H2 and formate, or the reduced form of F420 , respectively. Recently, an Fdh-Hdr complex was purified as a 3-MDa megacomplex that contained Fmd, and its three-dimensional structure was elucidated by cryo-electron microscopy. In contrast, the Mvh-Hdr complex has been characterized only as a complex without Fmd. Here, we report the isolation and characterization of a 1-MDa Mvh-Hdr-Fmd megacomplex from Methanothermobacter marburgensis. After anion-exchange and hydrophobic chromatography was performed, the proteins with Hdr activity eluted in the 1- and 0.5-MDa fractions during size exclusion chromatography. Considering the apparent molecular mass and the protein profile in the fractions, the 1-MDa megacomplex was determined to be a dimeric Mvh-Hdr-Fmd complex. The megacomplex fraction contained a polyferredoxin subunit MvhB, which contains 12 [4Fe-4S]-clusters. MvhB polyferredoxin has never been identified in the previously purified Mvh-Hdr and Fmd preparations, suggesting that MvhB polyferredoxin is stabilized by the binding between Mvh-Hdr and Fmd in the Mvh-Hdr-Fmd complex. The purified Mvh-Hdr-Fmd megacomplex catalyzed electron-bifurcating reduction of [13 C]-CO2 to form [13 C]-formylmethanofuran in the absence of extrinsic ferredoxin. These results demonstrated that the subunits in the Mvh-Hdr-Fmd megacomplex are electronically connected for the reduction of CO2 , which likely involves MvhB polyferredoxin as an electron relay.

Structural and mechanistic basis of the central energy-converting methyltransferase complex of methanogenesis.

Methanogenic archaea inhabiting anaerobic environments play a crucial role in the global biogeochemical material cycle. The most universal electrogenic reaction of their methane-producing energy metabolism is catalyzed by N    5-methyl-tetrahydromethanopterin: coenzyme M methyltransferase (MtrABCDEFGH), which couples the vectorial Na+ transport with a methyl transfer between the one-carbon carriers tetrahydromethanopterin and coenzyme M via a vitamin B12 derivative (cobamide) as prosthetic group. We present the 2.08 Šcryo-EM structure of Mtr(ABCDEFG)3 composed of the central Mtr(ABFG)3 stalk symmetrically flanked by three membrane-spanning MtrCDE globes. Tetraether glycolipids visible in the map fill gaps inside the multisubunit complex. Putative coenzyme M and Na+ were identified inside or in a side-pocket of a cytoplasmic cavity formed within MtrCDE. Its bottom marks the gate of the transmembrane pore occluded in the cryo-EM map. By integrating Alphafold2 information, functionally competent MtrA-MtrH and MtrA-MtrCDE subcomplexes could be modeled and thus the methyl-tetrahydromethanopterin demethylation and coenzyme M methylation half-reactions structurally described. Methyl-transfer-driven Na+ transport is proposed to be based on a strong and weak complex between MtrCDE and MtrA carrying vitamin B12, the latter being placed at the entrance of the cytoplasmic MtrCDE cavity. Hypothetically, strongly attached methyl-cob(III)amide (His-on) carrying MtrA induces an inward-facing conformation, Na+ flux into the membrane protein center and finally coenzyme M methylation while the generated loosely attached (or detached) MtrA carrying cob(I)amide (His-off) induces an outward-facing conformation and an extracellular Na+ outflux. Methyl-cob(III)amide (His-on) is regenerated in the distant active site of the methyl-tetrahydromethanopterin binding MtrH implicating a large-scale shuttling movement of the vitamin B12-carrying domain.

Acyl and CO Ligands in the [Fe]-Hydrogenase Cofactor Scramble upon Photolysis.

[Fe]-hydrogenase harbors the iron-guanylylpyridinol (FeGP) cofactor, in which the Fe(II) complex contains acyl-carbon, pyridinol-nitrogen, cysteine-thiolate and two CO as ligands. Irradiation with UV-A/blue light decomposes the FeGP cofactor to a 6-carboxymethyl-4-guanylyl-2-pyridone (GP) and other components. Previous in vitro biosynthesis experiments indicated that the acyl- and CO-ligands in the FeGP cofactor can scramble, but whether scrambling occurred during biosynthesis or photolysis was unclear. Here, we demonstrate that the [18 O1 -carboxy]-group of GP is incorporated into the FeGP cofactor by in vitro biosynthesis. MS/MS analysis of the 18 O-labeled FeGP cofactor revealed that the produced [18 O1 ]-acyl group is not exchanged with a CO ligand of the cofactor, indicating that the acyl and CO ligands are scrambled during photolysis rather than biosynthesis, which ruled out any biosynthesis mechanisms allowing acyl/CO ligands scrambling. Time-resolved infrared spectroscopy indicated that an acyl-Fe(CO)3 intermediate is formed during photolysis, in which scrambling of the CO and acyl ligands can occur. This finding also suggests that the light-excited FeGP cofactor has a higher affinity for external CO. These results contribute to our understanding of the biosynthesis and photosensitive properties of this unique H2 -activating natural complex.

[Fe]-Hydrogenase, Cofactor Biosynthesis and Engineering.

[Fe]-hydrogenase catalyzes the heterolytic cleavage of H2 and reversible hydride transfer to methenyl-tetrahydromethanopterin. The iron-guanylylpyridinol (FeGP) cofactor is the prosthetic group of this enzyme, in which mononuclear Fe(II) is ligated with a pyridinol and two CO ligands. The pyridinol ligand fixes the iron by an acyl carbon and a pyridinol nitrogen. Biosynthetic proteins for this cofactor are encoded in the hmd co-occurring (hcg) genes. The function of HcgB, HcgC, HcgD, HcgE, and HcgF was studied by using structure-to-function analysis, which is based on the crystal structure of the proteins and subsequent enzyme assays. Recently, we reported the catalytic properties of HcgA and HcgG, novel radical S-adenosyl methionine enzymes, by using an in vitro biosynthesis assay. Here, we review the properties of [Fe]-hydrogenase and the FeGP cofactor, and the biosynthesis of the FeGP cofactor. Finally, we discuss the expected engineering of [Fe]-hydrogenase and the FeGP cofactor.

Crystal structure of FAD-independent methylene-tetrahydrofolate reductase from Mycobacterium hassiacum.

FAD-independent methylene-tetrahydrofolate (methylene-H4 F) reductase (Mfr), recently identified in mycobacteria, catalyzes the reduction of methylene-H4 F to methyl-H4 F with NADH as hydride donor by a ternary complex mechanism. This biochemical reaction corresponds to that of the ubiquitous FAD-dependent methylene-H4 F reductase (MTHFR), although the latter uses a ping-pong mechanism with the prosthetic group as intermediate hydride carrier. Comparative genomics and genetic analyses indicated that Mfr is indispensable for the growth of Mycobacterium tuberculosis, which lacks the MTHFR encoding gene. Therefore, Mfr appears to be an excellent target for the design of antimycobacterial drugs. Here, we report the heterologous production, enzymological characterization, and the crystal structure of Mfr from the thermophilic mycobacterium Mycobacterium hassiacum (hMfr), which shows 78% sequence identity to Mfr from M. tuberculosis. Although hMfr and MTHFR have minor sequence identity and different catalytic mechanisms, their structures are highly similar, thus suggesting a divergent evolution of Mfr and MTHFR from a common ancestor. Most of the important active site residues of MTHFR are conserved and equivalently positioned in the tertiary structure of hMfr. The Glu9Gln variant of hMfr exhibits a drastic reduction of the catalytic activity, which supports the predicted function of the glutamate residue as proton donor in both hMfr and MTHFR. Thus, highly similar binding modes for the C1 -carriers and the reducing agents in hMfr and MTHFR are assumed.

The Function of Two Radical-SAM Enzymes, HcgA and HcgG, in the Biosynthesis of the [Fe]-Hydrogenase Cofactor.

In the biosynthesis of the iron-guanylylpyridinol (FeGP) cofactor, 6-carboxymethyl-5-methyl-4-hydroxy-2-pyridinol (1) is 3-methylated to form 2, then 4-guanylylated to form 3, and converted into the full cofactor. HcgA-G proteins catalyze the biosynthetic reactions. Herein, we report the function of two radical S-adenosyl methionine enzymes, HcgA and HcgG, as uncovered by in vitro complementation experiments and the use of purified enzymes. In vitro biosynthesis using the cell extract from the Methanococcus maripaludis ΔhcgA strain was complemented with HcgA or precursors 1, 2 or 3. The results suggested that HcgA catalyzes the biosynthetic reaction that forms 1. We demonstrated the formation of 1 by HcgA using the 3 kDa cell extract filtrate as the substrate. Biosynthesis in the ΔhcgG system was recovered by HcgG but not by 3, which indicated that HcgG catalyzes the reactions after the biosynthesis of 3. The data indicated that HcgG contributes to the formation of CO and completes biosynthesis of the FeGP cofactor.

In Vitro Biosynthesis of the [Fe]-Hydrogenase Cofactor Verifies the Proposed Biosynthetic Precursors.

In the FeGP cofactor of [Fe]-hydrogenase, low-spin FeII is in complex with two CO ligands and a pyridinol derivative; the latter ligates the iron with a 6-acylmethyl substituent and the pyridinol nitrogen. A guanylylpyridinol derivative, 6-carboxymethyl-3,5-dimethyl-4-guanylyl-2-pyridinol (3), is produced by the decomposition of the FeGP cofactor under irradiation with UV-A/blue light and is also postulated to be a precursor of FeGP cofactor biosynthesis. HcgC and HcgB catalyze consecutive biosynthesis steps leading to 3. Here, we report an in vitro biosynthesis assay of the FeGP cofactor using the cell extract of the ΔhcgBΔhcgC strain of Methanococcus maripaludis, which does not biosynthesize 3. We chemically synthesized pyridinol precursors 1 and 2, and detected the production of the FeGP cofactor from 1, 2 and 3. These results indicated that 1, 2 and 3 are the precursors of the FeGP cofactor, and the carboxy group of 3 is converted to the acyl ligand.

Influence of Divalent Cations in the Protein Crystallization Process Assisted by Lanthanide-Based Additives.

The use of lanthanide complexes as powerful auxiliaries for biocrystallography prompted us to systematically analyze the influence of the commercial crystallization kit composition on the efficiency of two lanthanide additives: [Eu(DPA)3]3- and Tb-Xo4. This study reveals that the tris(dipicolinate) complex presents a lower chemical stability and a strong tendency toward false positives, which are detrimental for its use in a high-throughput robotized crystallization platform. In particular, the crystal structures of (Mg(H2O)6)3[Eu(DPA)3]2·7H2O (1), {(Ca(H2O)4)3[Eu(DPA)3]2}n·10nH2O (2), and {Cu(DPA)(H2O)2}n (3), resulting from spontaneous crystallization in the presence of a divalent alkaline-earth cation and transmetalation, are reported. On the other hand, Tb-Xo4 is perfectly soluble in the crystallization media, stable in the presence of alkaline-earth dications, and slowly decomposes (within days) by transmetalation with transition metals. The original structure of [Tb4L4(H2O)4]Cl4·15H2O (4) is also described, where L represents a bis(pinacolato)triazacyclononane ligand. This paper also highlights a potential synergy of interactions between Tb-Xo4 and components of the crystallization mixtures, leading to the formation of complex adducts like {AdkA/Tb-Xo4/Mg2+/glycerol} in the protein binding sites. The observation of such multicomponent adducts illustrated the complexity and versatility of the supramolecular chemistry occurring at the surface of the proteins.

Three-megadalton complex of methanogenic electron-bifurcating and CO2-fixing enzymes.

The first reaction of the methanogenic pathway from carbon dioxide (CO2) is the reduction and condensation of CO2 to formyl-methanofuran, catalyzed by formyl-methanofuran dehydrogenase (Fmd). Strongly reducing electrons for this reaction are generated by heterodisulfide reductase (Hdr) in complex with hydrogenase or formate dehydrogenase (Fdh) using a flavin-based electron-bifurcation mechanism. Here, we report enzymological and structural characterizations of Fdh-Hdr-Fmd complexes from Methanospirillum hungatei. The complexes catalyze this reaction using electrons from formate and the reduced form of the electron carrier F420. Conformational changes in HdrA mediate electron bifurcation, and polyferredoxin FmdF directly transfers electrons to the CO2 reduction site, as evidenced by methanofuran-dependent flavin-based electron bifurcation even without free ferredoxin, a diffusible electron carrier between Hdr and Fmd. Conservation of Hdr and Fmd structures suggests that this complex is common among hydrogenotrophic methanogens.

Diversifying Metal-Ligand Cooperative Catalysis in Semi-Synthetic [Mn]-Hydrogenases.

The reconstitution of [Mn]-hydrogenases using a series of MnI complexes is described. These complexes are designed to have an internal base or pro-base that may participate in metal-ligand cooperative catalysis or have no internal base or pro-base. Only MnI complexes with an internal base or pro-base are active for H2 activation; only [Mn]-hydrogenases incorporating such complexes are active for hydrogenase reactions. These results confirm the essential role of metal-ligand cooperation for H2 activation by the MnI complexes alone and by [Mn]-hydrogenases. Owing to the nature and position of the internal base or pro-base, the mode of metal-ligand cooperation in two active [Mn]-hydrogenases is different from that of the native [Fe]-hydrogenase. One [Mn]-hydrogenase has the highest specific activity of semi-synthetic [Mn]- and [Fe]-hydrogenases. This work demonstrates reconstitution of active artificial hydrogenases using synthetic complexes differing greatly from the native active site.

Krypton-derivatization highlights O2-channeling in a four-electron reducing oxidase.

F420H2-oxidase (FprA) catalyses the four-electron reduction of O2 to 2H2O using the reduced form of F420 as electron donor. The hydrophobic O2-channel detected by Kr-derivatization and the concerted movement of a gating loop could contribute to prevent unwanted side-reaction between the catalytic intermediates and solvents, therefore preventing reactive oxygen species formation.

Structural Basis of Hydrogenotrophic Methanogenesis.

Most methanogenic archaea use the rudimentary hydrogenotrophic pathway-from CO2 and H2 to methane-as the terminal step of microbial biomass degradation in anoxic habitats. The barely exergonic process that just conserves sufficient energy for a modest lifestyle involves chemically challenging reactions catalyzed by complex enzyme machineries with unique metal-containing cofactors. The basic strategy of the methanogenic energy metabolism is to covalently bind C1 species to the C1 carriers methanofuran, tetrahydromethanopterin, and coenzyme M at different oxidation states. The four reduction reactions from CO2 to methane involve one molybdopterin-based two-electron reduction, two coenzyme F420-based hydride transfers, and one coenzyme F430-based radical process. For energy conservation, one ion-gradient-forming methyl transfer reaction is sufficient, albeit supported by a sophisticated energy-coupling process termed flavin-based electron bifurcation for driving the endergonic CO2 reduction and fixation. Here, we review the knowledge about the structure-based catalytic mechanism of each enzyme of hydrogenotrophic methanogenesis.

The Hydride Transfer Process in NADP-dependent Methylene-tetrahydromethanopterin Dehydrogenase.

NADP-dependent methylene-tetrahydromethanopterin (methylene-H4MPT) dehydrogenase (MtdA) catalyzes the reversible dehydrogenation of methylene-H4MPT to form methenyl-H4MPT+ by using NADP+ as a hydride acceptor. This hydride transfer reaction is involved in the oxidative metabolism from formaldehyde to CO2 in methylotrophic and methanotrophic bacteria. Here, we report on the crystal structures of the ternary MtdA-substrate complexes from Methylorubrum extorquens AM1 obtained in open and closed forms. Their conversion is accomplished by opening/closing the active site cleft via a 15° rotation of the NADP, relative to the pterin domain. The 1.08 Å structure of the closed and active enzyme-NADP-methylene-H4MPT complex allows a detailed geometric analysis of the bulky substrates and a precise prediction of the hydride trajectory. Upon domain closure, the bulky substrate rings become compressed resulting in a tilt of the imidazolidine group of methylene-H4MPT that optimizes the geometry for hydride transfer. An additional 1.5 Å structure of MtdA in complex with the nonreactive NADP+ and methenyl-H4MPT+ revealed an extremely short distance between nicotinamide-C4 and imidazoline-C14a of 2.5 Å, which demonstrates the strong pressure imposed. The pterin-imidazolidine-phenyl butterfly angle of methylene-H4MPT bound to MtdA is smaller than that in the enzyme-free state but is similar to that in H2- and F420-dependent methylene-H4MPT dehydrogenases. The concept of compression-driven hydride transfer including quantum mechanical hydrogen tunneling effects, which are established for flavin- and NADP-dependent enzymes, can be expanded to hydride-transferring H4MPT-dependent enzymes.

Methanogenesis involves direct hydride transfer from H2 to an organic substrate.

Certain anaerobic microorganisms evolved a mechanism to use H2 as a reductant in their energy metabolisms. For these purposes, the microorganisms developed H2-activating enzymes, which are aspirational catalysts in a sustainable hydrogen economy. In the case of the hydrogenotrophic pathway performed by methanogenic archaea, 8e- are extracted from 4H2 and used as reducing equivalents to convert CO2 into CH4. Under standard cultivation conditions, these archaea express [NiFe]-hydrogenases, which are Ni-dependent and Fe-dependent enzymes and heterolytically cleave H2 into 2H+ and 2e-, the latter being supplied into the central metabolism. Under Ni-limiting conditions, F420-reducing [NiFe]-hydrogenases are downregulated and their functions are predominantly taken over by an upregulated [Fe]-hydrogenase. Unique in biology, this Fe-dependent hydrogenase cleaves H2 and directly transfers H- to an imidazolium-containing substrate. [Fe]-hydrogenase activates H2 at an Fe cofactor ligated by two CO molecules, an acyl group, a pyridinol N atom and a cysteine thiolate as the central constituent. This Fe centre has inspired chemists to not only design synthetic mimics to catalytically cleave H2 in solution but also for incorporation into apo-[Fe]-hydrogenase to give semi-synthetic proteins. This Perspective describes the enzymes involved in hydrogenotrophic methanogenesis, with a focus on those performing the reduction steps. Of these, we describe [Fe]-hydrogenases in detail and cover recent progress in their synthetic modelling.

Methylofuran is a prosthetic group of the formyltransferase/hydrolase complex and shuttles one-carbon units between two active sites.

Methylotrophy, the ability of microorganisms to grow on reduced one-carbon substrates such as methane or methanol, is a feature of various bacterial species. The prevailing oxidation pathway depends on tetrahydromethanopterin (H4MPT) and methylofuran (MYFR), an analog of methanofuran from methanogenic archaea. Formyltransferase/hydrolase complex (Fhc) generates formate from formyl-H4MPT in two consecutive reactions where MYFR acts as a carrier of one-carbon units. Recently, we chemically characterized MYFR from the model methylotroph Methylorubrum extorquens and identified an unusually long polyglutamate side chain of up to 24 glutamates. Here, we report on the crystal structure of Fhc to investigate the function of the polyglutamate side chain in MYFR and the relatedness of the enzyme complex with the orthologous enzymes in archaea. We identified MYFR as a prosthetic group that is tightly, but noncovalently, bound to Fhc. Surprisingly, the structure of Fhc together with MYFR revealed that the polyglutamate side chain of MYFR is branched and contains glutamates with amide bonds at both their α- and γ-carboxyl groups. This negatively charged and branched polyglutamate side chain interacts with a cluster of conserved positively charged residues of Fhc, allowing for strong interactions. The MYFR binding site is located equidistantly from the active site of the formyltransferase (FhcD) and metallo-hydrolase (FhcA). The polyglutamate serves therefore an additional function as a swinging linker to shuttle the one-carbon carrying amine between the two active sites, thereby likely increasing overall catalysis while decreasing the need for high intracellular MYFR concentrations.

X-ray Crystallography and Vibrational Spectroscopy Reveal the Key Determinants of Biocatalytic Dihydrogen Cycling by [NiFe] Hydrogenases.

[NiFe] hydrogenases are complex model enzymes for the reversible cleavage of dihydrogen (H2 ). However, structural determinants of efficient H2 binding to their [NiFe] active site are not properly understood. Here, we present crystallographic and vibrational-spectroscopic insights into the unexplored structure of the H2 -binding [NiFe] intermediate. Using an F420 -reducing [NiFe]-hydrogenase from Methanosarcina barkeri as a model enzyme, we show that the protein backbone provides a strained chelating scaffold that tunes the [NiFe] active site for efficient H2 binding and conversion. The protein matrix also directs H2 diffusion to the [NiFe] site via two gas channels and allows the distribution of electrons between functional protomers through a subunit-bridging FeS cluster. Our findings emphasize the relevance of an atypical Ni coordination, thereby providing a blueprint for the design of bio-inspired H2 -conversion catalysts.

Protein crystal structure determination with the crystallophore, a nucleating and phasing agent.

Obtaining crystals and solving the phase problem remain major hurdles encountered by bio-crystallographers in their race to obtain new high-quality structures. Both issues can be overcome by the crystallophore, Tb-Xo4, a lanthanide-based molecular complex with unique nucleating and phasing properties. This article presents examples of new crystallization conditions induced by the presence of Tb-Xo4. These new crystalline forms bypass crystal defects often encountered by crystallographers, such as low-resolution diffracting samples or crystals with twinning. Thanks to Tb-Xo4's high phasing power, the structure determination process is greatly facilitated and can be extended to serial crystallography approaches.

A catalytically active [Mn]-hydrogenase incorporating a non-native metal cofactor.

Nature carefully selects specific metal ions for incorporation into the enzymes that catalyse the chemical reactions necessary for life. Hydrogenases, enzymes that activate molecular H2, exclusively utilize Ni and Fe in [NiFe]-, [FeFe]- and [Fe]-hydrogeanses. However, other transition metals are known to activate or catalyse the production of hydrogen in synthetic systems. Here, we report the development of a biomimetic model complex of [Fe]-hydrogenase that incorporates a Mn, as opposed to a Fe, metal centre. This Mn complex is able to heterolytically cleave H2 as well as catalyse hydrogenation reactions. The incorporation of the model into an apoenzyme of [Fe]-hydrogenase results in a [Mn]-hydrogenase with an enhanced occupancy-normalized activity over an analogous semi-synthetic [Fe]-hydrogenase. These findings demonstrate a non-native metal hydrogenase that shows catalytic functionality and that hydrogenases based on a manganese active site are viable.

Crystal structure of archaeal HMG-CoA reductase: insights into structural changes of the C-terminal helix of the class-I enzyme.

3-hydroxy-3-methylglutaryl-CoA reductase (HMGR) catalyses the last step in mevalonate biosynthesis. HMGR is the target of statin inhibitors that regulate cholesterol concentration in human blood. Here, we report the properties and structures of HMGR from an archaeon Methanothermococcus thermolithotrophicus (mHMGR). The structures of the apoenzyme and the NADPH complex are highly similar to those of human HMGR. A notable exception is C-terminal helix (Lα10-11) that is straight in both mHMGR structures. This helix is kinked and closes the active site in the human enzyme ternary complex, pointing to a substrate-induced structural rearrangement of C-terminal in class-I HMGRs during the catalytic cycle.