X-ray analysis of ferredoxin from Spirulina platensis. II. Chelate structure of active center.

A chloroplast-type ferredoxin from Spirulina platenis crystallized in an orthorhombic system, space group C2221, with cell dimensions a=62.32, b=28.51, and c=108.08 A. The electron density map at 2.8 A resolution was prepared by using the best phase angles determined by the single isomorphous replacement method coupled with the anomalous dispersion method. The chelating structure of the acitve center was revealed as follows. Of the six cysteinyl residues in the molecule, Cys 41, Cys 4k, Cys 49, and Cys 79 are involved in the active center. Cys 41 and Cys 46 are coordinated to one iron atom, and Cys 49 and Cys 79 to the other iron atom. Only one of these cysteinyl residues, Cys 79, is comparatively apart from the other three in the amino acids sequence of the molecule, as found in the case of bacterial ferredoxin. It appears that the NH....S hydrogen bonds are around the active center, as in other non-heme iron sulfur proteins.     

Dual modes of modification of hepatitis A virus 3C protease by a serine-derived beta-lactone: selective crystallization and formation of a functional catalytic triad in the active site

Hepatitis A virus (HAV) 3C proteinase is a member of the picornain cysteine proteases responsible for the processing of the viral polyprotein, a function essential for viral maturation and infectivity. This and its structural similarity to other 3C and 3C-like proteases make it an attractive target for the development of antiviral drugs. Previous solution NMR studies have shown that a Cys24Ser (C24S) variant of HAV 3C protein, which displays catalytic properties indistinguishable from the native enzyme, is irreversibly inactivated by N-benzyloxycarbonyl-l-serine-beta-lactone (1a) through alkylation of the sulfur atom at the active site Cys172. However, crystallization of an enzyme-inhibitor adduct from the reaction mixture followed by X-ray structural analysis shows only covalent modification of the epsilon2-nitrogen of the surface His102 by the beta-lactone with no reaction at Cys172. Re-examination of the heteronuclear multiple quantum coherence (HMQC) NMR spectra of the enzyme-inhibitor mixture indicates that dual modes of single covalent modification occur with a >/=3:1 ratio of S-alkylation of Cys172 to N-alkylation of His102. The latter product crystallizes readily, probably due to the interaction between the phenyl ring of the N-benzyloxycarbonyl (N-Cbz) moiety and a hydrophobic pocket of a neighboring protein molecule in the crystal. Furthermore, significant structural changes are observed in the active site of the 3C protease, which lead to the formation of a functional catalytic triad with Asp84 accepting one hydrogen bond from His44. Although the 3C protease modified at Cys172 is catalytically inactive, the singly modified His102 N(epsilon2)-alkylated protein displays a significant level of enzymatic activity, which can be further modified/inhibited by N-iodoacetyl-valine-phenylalanine-amide (IVF) (in solution and in crystal) or excessive amount of the same beta-lactone inhibitor (in solution). The success of soaking IVF into HAV 3C-1a crystals demonstrates the usefulness of this new crystal form in the study of enzyme-inhibitor interactions in the proteolytic active site.     

Cysteine reactivity in Thermoanaerobacter brockii alcohol dehydrogenase.

The free cysteine residues in the extremely thermophilic Thermoanaerobacter brockii alcohol dehydrogenase (TBADH) were characterized using selective chemical modification with the stable nitroxyl biradical bis(1-oxy-2,2,5,5-tetramethyl-3-imidazoline-4-yl)disulfide, via a thiol-disulfide exchange reaction and with 2[14C]iodoacetic acid, via S-alkylation. The respective reactions were monitored by electron paramagenetic resonance (EPR) and by the incorporation of the radioactive label. In native TBADH, the rapid modification of one cysteine residue per subunit by the biradical and the concomitant loss of catalytic activity was reversed by DTT. NADP protected the enzyme from both modification and inactivation by the biradical. RPLC fingerprint analysis of reduced and S-carboxymethylated lysyl peptides from the radioactive alkylated enzyme identified Cys 203 as the readily modified residue. A second cysteine residue was rapidly modified with both modification reagents when the catalytic zinc was removed from the enzyme by o-phenanthroline. This cysteine residue, which could serve as a putative ligand to the active-site zinc atom, was identified as Cys 37 in RPLC. The EPR data suggested a distance of < or 10 A between Cys 37 and Cys 203. Although Cys 283 and Cys 295 were buried within the protein core and were not accessible for chemical modification, the two residues were oxidized to cystine when TBADH was heated at 75 degrees C, forming a disulfide bridge that was not present in the native enzyme, without affecting either enzymatic activity or thermal stability. The status of these cysteine residues was verified by site directed mutagenesis.     

Alteration of the iron-sulfur cluster composition of Escherichia coli dimethyl sulfoxide reductase by site-directed mutagenesis.

We have used site-directed mutagenesis to alter the [Fe-S] cluster composition of Escherichia coli dimethyl sulfoxide (DMSO) reductase (DmsABC). The electron-transfer subunit (DmsB) of this enzyme contains 16 Cys residues arranged in 4 groups (I-IV) which provide ligands to 4 [4Fe-4S] clusters [Cammack, R., & Weiner, J. H. (1990) Biochemistry 29, 8410-8416]. Strong homologies exist between these Cys groups and the four Cys groups of the electron-transfer subunit (NarH) of E. coli nitrate reductase (NarGHJI), which contains a [3Fe-4S] cluster in addition to multiple [4Fe-4S] clusters. The Cys group primarily involved in providing ligands to the [3Fe-4S] cluster of NarH has a Trp residue at a position equivalent to Cys102 of DmsB. We have mutated Cys102 to Trp, Ser, Tyr, and Phe and have investigated the altered enzymes in terms of their enzymatic activities and EPR properties. The mutant enzymes do not support electron transfer from menaquinol to DMSO, although they retain high rates of electron transport from reduced benzyl viologen to DMSO. The mutations cause major changes in the EPR properties of the enzyme in the fully reduced and oxidized states. In the oxidized state, new species are observed in all the mutants; these have spectral features comprising a peak at g = 2.03 (gz) and a peak-trough at g = 2.00 (gxy). The temperature dependencies, microwave power dependencies, and spin quantitations of these species are consistent with the Trp102, Ser102, Phe102, and Tyr102 mutations causing conversion of one of the [4Fe-4S] clusters present in the wild-type enzyme into [3Fe-4S] clusters in the mutant enzymes.     

Thiaisoleucine and protein synthesis.

Thiaisoleucine is an isoleucine analogue having the gamma-methylene group of the valerianic carbon chain substituted by a sulphur atom. It has been demonstrated that thiaisoleucine is activated and transferred to tRNAIle by rat liver aminoacyl-tRNA synthetase and inhibits isoleucine incorporation into polypeptides in protein synthesizing systems from rat liver or rabbit reticulocytes, whereas it does not affect either leucine incorporation or ribosome run-off or polypeptide chain elongation rate. All tests were performed in comparison with O-methyl-threonine, an isoleucine analogue with the gamma-methylene group substituted by an oxygen atom. In all the reactions studied, both thiaisoleucine and O-methyl-threonine act as competitive inhibitors of isoleucine. With respect to O-methyl-threonine, thiaisoleucine shows higher activity as an isoleucine inhibitor.     

The reductive desulfurization of Met and Cys residues in bovine RNase A is associated with trans lipids formation in a mimetic model of biological membranes

Damage to bovine pancreatic RNase A, due to the H* atom and/or solvated electron attack at protein sulfur-containing residues, was investigated by Raman spectroscopy and mass spectrometry techniques. To the already known desulfurization process affecting Met residues, novel reactivity was observed involving disulfide moieties, leading to the chemical transformation of Cys into Ala residues. Mapping experiments demonstrated that desulfurization selectively affected Met79, Cys110, Cys58 and Cys72 during first stages of reaction. While this reaction was performed on protein species added to large unilamellar vescicles, desulfurization yielded sulfur radicals able to induce a cis-trans isomerization of lipids at the onset of irradiation. These findings reveal new scenarios on reactions generated by radical stressing conditions, suggesting the need for specific assays and for future investigations to detect these modifications in proteins and lipids within challenged cells.     

The structure of bovine liver rhodanese. II. The active site in the sulfur-substituted and the sulfur-free enzyme.

X-ray studies at 2.5 Å resolution show that the active site of bovine liver rhodanese is a depression between the two domains. In sulfur-substituted rhodanese the density of the essential Cys247 corresponds with that of a persulfide. Both sulfur atoms are interacting via hydrogen bonds with several peptide NH and side-chain OH groups. One side of the active site pocket contains mainly hydrophylic, the other side mainly hydrophobic residues. None of these hydrophylic or hydrophobic groups appears to interact strongly with the persulfide.Crystals of the sulfur-substituted enzyme were treated with cyanide, a sulfur acceptor. Subsequent difference Fourier studies show that the extra sulfur atom has been removed. Only minor conformational differences appear to exist between the two rhodanese species studied. These are a movement of the Sγ atom of Cys247 and some rearrangement of solvent molecules near the active site.The combination of these observations with the results of experiments performed by other investigators suggest a mechanism for sulfur transfer by rhodanese in which the thiol group of Cys247 is the essential nucleophile, whereas the positive charges on Arg186 and Lys249 act in various ways as “electrophilic assistants”. The transition state and the persulfide in the sulfur-substituted enzyme are stabilized by several hydrogen bonds.     

H-bonding maintains the active site of type 1 copper proteins: site-directed mutagenesis of Asn38 in poplar plastocyanin

Type I Cu proteins maintain a trigonal N2S coordination group (with weak axial ligation) in both oxidation states of the Cu2+/+ ion, thereby reducing the reorganization energy for electron transfer. Requirements for maintaining this coordination group were investigated in poplar plastocyanin (Pcy) by mutation of a conserved element of the type 1 architecture, an asparagine residue (Asn38) adjacent to one of the ligating histidines. The side chain of this asparagine forms an active site clasp via two H-bonds with the residue (Ser85) adjacent to the ligating cysteine (Cys84). In addition, the main chain NH of Asn38 donates an H-bond to the thiolate ligand. We have investigated the importance of these interactions by mutating Asn38 to Gln, Thr, and Leu. The mutant proteins are capable of folding and binding Cu2+, but the blue color fades; the rate of fading increases in the order Gln < Thr < Leu. The color is not restored by ferricyanide, showing that the protein is modified irreversibly, probably by oxidation of Cys84. The more stable mutants N38Q and N38T were characterized spectroscopically. The wild-type properties are slightly perturbed for N38Q, but N38T shows remarkable similarity to another type 1 Cu protein, azurin (Azu) from Pseudomonas aeruginosa. The Cu-S(Cys) bond is longer in Azu than in Pcy, and the NH H-bond to the ligating S atom is shorter. Molecular modeling suggests a similar effect for N38T because the threonine residue shifts toward Ser85 in order to avoid a steric clash and to optimize H-bonding. These results demonstrate that H-bonding adjacent to the type 1 site stabilizes an architecture which both modulates the electronic properties of the Cu, and suppresses side reactions of the cysteine ligand.     

Mechanism of human S-adenosylmethionine decarboxylase proenzyme processing as revealed by the structure of the S68A mutant

S-Adenosylmethionine decarboxylase (AdoMetDC) is a pyruvoyl-dependent enzyme that catalyzes the formation of the aminopropyl group donor in the biosynthesis of the polyamines spermidine and spermine. The enzyme is synthesized as a protein precursor and is activated by an autocatalytic serinolysis reaction that creates the pyruvoyl group. The autoprocessing reaction proceeds via an N --> O acyl rearrangement, generating first an oxyoxazolidine anion intermediate followed by an ester intermediate. A similar strategy is utilized in self-catalyzed protein splicing reactions and in autoproteolytic activation of protein precursors. Mutation of Ser68 to alanine in human AdoMetDC prevents processing by removing the serine side chain necessary for nucleophilic attack at the adjacent carbonyl carbon atom. We have determined the X-ray structure of the S68A mutant and have constructed models of the proenzyme and the oxyoxazolidine intermediate. Formation of the oxyoxazolidine intermediate is promoted by a hydrogen bond from Cys82 and stabilized by a hydrogen bond from Ser229. These observations are consistent with mutagenesis studies, which show that the C82S and C82A mutants process slowly and that the S229A mutant does not process at all. Donation of a proton by His243 to the nitrogen atom of the oxyoxazolidine ring converts the oxyoxazolidine anion to the ester intermediate. The absence of a base to activate the hydroxyl group of Ser68 suggests that strain may play a role in the cleavage reaction. Comparison of AdoMetDC with other self-processing proteins shows no common structural features. Comparison to histidine decarboxylase and aspartate decarboxylase shows that these pyruvoyl-dependent enzymes evolved different catalytic strategies for forming the same cofactor.     

Recombinant expression of Mus musculus myoglobin

The cDNA encoding for Mus musculus myoglobin (Mb) was amplified using standard RT-PCR techniques and cloned in an appropriate bacterial expression vector. For the first time, mouse Mb was recombinantly expressed in Escherichia coli cells, BL21(DE3), and purified in sufficient amounts to carry out a preliminary characterization. As shown by mass spectrometry, the protein is found in complex with glutathione, which binds the Cys residue in the topological position E9, in the proximity of the heme group. In recombinant murine Mb, azide affinities are only slightly dependent on the Cys(E9) oxidation state. This suggests that Cys(E9) does not provide a relevant contribution for the stabilization of ligands bound to the heme iron atom. Recombinant expression of M. musculus Mb might have an important role in order to investigate the eventual involvement of Cys(E9) in the new physiological roles proposed for Mb.     

The "rhodanese" fold and catalytic mechanism of 3-mercaptopyruvate sulfurtransferases: crystal structure of SseA from Escherichia coli

3-Mercaptopyruvate sulfurtransferases (MSTs) catalyze, in vitro, the transfer of a sulfur atom from substrate to cyanide, yielding pyruvate and thiocyanate as products. They display clear structural homology with the protein fold observed in the rhodanese sulfurtransferase family, composed of two structurally related domains. The role of MSTs in vivo, as well as their detailed molecular mechanisms of action have been little investigated. Here, we report the crystal structure of SseA, a MST from Escherichia coli, which is the first MST three-dimensional structure disclosed to date. SseA displays specific structural differences relative to eukaryotic and prokaryotic rhodaneses. In particular, conformational variation of the rhodanese active site loop, hosting the family invariant catalytic Cys residue, may support a new sulfur transfer mechanism involving Cys237 as the nucleophilic species and His66, Arg102 and Asp262 as residues assisting catalysis.     

The electrostatic driving force for nucleophilic catalysis in L-arginine deiminase: a combined experimental and theoretical study

L-arginine deiminase (ADI) catalyzes the hydrolysis of L-arginine to form L-citrulline and ammonia via two partial reactions. A working model of the ADI catalytic mechanism assumes nucleophilic catalysis by a stringently conserved active site Cys and general acid-general base catalysis by a stringently conserved active site His. Accordingly, in the first partial reaction, the Cys attacks the substrate guanidino C zeta atom to form a tetrahedral covalent adduct, which is protonated by the His at the departing ammonia group to facilitate the formation of the Cys- S-alkylthiouronium intermediate. In the second partial reaction, the His activates a water molecule for nucleophilic addition at the thiouronium C zeta atom to form the second tetrahedral intermediate, which eliminates the Cys in formation of the L-citrulline product. The absence of a basic residue near the Cys thiol suggested that the electrostatic environment of the Cys thiol, in the enzyme-substrate complex, stabilizes the Cys thiolate anion. The studies described in this paper explore the mechanism of stabilization of the Cys thiolate. First, the log(k(cat)/K(m)) and log k(cat) pH rate profiles were measured for several structurally divergent ADIs to establish the pH range for ADI catalysis. All ADIs were optimally active at pH 5, which suggested that the Cys pKa is strongly perturbed by the prevailing electrostatics of the ADI active site. The p K a of the Bacillus cereus ADI (BcADI) was determined by UV-pH titration to be 9.6. In contrast, the pKa determined by iodoacetamide Cys alkylation is 6.9. These results suggest that the negative electrostatic field from the two opposing Asp carboxylates perturbs the Cys pKa upward in the apoenzyme and that the binding of the iodoacetamide (a truncated analogue of the citrulline product) between the Cys thiol and the two Asp carboxylates shields the Cys thiol, thereby reducing its pKa. It is hypothesized that the bound positively charged guanidinium group of the L-arginine substrate further stabilizes the Cys thiolate. The so-called "substrate-assisted" Cys ionization, first reported by Fast and co-workers to operate in the related enzyme dimethylarginine dimethylaminohydrolase [Stone, E. M., Costello, A. L., Tierney, D. L., and Fast, W. (2006) Biochemistry 45, 5618-5630], was further explored computationally in ADI by using an ab initio quantum mechanics/molecular mechanics method. The energy profiles for formation of the tetrahedral intermediate in the first partial reaction were calculated for three different reaction scenarios. From these results, we conclude that catalytic turnover commences from the active configuration of the ADI(L-arginine) complex which consists of the Cys thiolate (nucleophile) and His imidazolium ion (general acid) and that the energy barriers for the nucleophilic addition of Cys thiolate to the thiouronium C zeta atom and His imidazolium ion-assisted elimination from the tetrahedral intermediate are small.     

Selenocysteine in proteins-properties and biotechnological use

Selenocysteine (Sec), the 21st amino acid, exists naturally in all kingdoms of life as the defining entity of selenoproteins. Sec is a cysteine (Cys) residue analogue with a selenium-containing selenol group in place of the sulfur-containing thiol group in Cys. The selenium atom gives Sec quite different properties from Cys. The most obvious difference is the lower pK(a) of Sec, and Sec is also a stronger nucleophile than Cys. Proteins naturally containing Sec are often enzymes, employing the reactivity of the Sec residue during the catalytic cycle and therefore Sec is normally essential for their catalytic efficiencies. Other unique features of Sec, not shared by any of the other 20 common amino acids, derive from the atomic weight and chemical properties of selenium and the particular occurrence and properties of its stable and radioactive isotopes. Sec is, moreover, incorporated into proteins by an expansion of the genetic code as the translation of selenoproteins involves the decoding of a UGA codon, otherwise being a termination codon. In this review, we will describe the different unique properties of Sec and we will discuss the prerequisites for selenoprotein production as well as the possible use of Sec introduction into proteins for biotechnological applications. These include residue-specific radiolabeling with gamma or positron emitters, the use of Sec as a reactive handle for electophilic probes introducing fluorescence or other peptide conjugates, as the basis for affinity purification of recombinant proteins, the trapping of folding intermediates, improved phasing in X-ray crystallography, introduction of 77Se for NMR spectroscopy, or, finally, the analysis or tailoring of enzymatic reactions involving thiol or oxidoreductase (redox) selenolate chemistry.     

Expression, purification, and characterization of a soluble form of the first extracellular domain of the human type 1 corticotropin releasing factor receptor.

The first extracellular domain (ECD-1) of the corticotropin releasing factor (CRF) type 1 receptor, (CRFR1), is important for binding of CRF ligands. A soluble protein, mNT-CRFR1, produced by COS M6 cells transfected with a cDNA encoding amino acids 1--119 of human CRFR1 and modified to include epitope tags, binds a CRF antagonist, astressin, in a radioreceptor assay using [(125)I-d-Tyr(0)]astressin. N-terminal sequencing of mNT-CRFR1 showed the absence of the first 23 amino acids of human CRFR1. This result suggests that the CRFR1 protein is processed to cleave a putative signal peptide corresponding to amino acids 1--23. A cDNA encoding amino acids 24--119 followed by a FLAG tag, was expressed as a thioredoxin fusion protein in Escherichia coli. Following thrombin cleavage, the purified protein (bNT-CRFR1) binds astressin and the agonist urocortin with high affinity. Reduced, alkylated bNT-CRFR1 does not bind [(125)I-D-Tyr(0)]astressin. Mass spectrometric analysis of photoaffinity labeled bNT-CRFR1 yielded a 1:1 complex with ligand. Analysis of the disulfide arrangement of bNT-CRFR1 revealed bonds between Cys(30) and Cys(54), Cys(44) and Cys(87), and Cys(68) and Cys(102). This arrangement is similar to that of the ECD-1 of the parathyroid hormone receptor (PTHR), suggesting a conserved structural motif in the N-terminal domain of this family of receptors.     

Biochemical discrimination between selenium and sulfur 2: mechanistic investigation of the selenium specificity of human selenocysteine lyase

Selenium is an essential trace element incorporated into selenoproteins as selenocysteine. Selenocysteine (Sec) lyases (SCLs) and cysteine (Cys) desulfurases (CDs) catalyze the removal of selenium or sulfur from Sec or Cys, respectively, and generally accept both substrates. Intriguingly, human SCL (hSCL) is specific for Sec even though the only difference between Sec and Cys is a single chalcogen atom.The crystal structure of hSCL was recently determined and gain-of-function protein variants that also could accept Cys as substrate were identified. To obtain mechanistic insight into the chemical basis for its substrate discrimination, we here report time-resolved spectroscopic studies comparing the reactions of the Sec-specific wild-type hSCL and the gain-of-function D146K/H389T variant, when given Cys as a substrate. The data are interpreted in light of other studies of SCL/CD enzymes and offer mechanistic insight into the function of the wild-type enzyme. Based on these results and previously available data we propose a reaction mechanism whereby the Sec over Cys specificity is achieved using a combination of chemical and physico-mechanical control mechanisms.     

Heterogeneous environment of the S-H group of Cys966 near the flavin chromophore in the LOV2 domain of Adiantum neochrome1

The primary photochemistry of the blue-light sensor protein, phototropin, is adduct formation between the C4a atom of the flavin mononucleotide (FMN) chromophore and a nearby, reactive cysteine (Cys966), following decay of the triplet excited state of FMN. The distance between the C4a position of FMN and the sulfur atom of Cys966 is 4.2 A in the LOV2 domain of Adiantum neochrome 1 (neo1-LOV2), a fusion protein of phototropin containing the phytochrome chromophoric domain. We previously reported the presence of an unreactive fraction in neo1-LOV2 at low temperatures, which presumably originated from the heterogeneous environment of Cys966 [Iwata, T., Nozaki, D., Tokutomi, S., Kagawa, T., Wada, M., and Kandori, H. (2003) Biochemistry 42, 8183-8191]. The present study showed that (i) 28% forms an adduct at 77 K (state I), (ii) 50% forms an adduct at 150 K but not at 77 K (state II), and (iii) 22% does not form an adduct at 150 K (state III). By Fourier transform infrared (FTIR) spectroscopy, we observed the S-H stretching frequencies at 2570 and 2562 cm-1 for state I and at 2563 cm-1 for state II, suggesting that the microenvironment of the S-H group of Cys966 determines the reactivity at low temperatures. Adduct formation is more efficient for state I than for states II and III. Molecular dynamics simulation strongly suggests that the observed multiple structures originate from the isomeric forms of Cys966. We thus concluded that there are multiple local structures of FMN and cysteine in neo1-LOV2, each of which is thermally converted by protein fluctuation at physiological temperatures.     

Biochemical discrimination between selenium and sulfur 1: a single residue provides selenium specificity to human selenocysteine lyase

Selenium and sulfur are two closely related basic elements utilized in nature for a vast array of biochemical reactions. While toxic at higher concentrations, selenium is an essential trace element incorporated into selenoproteins as selenocysteine (Sec), the selenium analogue of cysteine (Cys). Sec lyases (SCLs) and Cys desulfurases (CDs) catalyze the removal of selenium or sulfur from Sec or Cys and generally act on both substrates. In contrast, human SCL (hSCL) is specific for Sec although the only difference between Sec and Cys is the identity of a single atom. The chemical basis of this selenium-over-sulfur discrimination is not understood. Here we describe the X-ray crystal structure of hSCL and identify Asp146 as the key residue that provides the Sec specificity. A D146K variant resulted in loss of Sec specificity and appearance of CD activity. A dynamic active site segment also provides the structural prerequisites for direct product delivery of selenide produced by Sec cleavage, thus avoiding release of reactive selenide species into the cell. We thus here define a molecular determinant for enzymatic specificity discrimination between a single selenium versus sulfur atom, elements with very similar chemical properties. Our findings thus provide molecular insights into a key level of control in human selenium and selenoprotein turnover and metabolism.     

Crystal structures of cobalamin-independent methionine synthase complexed with zinc, homocysteine, and methyltetrahydrofolate

Cobalamin-independent methionine synthase (MetE) catalyzes the synthesis of methionine by a direct transfer of the methyl group of N5-methyltetrahydrofolate (CH3-H2PteGlun) to the sulfur atom of homocysteine (Hcy). We report here the first crystal structure of this metalloenzyme under different forms, free or complexed with the Hcy and folate substrates. The Arabidopsis thaliana MetE (AtMetE) crystals reveal a monomeric structure built by two (betaalpha)8 barrels making a deep groove at their interface. The active site is located at the surface of the C-terminal domain, facing the large interdomain cleft. Inside the active site, His647, Cys649, and Cys733 are involved in zinc coordination, whereas Asp605, Ile437, and Ser439 interact with Hcy. Opposite the zinc/Hcy binding site, a cationic loop (residues 507-529) belonging to the C-terminal domain anchors the first glutamyl residue of CH3-H4PteGlu5. The pterin moiety of CH3-H4PteGlu5 is stacked with Trp567, enabling the N5-methyl group to protrude in the direction of the zinc atom. These data suggest a structural role of the N-terminal domain of AtMetE in the stabilization of loop 507-529 and in the interaction with the poly-glutamate chain of CH3-H4PteGlun. Comparison of AtMetE structures reveals that the addition of Hcy does not lead to a direct coordination of the sulfur atom with zinc but to a reorganization of the zinc binding site with a stronger coordination to Cys649, Cys733, and a water molecule.