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Methenyltetrahydromethanopterin (methenyl-H(4)MPT(+)) cyclohydrolase (Mch) catalyzes the interconversion of methenyl-H(4)MPT(+) and formyl-H(4)MPT in the one-carbon energy metabolism of methanogenic, methanotrophic, and sulfate-reducing archaea and of methylotrophic bacteria. To understand the catalytic mechanism of this reaction, we kinetically characterized site-specific variants of Mch from Archaeoglobus fulgidus (aMch) and determined the X-ray structures of the substrate-free aMch(E186Q), the aMch:H(4)MPT complex, and the aMch(E186Q):formyl-H(4)MPT complex. (Formyl-)H(4)MPT is embedded inside a largely preformed, interdomain pocket of the homotrimeric enzyme with the pterin and benzyl rings being oriented nearly perpendicular to each other. The active site is primarily built up by the segment 93:95, Arg183 and Glu186 that either interact with the catalytic water attacking methenyl-H(4)MPT(+) or with the formyl oxygen of formyl-H(4)MPT. The catalytic function of the strictly conserved Arg183 and Glu186 was substantiated by the low enzymatic activities of the E186A, E186D, E186N, E186Q, R183A, R183Q, R183E, R183K, and R183E-E186Q variants. Glu186 most likely acts as a general base. Arg183 decisively influences the pK(a) value of Glu186 and the proposed catalytic water mainly by its positive charge. In addition, Glu186 appears to be also responsible for product specificity by donating a proton to the directly neighbored N(10) tertiary amine of H(4)MPT. Thus, N(10) becomes a better leaving group than N(5) which implies the generation of N(5)-formyl-H(4)MPT. For comparison, methenyltetrahydrofolate (H(4)F) cyclohydrolase produces N(10)-formyl-H(4)F in an analogous reaction. An enzymatic mechanism of Mch is postulated and compared with that of other cyclohydrolases.

Some N(2)-fixing bacteria prolong the functionality of nitrogenase in molybdenum starvation by a special Mo storage protein (MoSto) that can store more than 100 Mo atoms. The presented 1.6 Å X-ray structure of MoSto from Azotobacter vinelandii reveals various discrete polyoxomolybdate clusters, three covalently and three noncovalently bound Mo(8), three Mo(5-7), and one Mo(3) clusters, and several low occupied, so far undefinable clusters, which are embedded in specific pockets inside a locked cage-shaped (αβ)(3) protein complex. The structurally identical Mo(8) clusters (three layers of two, four, and two MoO(n) octahedra) are distinguishable from the [Mo(8)O(26)](4-) cluster formed in acidic solutions by two displaced MoO(n) octahedra implicating three kinetically labile terminal ligands. Stabilization in the covalent Mo(8) cluster is achieved by Mo bonding to Hisα156-N(ε2) and Gluα129-O(ε1). The absence of covalent protein interactions in the noncovalent Mo(8) cluster is compensated by a more extended hydrogen-bond network involving three pronounced histidines. One displaced MoO(n) octahedron might serve as nucleation site for an inhomogeneous Mo(5-7) cluster largely surrounded by bulk solvent. In the Mo(3) cluster located on the 3-fold axis, the three accurately positioned His140-N(ε2) atoms of the α subunits coordinate to the Mo atoms. The formed polyoxomolybdate clusters of MoSto, not detectable in bulk solvent, are the result of an interplay between self- and protein-driven assembly processes that unite inorganic supramolecular and protein chemistry in a host-guest system. Template, nucleation/protection, and catalyst functions of the polypeptide as well as perspectives for designing new clusters are discussed.

Flavohemoglobins (flavoHbs) are enzymes that operate primarily as nitric oxide dioxygenases and shuttle thereby electrons among NAD(P)H, FAD, heme, and a ligated redox-active substrate such as O(2). They function in the bacterial defense against nitrosative stress and are therefore considered as targets for new antibiotic drugs. Recently, azole derivatives were proven to be attractive nitric oxide dioxygenase inhibitors, and to explore their binding characteristics, we determined the X-ray structure of the flavoHb from Ralstonia eutropha in a complex with miconazole (FHP(M)), econazole (FHP(E)), and ketoconazole (FHP(K)). In agreement with UV-vis spectroscopic data, one azole compound binds inside the distal heme pocket and ligates to the heme iron by its imidazole substituent. The two additional substituents, mostly chlorinated phenyl groups, form a series of van der Waals contacts with the protein matrix. Both interactions explain their high affinity for flavoHbs, the binding constants being 2.6, 1.2, and 11.6 μM for miconazole, econazole, and ketoconazole, respectively. The FHP(M) and FHP(Lip) (flavoHbs originally loaded with a phospholipid) structures share an "open" state and the FHP(E) and FHP(K) structures a "closed" state. Although the azole compounds were able to push the lipid out of its binding site, a fatty acid fragment is still bound inside the heme pocket of FHP(E) and FHP(K) and dictates the state of the protein. The ligand-induced open-to-closed transition involves a reorientation of the NADH domain accompanied by conformational changes in the C-terminal arm, helix E, and the CE loop resulting in an encapsulation of the heme-binding pocket. Implications of the observed open-to-closed process on the catalytic cycle are discussed.

Glutaryl-coenzyme A dehydrogenases (GDHs) involved in amino acid degradation were thought to catalyze both the dehydrogenation and decarboxylation of glutaryl-coenzyme A to crotonyl-coenzyme A and CO(2). Recently, a structurally related but nondecarboxylating, glutaconyl-coenzyme A-forming GDH was characterized in the obligately anaerobic bacteria Desulfococcus multivorans (GDH(Des)) which conserves the free energy of decarboxylation by a Na(+)-pumping glutaconyl-coenzyme A decarboxylase. To understand the distinct catalytic behavior of the two GDH types on an atomic basis, we determined the crystal structure of GDH(Des) with and without glutaconyl-coenzyme A bound at 2.05 and 2.1 A resolution, respectively. The decarboxylating and nondecarboxylating capabilities are provided by complex structural changes around the glutaconyl carboxylate group, the key factor being a Tyr --> Val exchange strictly conserved between the two GDH types. As a result, the interaction between the glutaconyl carboxylate and the guanidinium group of a conserved arginine is stronger in GDH(Des) (short and planar bidentate hydrogen bond) than in the decarboxylating human GDH (longer and monodentate hydrogen bond), which is corroborated by molecular dynamics studies. The identified structural changes prevent decarboxylation (i) by strengthening the C4-C5 bond of glutaconyl-coenzyme A, (ii) by reducing the leaving group potential of CO(2), and (iii) by increasing the distance between the C4 atom (negatively charged in the dienolate transition state) and the adjacent glutamic acid.

F(420)-dependent methylenetetrahydromethanopterin (methylene-H(4)MPT) dehydrogenase (Mtd) of Methanopyrus kandleri is an enzyme of the methanogenic energy metabolism that catalyzes the reversible hydride transfer between methenyl-H(4)MPT(+) and methylene-H(4)MPT using coenzyme F(420) as hydride carrier. We determined the structures of the Mtd-methylene-H(4)MPT, Mtd-methenyl-H(4)MPT(+), and the Mtd-methenyl-H(4)MPT(+)-F(420)H(2) complexes at 2.1, 2.0, and 1.8 A resolution, respectively. The pterin-imidazolidine-phenyl ring system is present in a new extended but not planar conformation which is virtually identical in methenyl-H(4)MPT(+) and methylene-H(4)MPT at the current resolution. Both substrates methenyl-H(4)MPT(+) and F(420)H(2) bind in a face to face arrangement to an active site cleft, thereby ensuring a direct hydride transfer between their C14a and C5 atoms, respectively. The polypeptide scaffold does not reveal any significant conformational change upon binding of the bulky substrates but in turn changes the conformations of the substrate rings either to avoid clashes between certain ring atoms or to adjust the rings involved in hydride transfer for providing an optimal catalytic efficiency.

Molecular evolution has evolved two metabolic routes for isoprenoid biosynthesis: the mevalonate and the 2-C-methyl-D-erythritol-4-phosphate (MEP) pathway. The MEP pathway is used by most pathogenic bacteria and some parasitic protozoa (including the malaria parasite, Plasmodium falciparum) as well as by plants, but is not present in animals. The terminal reaction of the MEP pathway is catalyzed by (E)-4-hydroxy-3-methyl-but-2-enyl diphosphate (HMBPP) reductase (LytB), an enzyme that converts HMBPP into isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP). Here, we present the structure of Aquifex aeolicus LytB, at 1.65 A resolution. The protein adopts a cloverleaf or trefoil-like structure with each monomer in the dimer containing three alpha/beta domains surrounding a central [Fe3S4] cluster ligated to Cys13, Cys96, and Cys193. Two highly conserved His (His 42 and His 124) and a totally conserved Glu (Glu126) are located in the same central site and are proposed to be involved in ligand binding and catalysis. Substrate access is proposed to occur from the front-side face of the protein, with the HMBPP diphosphate binding to the two His and the 4OH of HMBPP binding to the fourth iron thought to be present in activated clusters, while Glu126 provides the protons required for IPP/DMAPP formation.