Metal-cation regulation of enzyme dynamics is a key factor influencing the activity of S-adenosyl-L-homocysteine hydrolase from Pseudomonas aeruginosa.

S-adenosyl-L-homocysteine hydrolase from Pseudomonas aeruginosa (PaSAHase) coordinates one K+ ion and one Zn2+ ion in the substrate binding area. The cations affect the enzymatic activity and substrate binding but the molecular mechanisms of their action are unknown. Enzymatic and isothermal titration calorimetry studies demonstrated that the K+ ions stimulate the highest activity and strongest ligand binding in comparison to other alkali cations, while the Zn2+ ions inhibit the enzyme activity. PaSAHase was crystallized in the presence of adenine nucleosides and K+ or Rb+ ions. The crystal structures show that the alkali ion is coordinated in close proximity of the purine ring and a 23Na NMR study showed that the monovalent cation coordination site is formed upon ligand binding. The cation, bound in the area of a molecular hinge, orders and accurately positions the amide group of Q65 residue to allow its interaction with the ligand. Moreover, binding of potassium is required to enable unique dynamic properties of the enzyme that ensure its maximum catalytic activity. The Zn2+ ion is bound in the area of a molecular gate that regulates access to the active site. Zn2+ coordination switches the gate to a shut state and arrests the enzyme in its closed, inactive conformation.

S-adenosyl-L-homocysteine hydrolase from a hyperthermophile (Thermotoga maritima) is expressed in Escherichia coli in inactive form - Biochemical and structural studies.

Thermotoga maritima is a hyperthermophilic bacterium but its genome encodes a number of archaeal proteins including S-adenosyl-L-homocysteine hydrolase (SAHase), which regulates cellular methylation reactions. The question of proper folding and activity of proteins of extremophilic origin is an intriguing problem. When expressed in E.coli and purified (as a homotetramer) at room temperature, the hyperthermophilic SAHase from T.maritima was inactive. ITC study indicated that the protein undergoes heat-induced conformational changes, and enzymatic activity assays demonstrated that these changes are required to attain enzymatic activity. To explain the mechanism of thermal activation, two crystal structures of the inactive form of T. maritima SAHase (iTmSAHase) were determined for an incomplete binary complex with the reduced cofactor (NADH), and in a mixture of binary complexes with NADH and with adenosine. In contrast to active SAHases, in iTmSAHase only two of the four subunits contain a bound cofactor, predominantly in its non-reactive, reduced state. Moreover, the closed-like conformation of the cofactor-containing subunits precludes substrate delivery to the active site. The two other subunits cannot be involved in the enzymatic reaction either; although they have an open-like conformation, they do not contain the cofactor, whose binding site may be occupied by an adenosine molecule. The results suggest that this enzyme, when expressed in mesophilic cells, is arrested in the activity-incompatible conformation revealed by its crystal structures.

Tetra-ethyl-ammonium toluene-4-sulfon-ate.

There are two tetra-ethyl-ammonium cations and two toluene-4-sulfate anions in the asymmetric unit of the title salt, C8H20N(+)·C7H7O3S(-). One of the anions is disordered over two positions, with refined occupancies of 0.447 (3) and 0.553 (3). In the crystal, the cations and anions are linked by C-H⋯O hydrogen bonds, forming ribbons along [10-1]. The ribbons are linked via C-H⋯O hydrogen bonds, forming a two-dimensional network lying parallel to (10-1).

Tris(1,10-phenanthroline-κ(2) N,N')ruthenium(II) bis-(perchlorate).

The asymmetric unit of the title compound, [Ru(C12H8N2)3](ClO4)2, contains one octahedrally coordinated Ru(II) cation of the ruthenium-phenanthroline complex and three differently occupied perchlorate anions: two, denoted A and B, are located on the twofold axis while another, denoted C, is positioned in the proximity of the twofold screw axis. Perchlorate anions B and C are severely disordered. The occupancies of the two major conformers of anion B refined to 0.302 (6) and 0.198 (6). Perchlorate ion C was modeled in two alternate conformations which refined to occupancies of 0.552 (10) and 0.448 (10).