Genetically Engineered Zinc-chelating Adenylate Kinase fromEscherichia coli with Enhanced Thermal Stability

Abstract

149(corresponding to Cys 130 , Cys 133 , Cys 150 , and Cys 153 in adenylate kinase from Bacillus stearothermophilus) in Escherichia coli adenylate kinase was undertaken for determining whether the presence of Cys residues is the only prerequisite to bind zinc or (possible) other cations. A number of variants of adenylate kinase from E. coli, containing 1-4 Cys residues were obtained, purified, and analyzed for metal content, structural integrity, activity, and thermodynamic stability. All mutants bearing 3 or 4 cysteine residues acquired zinc binding properties. Moreover, the quadruple mutant exhibited a remarkably high thermal stability as compared with the wild-type form with preservation of the kinetic parameters of the parent enzyme.Adenylate kinase (AK, 1 ATP:AMP phosphotransferase, EC 2.7.4.3) is a ubiquitous enzyme which contributes to homeostasis of adenine nucleotides in living cells (1). Three classes of AKs, differing in size, subcellular localization, and substrate specificity were identified in vertebrates, AK 1 in the cytosol, AK 2 in the mitochondrial intermembrane space, and AK 3 (called also GTP:AMP phosphotransferase) in the mitochondrial matrix. Only one form of AK was identified in bacteria. Mitochondrial adenylate kinases (AK 2 , AK 3 ) and the vast majority of bacterial adenylate kinases belong to the class of long forms. They differ from AK 1 and some bacterial AKs, the short variants, by a 28-residue long insertion organized into a small domain (2) called LID well exposed to the solvent (Fig. 1A) and undergoing a large movement during catalysis (3, 4).AKs from Gram-positive bacteria contain a structural zinc atom (5-7), a property which is due to the presence of 3 or 4 cysteine residues in the LID domain. Sequence alignment of AKs from Gram-positive and Gram-negative organisms, devoid of metal, showed that in the latter species the Cys residues are substituted with four other highly conserved amino acids, His, Ser, Asp, and Thr (Fig. 1B). This conservation suggests that these particular residues have some essential function, but different in the enzyme from the , and Thr 149 in Escherichia coli adenylate kinase with cysteine residues. Our aim was to know whether a motif composed of 3 or 4 Cys residues generates a metal-binding site in AK or whether other structural factors contribute to the specificity (zinc versus iron or any other metal) or to the strength of the protein/metal interaction. On the other hand, we wanted to know the relevance of the metal binding for catalysis or stability of AK. A number of variants of AK e containing one to four cysteine residues were thus obtained. In agreement with previous studies on zinc-binding AKs, we found that the 3 and 4 cysteine modified forms of AK e acquired zinc binding properties. Moreover, the 4 cysteine-containing AK e exhibited an increased stability against thermal denaturation as compared with the wild-type form, with full conservation of its catalytic properties. EXPERIMENTAL PROCEDURESMaterials-Adenine nucleotides, coupling enzymes, ...

Crystal structure of the zinc-, cobalt-, and iron-containing adenylate kinase from Desulfovibrio gigas: a novel metal-containing adenylate kinase from Gram-negative bacteria

“…Apart from these, several close interactions between the residues of the LID and the core domains and also with its symmetry mates contribute to the stabilization of the open conformation in the crystal. These strong interactions along with the metal chelation in the LID domain may enhance the thermal stability of the enzymes and also give it the structural integrity that makes it move as a rigid block towards the ATP binding pocket at the time of catalysis [14]. Binding of a metal ion (Zn, Co, or Fe) to the LID domain, as was shown previously, was found to provide a thermal stabilizing effect on the protein [18].…”

“…Nevertheless, the topology of the LID domain is very similar. All of these share the two reverse turns, a characteristic feature of the zinc-containing AK from Gram-negative bacteria [14], which facilitates the zinc coordination. The corresponding Arg residues (Arg124 and Arg166) in these three enzymes also show a 4-Å shift in the substrate-bound form as compared with Zn-AK.…”

“…as a stabilizing feature in AK. The removal of metal ions does not affect the phosphorylating activity of apoproteins from Paracoccus denitrificans, E. coli (quadruple Cys mutant AK e C 4 ), B. stearothermophilus, and T. neapolitana [10,14,16,17].…”

“…In AK from Gram-positive bacteria, zinc seems to stabilize the LID domain more readily than does the hydrogen-bond network present in Gram-negative bacteria and in all of the eukaryotic longform AK determined so far [4,13]. Several studies have shown that the presence of a metal ion in the LID domain causes considerable differences in the overall stability of AK and its removal leads to partial or complete loss of catalytic activity [7,12,14,[16][17][18].…”

“…The sulfur positions of the four Cys side chains of Gram-positive bacteria are geometrically optimized to bind the metal ion [13]. In AK from Gram-negative bacteria, the four Cys residues are substituted by four highly conserved amino acids, His, Ser, Asp, and Thr, respectively, suggesting that these residues might play a similar structural role [14]. The crystal structure of AK from Escherichia coli shows that the LID domain forms a single distorted antiparallel b-sheet, two turns, and one loop [15].…”