METAL CHELATING PROPERTIES OF ADENYLATE KINASE FROM PARACOCCUS-DENITRIFICANS

Zinc, a common element of adenylate kinases from Grampositive bacteria , binds to a structural motif consisting of three or four cysteine res idues, Cys-X-2-Cys-X-16-Cys-X-2-Cys/Asp. The enzyme from Paracoccus de nitrificans, a Gram-negative bacterium, has structural features much s imilar to those of adenylate kinases from Gram-positive organisms [Spu rgin,P., Tomasselli,A.G., and Schiltz,E. (1989) Eur. J. Biochem., 179, 621-628]. However, adenylate kinase isolated from this bacterium was not reported to bind metal. These findings prompted us to clone the co rresponding gene of P.denitrificans, and to characterize the enzyme ov erproduced in Escherichia coli. The deduced primary structure of adeny late kinase from P.denitrificans revealed two differences from that pr eviously published: Cys was found at position 130 instead of His, and His was found at position 138 instead of Gly. The recombinant enzyme i s a dimer which binds either zinc or iron, in a metal/monomer ratio of one. The dissociating sulfhydryl reagent, p-(hydroxy-mercuri)phenylsu lfonate, released the metal from the protein, confirming that thiols a re involved in zinc- or iron-binding. The iron-chelated form of recomb inant P.denitrificans adenylate kinase, which is essentially under red uced form, transfers electrons to the oxidized cytochrome c. In conclu sion, the absence of metal in the enzyme isolated from P.denitrificans is not related to the protein structure but most probably due to the physiological properties of the host organism.