Bacterial cadmium-binding proteins

Abstract

There is much current interest in the metallothioneins. These are low molecular weight proteins (6–7,000 Daltons) which have a high cysteine (33%) and metal content (7 g atoms/mol). This, together with the absence of aromatic amino acids and histidine, makes this protein very unusual [1]. Metallothioneins have no defined functions, although many possible roles have been suggested including detoxification [2], and regulation of zinc [3] and copper metabolism [4]. Metallothioneins are widespread in mammals [1] and have also been isolated from fish [5], invertebrates [6], plants [7] and eukaryotic microorganismas such as yeast [8] and Neurosporra crassa [9]. ▪ N. crassa protein has been sequenced and found to have extensive homology with mammalian metallothionein [9]. Metallothioneins or similar proteins have never been isolated from bacteria. We have studied the mechanism of cadmium resistance in a gram negative bacterium and isolated novel cadmium binding proteins. The study of these bacterial proteins may provide new insight into the metabolism and probable function of metallothioneins. Three different cadmium-binding-proteins are produced by a cadmium-resistant strain of Pseudomonas putida, isolated from sewage sludge. The bacterium accumulates cadmium from the environment, and the internal concentration can reach 9 m M. Up to 30% of this cadmium is found in the cytoplasm. Three cadmium binding proteins were isolated from the cytoplasm of P. putida (fig.1). Each protein is produced during a different phase of the growth cycle (Fig2). The proteins have a high cysteine content and bind cadmium, copper and zinc. They are induced ▪ by the presence of Cadmium-binding-protein 1, molecular weight 7,000 (CdBP 1 binds 7 g atoms metal/mol/, and CdBP 2 (7,000 daltons) binds 3 g atoms metal/mol. CdBP 3, is a much smaller molecule (3,800 daltons) and binds cadmium and copper (4.5 g atoms metal/mol). All three cadmium-binding proteins contain large amounts of cysteine, but only about half that commonly found in metallothioneins. CdBP 3 has the highest cysteine content of 22%. Also typical of the metallothioneins is the high serine, glycine, and alanine contents of CDBP 1 and CdBP 2. CadBP 2 also contains substantial amounts of lysine. Both CdBP 2 and CdBP 3 have few or no residues of arginine, leucine, isoleucine, or the aromatic amino acids. The major differences from metallothionein are the high glutamate content of CdBP 2 and CdBP 3. CdBP 1 also contains substantial amounts of leucine, isoleucine and also four residues of aromatic amino acids. 1H NMR experiments at 400 MHz support the amino acid analysis, revealing the lack of aromatic amino acids in CdBP 2 as well as the high cysteine content. The high metal-binding capacity of these proteins, but the lower content of cysteine, suggests that in addition to cysteine-SH other protein ligands may be involved in cadmium binding.