A Novel Histidine-Rich CPx-ATPase from the Filamentous Cyanobacterium Oscillatoria brevis Related to Multiple-Heavy-Metal Cotolerance

Abstract

A novel gene related to heavy-metal transport was cloned and identified from the filamentous cyanobacterium Oscillatoria brevis. Sequence analysis of the gene (the Bxa1 gene) showed that its product possessed high homology with heavy-metal transport CPx-ATPases. The CPC motif, which is proposed to form putative cation transduction channel, was found in the sixth transmembrane helix. However, instead of the CXXC motif that is present in the N termini of most metal transport CPx-ATPases, Bxa1 contains a unique Cys-Cys (CC) sequence element and histidine-rich motifs as a putative metal binding site. Northern blotting and real-time quantitative reverse transcription-PCR showed that expression of Bxa1 mRNA was induced in vivo by both monovalent (Cu ؉ and Ag ؉ ) and divalent (Zn 2؉ and Cd 2؉ ) heavy-metal ions at similar levels. Experiments on heavy-metal tolerance in Escherichia coli with recombinant Bxa1 demonstrated that Bxa1 conferred resistance to both monovalent and divalent heavy metals. This is the first report of a CPx-ATPase responsive to both monovalent and divalent heavy metals.Organisms usually utilize emergency mechanisms such as reduced uptake, facilitated efflux, sequestration, and modification to achieve resistance to the toxicity of heavy metals (5, 22, 27, 37). Heavy-metal transport P-type ATPases (or CPx-ATPases) are thought to act as a potential key heavy-metal transporter involved not only in metal ion homeostasis but also in the overall strategy for heavy-metal tolerance (3, 37).CPx-ATPases are a new subgroup of P-type ATPases and possess all the common characteristics of P-type ATPases (25, 37). One significant difference, however, is the presence of a conserved intramembranous cysteine-proline-cysteine/histidine/serine (CPx) motif. P-type ATPases with this unique characteristic are therefore termed CPx-ATPases. Another unique feature of CPx-ATPases is the N-terminal metal binding domain. In most cases, the heavy-metal binding domain consists of one or several cysteine-rich (CXXC) motifs. In certain cases, the cysteine-rich N terminus is replaced by a short sequence containing histidine.There is currently great interest in the N-terminal-cysteinerich CPx-ATPases (1, 6, 9, 17, 31); however, little interest has been paid to the N-terminal-histidine-rich CPx-ATPases. Recently, genomic DNA programs have revealed that the Nterminal-histidine-rich CPx-ATPases are also distributed widely among organisms (8,10,11,37,38), especially plants, although the true roles of these CPx-ATPases are not yet known.Although the detailed characterization of CPx-ATPases is still awaited, the main physiological function of these enzymes is thought to be the transport of heavy-metal ions across biological membranes to maintain the intracellular homeostasis of essential or nonessential heavy metals and deliver specific metal ions to target enzymes. CPx-ATPases have been found to have high specificity for the heavy-metal ions they transport. It has been demonstrated that the type of transport substrate is restricted by he...

A novel CPx‐ATPase from the cadmium hyperaccumulator Thlaspi caerulescens

“…Although similar motifs were found in TcHMA4-C and AtHMA4-C, this domain is also the most divergent part between the two sequences. CC dipeptides were only found, together with a His-rich domain, in the N-terminal region of bacterial CPx-ATPases and were associated with Cd 2þ tolerance [28,29]. The function of these putative heavy metal binding motifs remains to be elucidated.…”

“…All these three peptides also contained three additional cysteine motifs -C(x) 4 C, C(x) 3-5 CC, CC -and a His rich domain within their extended C-terminus which could be involved in heavy metal binding. Further examination of the two putative HMA4 COOH tails revealed that the C(x) 3 CC motifs were in a larger cysteine motif, C(x) [14][15][16][17][18][19][20][21][22][23][24][25][26][27][28] C(x) 3 CC for Thlaspi and C(x) [20][21][22][23][24][25][26][27][28][29][30][31][32][33][34][35] C(x) 3 CC for Arabidopsis. This sequence is similar to the recently described TRASH domain, C(x) [19][20][21][22] C(x) 3 C, predicted to be involved in heavy metal sensing, trafficking and resistance [25].…”

HMA1, a New Cu-ATPase of the Chloro plast Envelope, Is Essential for Growth under Adverse Light Conditions

“…HMA1 lacks the N-terminal Heavy Metal Associated regulatory domain usually found in P 1B -ATPases. Instead, it has a long His stretch reminding the N-terminal domain of the P 1B-2 Bxa1 from Oscillatoria brevis that was described as transporting both copper and zinc (41).…”

Identification of Thlaspi caerulescens Genes That May Be Involved in Heavy Metal Hyperaccumulation and Tolerance. Characterization of a Novel Heavy Metal Transporting ATPase

“…The P 1B subfamily of ATPases is believed to be involved in the transport of heavy metals such as Zn, Cu, Co, Cd, Pb, and silver that are either essential micronutrients or noessential toxic metals, although only a few members of this subfamily have been well characterized. Genome sequencing efforts as well as isolation and analysis of individual members of this subgroup from different organisms indicate these heavy metal ATPases are expressed in a wide range of organisms, ranging from the archaea and bacteria to eukaryotic organisms including Arabidopsis (see, for example, Rensing et al, 1997;Palmgren, 1998, 2001;Tong et al, 2002;Mills et al, 2003).…”