Biochemical and Structural Characterization of a Novel Family of Cystathionine β-Synthase Domain Proteins Fused to a Zn Ribbon-Like Domain

Abstract

We have identified a novel family of proteins, in which the N-terminal Cystathionine Beta-Synthase (CBS) domain is fused to the C-terminal Zn ribbon domain. Four proteins were over-expressed in E. coli and purified: TA0289 from Thermoplasma acidophilum, TV1335 from Thermoplasma vulcanum, PF1953 from Pyrococcus furiosus, and PH0267 from Pyrococcus horikoshii. The purified proteins had red/purple color in solution and an absorption spectrum typical of rubredoxins. Metal analysis of purified proteins revealed the presence of several metals with iron and zinc being the most abundant metals (2 to 67% of iron and 12 to 74% of zinc). Crystal structures of both mercuryand iron-bound TA0289 (1.5-2.0 Å resolution) revealed a dimeric protein whose inter-subunit contacts are formed exclusively by the α helices of two CBS sub-domains, whereas the C-terminal domain has a classical Zn-ribbon planar architecture. All proteins were reversibly reduced by chemical reductants (ascorbate or dithionite) or by the general rubredoxin reductase NorW from E. coli in the presence of NADH. Reduced TA0289 was found to be able to transfer electrons to cytochrome C from horse heart. Likewise, the purified Zn ribbon protein KTI11 from Saccharomyces cerevisiae had purple color in solution and a rubredoxin-like absorption spectrum, contained both iron and zinc, and was reduced by the rubredoxin reductase NorW from E. coli. Thus, recombinant Zn ribbon domains from archaea and yeast demonstrate a rubredoxin-like electron carrier activity in vitro. We suggest that in vivo some Zn ribbon domains might also bind iron and therefore possess an electron carrier activity adding another physiological role to this large family of important proteins.

Polyphosphate-dependent synthesis of ATP and ADP by the family-2 polyphosphate kinases in bacteria

“…The cloning of the genes encoding PA3455, SMc02148, and other PPK2 proteins (PAPTO1640, PA2428, SMa0172, SMa0670, Atu0418, and RPA4569) into the modified pET15b was carried out as described (23). The proteins were expressed as a fusion with an N-terminal His6 tag in the E. coli strain BL21 (DE3) and purified as described (23,24). Seleno-methionine (SeMet)-labeled proteins were produced by overexpression in the E. coli methionine auxotroph strain B834 (DE3) (Novagen).…”

“…Seleno-methionine (SeMet)-labeled proteins were produced by overexpression in the E. coli methionine auxotroph strain B834 (DE3) (Novagen). Alanine replacement mutagenesis of PA3455 and SMc02148 was performed as described (24).…”

A Novel Family of Sequence-specific Endoribonucleases Associated with the Clustered Regularly Interspaced Short Palindromic Repeats

“…The proteins were expressed as a fusion with an N-terminal His 6 tag in Escherichia coli strain BL21 (DE3) and purified to more than 95% homogeneity using metal-chelate affinity chromatography on nickel affinity resin and gel filtration on a Superdex 200 26/60 column (Amersham Biosciences) as described before (20,21). Site-directed mutagenesis of SSO1404 was performed as described previously (21) using a protocol based on the QuikChange site-directed mutagenesis kit (Stratagene).…”

Engineering of Ion Sensing by the Cystathionine β-Synthase Module of the ABC Transporter OpuA

“…Most of the CBS domains are found as tandem repeats but data base searches have also revealed tetra-repeat units (5). The crystal structures of several tandem CBS domains have been elucidated (7)(8)(9)32), and in a number of cases it has been shown that two tandem CBS domains form dimeric structures with a total of four CBS domains per structural module (hereafter referred to as CBS module). The crystal structures of the full-length MgtE Mg 2ϩ transporter confirm the dimeric configuration and show that the CBS domains undergo large conformational changes upon Mg 2ϩ binding or release (10, 11).…”