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.
Fructose-1,6-bisphosphatase (FBPase), a key enzyme of gluconeogenesis and photosynthetic CO 2 fixation, catalyzes the hydrolysis of fructose 1,6-bisphosphate (FBP) to produce fructose 6-phosphate, an important precursor in various biosynthetic pathways. All known FBPases are metal-dependent enzymes, which are classified into five different classes based on their amino acid sequences. Eukaryotes are known to contain only the type-I FBPases, whereas all five types exist in various combinations in prokaryotes. Here we demonstrate that the uncharacterized protein YK23 from Saccharomyces cerevisiae efficiently hydrolyzes FBP in a metal-independent reaction. YK23 is a member of the histidine phosphatase (phosphoglyceromutase) superfamily with homologues found in all organisms. The crystal structure of the YK23 apo-form was solved at Fructose-1,6-bisphosphatase (FBPase) 2 (EC 3.1.3.11) catalyzes the removal of phosphate 1 from fructose 1,6-bisphosphate to produce fructose 6-phosphate, an important precursor in various biosynthetic pathways. FBPase is a key, rate-controlling enzyme of gluconeogenesis, an important metabolic pathway that allows the cells to synthesize glucose and grow on non-carbohydrate carbon sources, such as glycerol, organic acids, and amino acids (1). Gluconeogenesis is also responsible for excessive glucose production found in type 2 diabetes, which is a growing worldwide health concern (2). Because FBPases represent the major control point and function only in gluconeogenesis in mammals, they are recognized as an attractive target for the development of drugs for the treatment of type 2 diabetes (3). FBPase also functions at the branch point between the regenerative phase of the photosynthetic CO 2 fixation cycle (Calvin), which is the primary pathway of carbon fixation, and the starch biosynthesis (4). In addition, FBPase is required for virulence in Mycobacterium tuberculosis and Leishmania major and plays an important role in the production of lysine and glutamate by Corynebacterium glutamicum (5, 6).Most characterized FBPases belong to the superfamily of lithium-sensitive metal-dependent phosphatases that also includes three families of inositol phosphatases (clan CL0171, Pfam data base) (7). Based on the amino acid sequence, FBPases can be assigned to one of the five proposed classes (8 -10). All known FBPases require a divalent metal cation for activity (Mg 2ϩ or Mn 2ϩ ), are inhibited by Li ϩ , and their activity is often regulated by AMP, fructose 2,6-bisphosphate, and phosphoenolpyruvate (11-13). Types I, II, and IV FBPases and inositol monophosphatases have similar three-dimensional structures with a sugar phosphatase-fold (␣␣␣) suggesting a common evolutionary origin and catalytic mechanism (14 -16). All five types of FBPases are normally found in prokaryotes, where types I, II, and III are more common to bacteria (8), type IV are mostly found in archaea (10), and type V in thermophiles from both domains (9). The majority of organisms have more than one FBPase, mostly the combinatio...
The Archaeoglobus fulgidus gene af0721 encodes CbiX(S), a small cobaltochelatase associated with the anaerobic biosynthesis of vitamin B12 (cobalamin). The protein was shown to have activity both in vivo and in vitro, catalyzing the insertion of Co2+ into sirohydrochlorin. The structure of CbiX(S) was determined in two different crystal forms and was shown to consist of a central mixed beta-sheet flanked by four alpha-helices, one of which originates in the C-terminus of a neighboring molecule. CbiX(S) is about half the size of other Class II tetrapyrrole chelatases. The overall topography of CbiX(S) exhibits substantial resemblance to both the N- and C-terminal regions of several members of the Class II metal chelatases involved in tetrapyrrole biosynthesis. Two histidines (His10 and His74), are in similar positions as the catalytic histidine residues in the anaerobic cobaltochelatase CbiK (His145 and His207). In light of the hypothesis that suggests the larger chelatases evolved via gene duplication and fusion from a CbiX(S)-like enzyme, the structure of AF0721 may represent that of an "ancestral" precursor of class II metal chelatases.
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