Crystal Structure of the Tetrameric Cytidine Deaminase from Bacillus subtilis at 2.0 Å Resolution^,

Abstract

Cytidine deaminases (CDA, EC 3.5.4.5) are zinc-containing enzymes in the pyrimidine salvage pathway that catalyze the formation of uridine and deoxyuridine from cytidine and deoxycytidine, respectively. Two different classes have been identified in the CDA family, a homodimeric form (D-CDA) with two zinc ions per dimer and a homotetrameric form (T-CDA) with four zinc ions per tetramer. We have determined the first structure of a T-CDA from Bacillus subtilis. The active form of T-CDA is assembled of four identical subunits with one active site apiece. The subunit of D-CDA is composed of two domains each exhibiting the same fold as the T-CDA subunits, but only one of them contains zinc in the active site. The similarity results in a conserved structural core in the two CDA forms. An intriguing difference between the two CDA structures is the zinc coordinating residues found at the N-terminal of two alpha-helices: three cysteine residues in the tetrameric form and two cysteine residues and one histidine residue in the dimeric form. The role of the zinc ion is to activate a water molecule and thereby generate a hydroxide ion. How the zinc ion in T-CDA surrounded with three negatively charged residues can create a similar activity of T-CDA compared to D-CDA has been an enigma. However, the structure of T-CDA reveals that the negative charge caused by the three ligands is partly neutralized by (1) an arginine residue hydrogen-bonded to two of the cysteine residues and (2) the dipoles of two alpha-helices.

The structure of a yeast RNA-editing deaminase provides insight into the fold and function of activation-induced deaminase and APOBEC-1

“…1B), the noncatalytic C-terminal domain (NCCTD; EcCDA residues 189-294), and the spatial arrangement between domains. Conservation of the subunit interfaces between tetramers and dimers implied that the latter arose through gene duplication of an ancestral monomer that adopted a tetrameric quaternary fold (21,29). This observation accounts for the maintenance of the fundamental ␤-triangle fold in both N-and Cterminal domains of dimeric EcCDA (28).…”

“…The tertiary fold of the CDD1 monomer (Fig. 1B) exhibited high structural homology to the catalytic domains of known bacterial CDAs from E. coli and B. subtilis (29), as well as ScCD (30), despite modest sequence identity; respective rms deviation values from superpositions were 1.32 Å (28% identity), 0.95 Å (43%), and 1.42 Å (16%). The tertiary fold of the deaminase catalytic domain was a triangular ␤-sheet comprising five core strands flanked on either broad face by three ␣-helices (Fig.…”

“…A comparison of the dimeric E. coli and tetrameric B. subtilis CDA structures revealed that their subunit interfaces are conserved, despite differences in quaternary structure (29). The CDD1 subunit interface adopted a similar structure whereby each tetramer could be generated from a single monomer (Fig.…”

“…An alignment template was constructed by use of a quadruple C ␣ superposition of CDAs including: ScCDD1 (this study), E. coli (Ec)CDA (28), Bacillus subtilis (Bs) CDA (29), and the ScCD monomer (30). Pairwise matches of tetrameric ScCDD1 atoms with the tetrameric enzyme of B. subtilis (29) and the dimeric enzyme from E. coli (28) produced rms deviation values of 1.14 and 1.54 Å for 88% and 57% of spatially matched atoms within the CDA core. Differences in the subunit interface of the ScCD dimer precluded superpositions with known CDAs.…”

“…NP 065712) with the four known structures were prepared before modeling. During calculation of the substrate position at the site of deamination, C or U nucleotides were restrained by distances derived from the coordinates of known CDA crystal structures bound to nucleoside substrate or analogs thereof (28,29,31). Details of template assembly and utilization were as described in the MODELLER manual.…”