Photon Factory Activity Report 2005 #23 Part B (2006)
Biological Science

6A, 5A, NW12/2004G342

Crystal Structure of A Bifunctional Deaminase and Reductase from Bacillus
subtilis Involved in Riboflavin Biosynthesis
Sheng-Chia Chen, Yuan-Chih Chang and Liaw, Shwu-Huey
Structural Biology Program, Faculty of Life Sciences, National Yang-Ming University, Taiwan
Introduction
Flavin coenzymes are ubiquitous in all organisms
because of their involvements in central metabolic
pathways. Therefore, the enzymes involved in riboflavinbiosynthesis have the potential to become attractive
candidates for the design of new defenses against
antibiotic-resistant pathogens. Most eubacteria contain abifunctional protein involved in the second and third steps
in the riboflavin biosynthesis; for instance the Bacillus
subtilis RibG (BsRibG) is composed of an N-terminal
deaminase domain (D domain) and a C-terminal
reductase domain (R domain). The D domain of bacterial
RibG was expected to belong to the cytidine deaminase(CDA) superfamily. The CDA superfamily consists of themononucleotide deaminases involved in nucleotide
metabolism, and the RNA (DNA)-editing deaminasesinvolved in gene diversity and in anti-virus defense.
These deaminases catalyze the hydrolytic deamination ofcytosine, guanine, adenine moieties and several of their
therapeutically useful analogues. Furthermore, thestructural fold of the R domain was predicted to be
similar to dihydrofolate reductase (DHFR). DHFR
catalyzes the NADPH-utilizing reduction of DHF totetrahydrofolate. Many DHFR inhibitors such as
methotrexate, pyrimethamine and trimethoprim, havelong been used clinically in the treatment of cancer,
rheumatoid arthritis, malaria, and bacterial and fungalinfection. To gain structural insights into the inhibitor
design, substrate specificity and evolution, we have
solved the BsRibG structure at 2.41-Å resolution.
Results and Discussion
Analytical ultracentrifugation experiments clearly
demonstrated that BsRibG exists as a tetramer in solution
as well as in crystal form, where the enzyme forms atetrameric ring-like structure. Molecules A and B interact
with each other through their D domains, while molecule
A makes extensive contacts with molecule C throughtheir R domains. The active site of the D domain contains
one tightly bound endogenous zinc ion, of which theanomalous data provided sufficient phase information forstructure determination. The active-site architecture
resembles those of the CDA members. The C-terminal
segment beyond the β4 strand, is quite diverse and may
make a major contribution to the structural plasticity andfunctional diversity among the CDA members. A
structure-based sequence alignment of the CDA members
was constructed, revealing not only the unique signaturesin each family member for gene annotation, but also
putative substrate-interacting residues for RNA-editing
deaminases such as the loss-of-function point mutants of

acid-induced deaminase in patients with hyper-IgM
syndrome type 2.
The strong conservation of the tertiary structures of
DHFRs and BsRibG suggests that the two reductases
involved in the riboflavin and folate biosyntheses aredescended from a single ancestral gene and thereby
define a new superfamily. They share four conserved
regions for NADPH binding, and the binding architectureof the nicotinamide ring is virtually identical, and hencewith a similar enzymatic mechanism. There is noevidence for any dependence of the active sites within theRibG tetramer. Previous deletion mutants havedemonstrated that the N-terminal 147 residues and the Cterminal 248 residues of BsRibG were sufficient for theirrespective enzyme activities. However, these truncatedproteins could not be isolated due to poor stability.
Therefore, even though the deaminase and the reductase
are separate functional domains, the domain fusion is
crucial for the enzyme activities through formation of astable tetrameric structure.

Fig. 1 The tetrameric BsRibG structure with the zinc ion
in the D domain, the cofactor NADPH in the R domain,
and the inter-domain linker highlighted in magenta.
References[1]T. Ko et al., J. Biol. Chem. 278, 19111 (2003).
[2] S. Liaw et al., J. Biol. Chem. 279, 35479 (2004).
[3] S. Chen et al., J. Biol. Chem. 281, 7605 (2006).
* shliaw@ym.edu.tw

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