The crystal structure reveals the molecular mechanism of bifunctional 3,4-dihydroxy-2-butanone 4-phosphate synthase/GTP cyclohydrolase II (Rv1415) fromMycobacterium tuberculosis

Abstract

The enzymes 3,4-dihydroxy-2-butanone 4-phosphate synthase (DHBPS) and GTP cyclohydrolase II (GCHII) catalyze the initial steps of both branches of the bacterial riboflavin-biosynthesis pathway. The structures and molecular mechanisms of DHBPS and GCHII as separate polypeptides are known; however, their organization and molecular mechanism as a bifunctional enzyme are unknown to date. Here, the crystal structure of an essential bifunctional DHBPS/GCHII enzyme from Mycobacterium tuberculosis (Mtb-ribA2) is reported at 3.0 Å resolution. The crystal structure revealed two conformationally different molecules of Mtb-ribA2 in the asymmetric unit that form a dimer via their GCHII domains. Interestingly, analysis of the crystal packing revealed a long `helical-like oligomer' formed by DHBPS and GCHII functional homodimers, thus generating an `open-ended' unit-cell lattice. However, size-exclusion chromatography studies suggest that Mtb-ribA2 exists as a dimer in solution. To understand the discrepancy between the oligomerization observed in solution and in the crystal structure, the DHBPS (Mtb-DHBPS) and GCHII (Mtb-GCHII) domains of Mtb-ribA2 have been cloned, expressed and purified as His-tagged proteins. Size-exclusion chromatography studies indicated that Mtb-GCHII is a dimer while Mtb-DHBPS exists as a monomer in solution. Moreover, kinetic studies revealed that the GCHII activities of Mtb-ribA2 and Mtb-GCHII are similar, while the DHBPS activity of Mtb-ribA2 is much higher than that of Mtb-DHBPS alone. Taken together, the results strongly suggest that Mtb-ribA2 exists as a dimer formed through its GCHII domains and requires full-length Mtb-ribA2 for optimal DHBPS activity.

Structural Basis for Competitive Inhibition of 3,4-Dihydroxy-2-butanone-4-phosphate Synthase from Vibrio cholerae

“…In most of Gram-negative bacteria, DHBPS exists as monofunctional form, whereas in Gram-positive bacteria including Mycobacterium tuberculosis DHBPS co-exists with GTP cyclohydrolase II and thus is found in a bifunctional form (9,16,(27)(28)(29). Crystal structures of DHBPS from E. coli (30,31), Magnaporthe grisea (32), Methanococcus jannaschii (33), Candida albicans (34), Salmonella typhimurium (35), M. tuberculosis (28,36), and Streptococcus pneumoniae (27) have been reported. All these structures reveal that DHBPS is a homodimer where each monomer forms an ␣ ϩ ␤ fold and that its active site is located at two topologically equivalent positions at the dimeric interface of each monomer (27, 28, 30 -36).…”

“…The DHBPS structure complexed with substrate and metal ions indicates that an acidic active site loop (Loop1) and another loop (Loop2) undergo a conformational change upon substrate and/or metal binding (32,33,35). Furthermore, these structures also reveal the amino acids involved in the proposed reaction mechanism of DHBPS (27,28,(32)(33)(34)(35)(36). Nevertheless, certain differences observed in the existing structures prevent us from predicting the molecular mechanism of DHBPS completely.…”

“…However, the DHBPS domain is observed as a dimer in the crystal structure of M. tuberculosis RibA2 (36). Thus, it is speculated that in the presence of substrate the DHBPS domain may transiently form a dimer in solution; however, the exact mechanism is still unknown (28).…”

Structural and biochemical characterization of GTP cyclohydrolase II from Helicobacter pylori reveals its redox dependent catalytic activity

“…Crystal structure of GCHII has been reported in mono-functional and bi-functional form from E. coli (eGCHII) (Ren et al, 2005) and Mycobacterium tuberculosis (M. tuberculosis) (mGCHII) (Singh et al, 2013) respectively. The E. coli GCHII crystal structure complexed with substrate analog guanosine-5 0 -[(a,b)-m ethyleno]-triphosphate (GMPCPP) revealed the key residues that are part of the active site (Ren et al, 2005).…”

“…In yeast and in most Gramnegative bacteria GCHII is encoded by a single gene designated as ribA (Oltmanns et al, 1969;Shavlovskiĭ et al, 1979) which is mono-functional. However, in plants and most Gram-positive bacteria, GCHII (C-terminal) exists as a bi-functional enzyme along with 3,4-dihydroxy-2-butanone 4-phosphate synthase (DHBPS/ ribB; N-terminal) (Herz et al, 2000;Mironov et al, 1994;Shavlovskiĭ et al, 1979;Singh et al, 2013).…”