title: The crystal structure reveals the molecular mechanism of bifunctional 3,4-dihydroxy-2-butanone 4-phosphate synthase/GTP cyclohydrolase II (Rv1415) from Mycobacterium 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.
authors.affiliation: CSIR - Institute of Microbial Technology, Council of Scientific and Industrial Research (CSIR), Sector 39-A, Chandigarh 160 036, India.
authors.forename: Mirage
authors.initials: M
authors.lastname: Singh
authors.forename: Pankaj
authors.initials: P
authors.lastname: Kumar
authors.forename: Savita
authors.initials: S
authors.lastname: Yadav
authors.forename: Ruchi
authors.initials: R
authors.lastname: Gautam
authors.forename: Nidhi
authors.initials: N
authors.lastname: Sharma
authors.forename: Subramanian
authors.initials: S
authors.lastname: Karthikeyan
mesh_terms: D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry* / Q000502:physiology; D018360:Crystallography, X-Ray; D006136:GTP Cyclohydrolase / Q000737:chemistry* / Q000502:physiology; D019751:Intramolecular Transferases / Q000737:chemistry* / Q000502:physiology; D064251:Multifunctional Enzymes / Q000737:chemistry* / Q000502:physiology; D009169:Mycobacterium tuberculosis / Q000201:enzymology*; D055503:Protein Multimerization; D017434:Protein Structure, Tertiary; D016415:Sequence Alignment
chemical_list: D001426:Bacterial Proteins; D064251:Multifunctional Enzymes; D006136:GTP Cyclohydrolase; D019751:Intramolecular Transferases; C066493:L-3,4-dihydroxy-2-butanone-4-phosphate synthase
keywords: DHBPS; FAD; FMN; GCHII; GTP; antimicrobial targets; ribA2; riboflavin biosynthesis