Abstract
Deoxythymidine diphosphate (dTDP)-4-keto-6-deoxy-d-hexulose 3, 5-epimerase (RmlC) is involved in the biosynthesis of dTDP-l-rhamnose, which is an essential component of the bacterial cell wall. The crystal structure of RmlC from Methanobacterium thermoautotrophicum was determined in the presence and absence of dTDP, a substrate analogue. RmlC is a homodimer comprising a central jelly roll motif, which extends in two directions into longer beta-sheets. Binding of dTDP is stabilized by ionic interactions to the phosphate group and by a combination of ionic and hydrophobic interactions with the base. The active site, which is located in the center of the jelly roll, is formed by residues that are conserved in all known RmlC sequence homologues. The conservation of the active site residues suggests that the mechanism of action is also conserved and that the RmlC structure may be useful in guiding the design of antibacterial drugs.
Highlights
Rsym ϭ ¥͉IϪ multiple measurements, and the summation is over all observed reflections
The dTDP complex model was refined against 1.75-Å resolution data to a working Rfactor of 0.195 and a free R-factor of 0.224
Addendum—While this manuscript was under review, the structure of RmlC from Salmonella typhimurium was published [16]
Summary
Protein Expression and Purification—The RmlC gene from M. thermoautotrophicum genomic DNA was amplified by polymerase chain reaction and cloned into the pET15b expression vector (Novagen). Recombinant dTDP-4-keto-6-deoxy-D-hexulose epimerase (RmlC) was expressed in Escherichia coli BL21 Gold (DE3) cells (Stratagene) harboring a plasmid encoding three rare E. coli tRNA genes (AGG and AGA for Arg and ATA for Ile). For the preparation of selenomethionine (Se-Met) protein, RmlC was expressed in a methionine auxotroph strain B834(DE3) (Novagen) and purified under the same conditions as native RmlC with the addition of 5 mM -mercaptoethanol in all buffers. Gel Filtration—Gel filtration of RmlC was performed with a Superdex 200 prep 16/60 (Amersham Pharmacia Biotech) column equili-.
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
