IntroductionThe crystal structure of YteR, a 42.9-kDa cytosolic protein of unknown function from Bacillus subtilis, has been determined at 1.60 Å by the multiwavelength anomalous dispersion (MAD) method. YteR represents a new protein fold, as at the time of deposition there were no homologous structures in the Protein Data Bank (PDB) according to BLAST 1 and DALI 2 searches. The 71 known members of YteR family are found in bacteria and fungi. B. subtilis YteR is so far the only member with known structure of the six-α-hairpin glycosyltransferases superfamily of the SCOP 1.65 database. 3 Other members of this SCOP superfamily include glucoamylase, cellulase catalytic domain, N-acyl-D-glucosamine 2-epimerase, the central domain of Lactobacillus maltose phosphorylase, and the C-terminal domain of bacterial glucoamylase. The nearest genomic neighbors of YteR are hypothetical protein BH0485 from B. halodurans (50% identity) and hypothetical conserved protein (Q8EPL5, 50% sequence identity) from Oceanobacillus iheyensis HTE831.
Materials and Methods
Protein cloning expression and purificationThe ORF of YteR was amplified by PCR from Escherichia coli genomic DNA (ATCC). The gene was cloned into the pMCSG7 expression vector. This construct provides for an N-terminal His 6 tag separated from the gene by a TEV protease recognition site (ENLYFQ G). The fusion protein was overexpressed in E. coli BL21-Gold (DE3) (Stratagene) harboring an extra plasmid encoding three rare tRNAs (AGG and AGA for Arg, ATA for Ile). The cells were grown in LB media at 37°C to an OD 600 of ~0.6 and protein expression induced with 0.4 mM IPTG. After induction, the cells were incubated overnight with shaking at 15°C. The harvested cells were resuspended in binding buffer (500 mM NaCl, 5% glycerol, 50 mM HEPES pH 7.5, 5 mM imidazole), flash-frozen in liquid N 2 and stored at 77 K. The thawed cells were incubated for 30 min on ice with lysozyme (Sigma) at 1 mg/mL and protease inhibitors (Sigma, P8849) at 0.05 μL/1 g of cells and lysed by the sonication (6 × 30 s, on ice). The lysate was clarified *Correspondence to: Andrzej Joachimiak, Structural Biology Center, Midwest Center for Structural Genomics, Biosciences, Argonne National Laboratory, 9700 South Cass Ave., Bldg. 202, Argonne, IL 60439. andrzejj@anl.gov. S. Korolev's present address is Saint Louis University Medical School, 1402 South Grand Blvd., St. Louis, MO 63104.The submitted manuscript has been created by the University of Chicago as Operator of Argonne National Laboratory ("Argonne") under Contract No. W-31-109-ENG-38 with the U.S. Department of Energy. The U.S. Government retains for itself, and others acting on its behalf, a paid-up, nonexclusive, irrevocable worldwide licence in said article to reproduce, prepare derivative works, distribute copies to the public, and perform publicly and display publicly, by or on behalf of the Government. by centrifugation (30 min at 27,000 × g, RC5C-Plus centrifuge, Sorval) for 20 min followed by filtration through 0.4 μm and 0.22 μm in-line filte...
