Crystal structure of the Escherichia coli glucose‐inhibited division protein B (GidB) reveals a methyltransferase fold

Introduction. The E. coli gidB gene is the promoterdistal member of the two-gene gid operon located in the vicinity of the bacterial origin of replication oriC. It has been suggested that coupled transcription from the gid and myoC promoters activates initiation of chromosome replication. Transcription from the gid promoter oscillates in the same manner as that of the dnaA gene and decreases dramatically after the onset of DNA replication. E. coli GidB is representative of a large family of proteins encountered in gram-positive and gram-negative bacteria that are thought to function as S-adenosyl-Lmethionine (SAM)-dependent methyltransferases in cell division or chromosome replication. GidB has been classified as a member of the minimal gene set for cellular life based on a comparison of completely sequenced genomes of two parasitic bacteria: Haemophilus influenzae and Mycoplasma genitalium. It was believed that because GidA and GidB homologs were found in M. genitalium, the smallest known self-sustaining living organism with a complement of roughly 517 genes, the genes encoding both protein products are essential. In fact, it has been reported that gidA is essential in Helicobacter pylori in which it is cotranscribed with the dapE gene encoding an N-succinyl-diaminopimelate deacylase. A more unexpected finding came from a study involving a global transposon mutagenesis of the minimal M. genitalium and Mycoplasma pneumoniae genomes. This work identified a single, possibly disruptive insertion in the gidB gene, suggesting that gidB is dispensable. GidB is a single / domain protein (molecular dimensions 45 Å 46 Å 44 Å) with a seven-stranded -sheet of mixed polarity (parallel 32145 followed by antiparallel 76) flanked by seven -helices [Fig. 1(a)]. The linear arrangement of the secondary structural elements is A, B, C, 1, 1, D, 2, E, 3, 4, F, 5, G, 6, and 7 (Fig. 2). In many, but not all, methyltransferases, the segment connecting 3 and 4 is an -helix. In GidB, as in H. influenzae YecO methyltransferase, the 34 linker is random coil. A DALI search of the Protein Data Bank (http:// www.rcsb.org/, December 2001) with the coordinates of GidB identified 303 structurally similar proteins with Z-scores 2.0. Only 15 of these proteins gave Z-scores 10.0 (Table I). The putative SAM-binding site includes the C-terminal portion of 1 and the loop connecting 1 and D. This 11-residue segment, starting at position 71, matches the SAM-binding consensus sequence (Asp-Val/Ile/ Leu-Gly-Thr/Ser/Ala-Gly-X-Gly-X-Pro-Gly/Ala/Ser-Ile/Leu/ Val, where X represents any residue). A superposition of GidB and rat catechol O-methyltransferase (PDB ID 1VID, Table I) permitted docking of SAM and identification of GidB residues likely to stabilize cofactor binding [Fig. 1(b)]. Automated homology modeling with MODPIPE using GidB as a template yielded 636 models of both prokaryotic and eukaryotic proteins (model score 0.7, model length 104 residues) reflecting the highly conserved methyltransferase fold. Models with 30 – 100% sequence identity to the template sequence were limited to GidB family members. The vast majority of the other models represent known methyltransferases with various substrate specificities or larger methyltransferase domain-containing proteins. Detailed inspection of the more stringent MODPIPE/PSI-BLAST results suggests that GidB is specific for sterol and/or lipid substrates. Of the four “best” models (length 180 residues, model score 0.9, PSI-BLAST E-value 1e-20), one is Salmonella enterica GidB (GI:16762453; E-value 9e-21; 86% identity to target residues 1–207) with the remainder including Nostoc sp. PCC 7120 -tocopherol methyltransferase (GI: 17229295; E-value 1e-23; 15% identity to target residues 1–194), Saccharomyces cerevisiae (24)-sterol C-methyltransferase (GI: 462024; E-value 3e-22; 15% identity to target residues 76 –264), and Vibrio cholerae cyclopropane-fatty-acyl-