The 1.6-Å resolution crystal structure of NovW: A 4-keto-6-deoxy sugar epimerase from the novobiocin biosynthetic gene cluster of Streptomyces spheroides

In RmlC-like sequences, the most conserved stretch of continuous sequence is the penta-peptide VX1RGX2H comprising most of β-strand 6, where the His is the catalytic base (His62 in NovW) and the Arg side-chain projects into the active site (Arg59 in NovW). The latter is implicated in substrate binding, forming hydrogen bonds to phosphate groups of bound ligands in four of the RmlC structures (1DZT, 1EPZ, 1NYW, and 1NZC); to a sulfate in NovW and S. typhimurium RmlC (1DZT); to a tartrate in P. aeruginosa RmlC (1RTV); and to glycerol in EvaD (1OFN). Both X1 and X2 are Leu in authentic RmlCs, but are more variable in NovW, CloW, CouW, and EvaD: X1 varies between Val, Ile, and Leu, and X2 can be either Val or Ile. However, the significance of this observation is unclear as the side-chains of residues X1 and X2 point away from the active center, and are thus unlikely to have a direct influence on substrate binding or the catalytic cycle. Also of note in this region is the G-X2 peptide bond. In NovW, EvaD, and the majority of RmlC structures, this is built in the cis configuration. In the structures where this bond is not cis (1DZR, 1DZT, 1NZC, and 1RTV), there appears to be significant local distortion of the geometry, which could be resolved by rebuilding as cis.29 Nonproline cis peptides are relatively uncommon in protein structures and frequently occur in functionally important regions32; thus, the occurrence of this one adjacent to the active site is perhaps no coincidence. However, its role is not immediately apparent, although it has been suggested that it may be “required to orient catalytic residues found on β6 in the active site”.33 Indeed, in NovW and EvaD, and all RmlC structures with the exception of one (1PM7), the side-chain of His62 stacks against the protein backbone of the Gly (Gly60 in NovW). From a purely structural perspective, the cis peptide helps to maintain the continuity of β-sheet 2. β6 is sandwiched between β11 and β13, which lie at an angle of approximately 55° relative to one another, looking down the β-barrel axis [Fig. 1(B)]. The cis peptide puts a 25° kink into β6 such that the N-terminal half (residues 58–60) is juxtaposed with β11, and the C-terminal half (residues 61–64) is juxtaposed with β13. Two further cis peptide bonds are present in the NovW structure, but both of these precede Pro residues. They are adjacent in sequence (Val66-Pro67 and Pro67-Pro68), lying in the surface loop connecting strands β6 and β7. These are only conserved in the structures of the homologs with the greatest structural similarity to NovW, namely, in EvaD (1OFN, 1OI6), M. tuberculosis RmlC (1PM7, 1UPI), and S. tokodaii RmlC (1WLT; but only the first cis Pro is present). Thus, an important structural or functional role for this motif seems unlikely.