J Michael Sauder - Functional identification of incorrectly annotated prolidases from the amidohydrolase superfamily of enzymes

Version 1

      Publication Details (including relevant citation   information):

      Biochemistry (2009) 48: 3730-3742.

      Xiang DF, Patskovsky Y, Xu C, Meyer AJ, Sauder JM, Burley SK,   Almo SC, Raushel FM

      Abstract:

      The substrate profiles for two proteins from Caulobacter   crescentus CB15  (Cc2672 and Cc3125) and one protein   (Sgx9359b) derived from a DNA  sequence ( gi|44368820 )   isolated from the Sargasso Sea were determined  using   combinatorial libraries of dipeptides and N-acyl derivatives   of  amino acids. These proteins are members of the   amidohydrolase  superfamily and are currently misannotated   in NCBI as catalyzing the  hydrolysis of l-Xaa-l-Pro   dipeptides. Cc2672 was shown to catalyze the  hydrolysis of   l-Xaa-l-Arg/Lys dipeptides and the N-acetyl and N-formyl    derivatives of lysine and arginine. This enzyme will also   hydrolyze  longer peptides that terminate in either lysine   or arginine. The  N-methyl phosphonate derivative of   l-lysine was a potent competitive  inhibitor of Cc2672 with   a K(i) value of 120 nM. Cc3125 was shown to  catalyze the   hydrolysis of l-Xaa-l-Arg/Lys dipeptides but will not    hydrolyze tripeptides or the N-formyl and N-acetyl derivatives of   lysine  or arginine. The substrate profile for Sgx9359b is   similar to that of  Cc2672 except that compounds with a   C-terminal lysine are not recognized  as substrates. The   X-ray structure of Sgx9359b was determined to a  resolution   of 2.3 A. The protein folds as a (beta/alpha)(8)-barrel and    self-associates to form a homooctamer. The active site is   composed of a  binuclear metal center similar to that found   in phosphotriesterase and  dihydroorotase. In one crystal   form, arginine was bound adventitiously  to the eight active   sites within the octamer. The orientation of the  arginine   in the active site identified the structural determinants   for  recognition of the alpha-carboxylate and the positively   charged side  chains of arginine-containing substrates. This   information was used to  identify 18 other bacterial   sequences that possess identical or similar  substrate   profiles.

      Address (URL): http://pubs.acs.org/doi/abs/10.1021/bi900111q