J Michael Sauder - Functional Identification and Structure Determination of Two Novel Prolidases from cog1228 in the Amidohydrolase Superfamily

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      Publication Details (including relevant citation   information):

      Biochemistry (2010)


      Two uncharacterized enzymes from the amidohydrolase   superfamily  belonging to cog1228 were cloned, expressed and   purified to homogeneity.  The two proteins, Sgx9260c   (gi|44242006) and Sgx9260b (gi|44479596),  were derived from   environmental DNA samples originating from the  Sargasso   Sea. The catalytic function and substrate profiles for   Sgx9260c  and Sgx9260b were determined using a comprehensive   library of  dipeptides and N-acyl derivative of L-amino   acids. Sgx9260c catalyzes  the hydrolysis of Gly-L-Pro,   L-Ala-L-Pro and N-acyl derivatives of  L-Pro. The best   substrate identified to date is N-acetyl-L-Pro with a  value   of kcat/Km of 3 x 105 M-1 s-1. Sgx9260b catalyzes the   hydrolysis  of L-hydrophobic L-Pro dipeptides and N-acyl   derivatives of L-Pro. The  best substrate identified to date   is N-propionyl-L-Pro with a value of  kcat/Km of 1 x 105 M-1   s-1. Three dimensional structures of both  proteins were   determined by X-ray diffraction methods (PDB codes: 3MKV    and 3FEQ). These proteins fold as distorted (beta/alpha)8-barrels   with  two divalent cations in the active site. The structure   of Sgx9260c was  also determined as a complex with the   N-methyl phosphonate derivative of  L-Pro (PDB code: 3N2C).   In this structure the phosphonate moiety  bridges the   binuclear metal center and one oxygen atom interacts with    His-140. The alpha-carboxylate of the inhibitor interacts with   Tyr-231.  The proline side chain occupies a small substrate   binding cavity formed  by residues contributed from the loop   that follows beta-strand 7 within  the (beta/alpha)8-barrel.   A total of 38 other proteins from cog1228 are  predicted to   have the same substrate profile based on conservation of    the substrate binding residues. The structure of an   evolutionarily  related protein, Cc2672 from Caulobacter   crecentus, was determined as a  complex with the N-methyl   phosphonate derivative of L-arginine (PDB  code: 3MTW).

      Address (URL): http://pubs.acs.org/doi/abs/10.1021/bi100897u