J Michael Sauder - Functional annotation of two new carboxypeptidases from the amidohydrolase superfamily of enzymes

Version 1

      Publication Details (including relevant citation   information):

      Biochemistry (2009) 48: 4567-4576.

      Xiang DF, Xu C, Kumaran D, Brown AC, Sauder JM, Burley SK,   Swaminathan S, Raushel FM

      Abstract:

      Two proteins from the amidohydrolase superfamily of enzymes were   cloned,  expressed, and purified to homogeneity. The first   protein, Cc0300, was  from Caulobacter crescentus CB-15   (Cc0300), while the second one  (Sgx9355e) was derived from   an environmental DNA sequence originally  isolated from the   Sargasso Sea ( gi|44371129 ). The catalytic functions  and   the substrate profiles for the two enzymes were determined with   the  aid of combinatorial dipeptide libraries. Both enzymes   were shown to  catalyze the hydrolysis of l-Xaa-l-Xaa   dipeptides in which the amino  acid at the N-terminus was   relatively unimportant. These enzymes were  specific for   hydrophobic amino acids at the C-terminus. With Cc0300,    substrates terminating in isoleucine, leucine, phenylalanine,   tyrosine,  valine, methionine, and tryptophan were   hydrolyzed. The same specificity  was observed with   Sgx9355e, but this protein was also able to hydrolyze    peptides terminating in threonine. Both enzymes were able to   hydrolyze  N-acetyl and N-formyl derivatives of the   hydrophobic amino acids and  tripeptides. The best   substrates identified for Cc0300 were l-Ala-l-Leu  with   k(cat) and k(cat)/K(m) values of 37 s(-1) and 1.1 x 10(5)   M(-1)  s(-1), respectively, and N-formyl-l-Tyr with k(cat)   and k(cat)/K(m)  values of 33 s(-1) and 3.9 x 10(5) M(-1)   s(-1), respectively. The best  substrate identified for   Sgx9355e was l-Ala-l-Phe with k(cat) and  k(cat)/K(m) values   of 0.41 s(-1) and 5.8 x 10(3) M(-1) s(-1). The    three-dimensional structure of Sgx9355e was determined to a   resolution  of 2.33 A with l-methionine bound in the active   site. The  alpha-carboxylate of the methionine is ion-paired   to His-237 and also  hydrogen bonded to the backbone amide   groups of Val-201 and Leu-202. The  alpha-amino group of the   bound methionine interacts with Asp-328. The  structural   determinants for substrate recognition were identified and    compared with other enzymes in this superfamily that   hydrolyze  dipeptides with different specificities.

      Address (URL): http://pubs.acs.org/doi/abs/10.1021/bi900453u