Domain substrate specificity

Predicting Modular Polyketide Synthase Acyl Transferase Domain Substrate Specificity... 

Figure 4 Partial organization of the poiyketide synthase (PKS) system for which AT domain substrate specificity and coiinearity ruie-based prediction has ied to the discovery of the haistoctacosanoiides. AT domains highiighted in green were predicted to specificaiiy ioad a methyimaionyi-CoA unit whereas the ones highiighted in biue were predicted to ioad maionyi-CoA. The actuai structure of the haistoctacosanoiides isoiated from Streptomyces halstedii is represented in the bottom ieft corner.

Figure 7 Predicted chemical structures of the nonribosomal peptide coelichelin based on A domain substrate specificity and colinearity rule (at the bottom) and experimentally determined structure of coelichelin (on the right).

Serine proteinases such as chymotrypsin and subtilisin catalyze the cleavage of peptide bonds. Four features essential for catalysis are present in the three-dimensional structures of all serine proteinases a catalytic triad, an oxyanion binding site, a substrate specificity pocket, and a nonspecific binding site for polypeptide substrates. These four features, in a very similar arrangement, are present in both chymotrypsin and subtilisin even though they are achieved in the two enzymes in completely different ways by quite different three-dimensional structures. Chymotrypsin is built up from two p-barrel domains, whereas the subtilisin structure is of the a/p type. These two enzymes provide an example of convergent evolution where completely different loop regions, attached to different framework structures, form similar active sites. 

Important members of this toxin family are Clostridium difficile toxins A and B, which are implicated in antibiotics-associated diarrhea and pseudomembranous colitis. The large clostridial cytotoxins are single-chain toxins with molecular masses of 250-308 kDa. The enzyme domain is located at the N terminus. The toxins are taken up from an acidic endosomal compartment. They glucosylate RhoA at Thr37 also, Rac and Cdc42 are substrates. Other members of this toxin family such as Clostridium sordellii lethal toxin possess a different substrate specificity and modify Rac but not Rho. In addition, Ras subfamily proteins (e.g., Ras, Ral, and Rap) are modified. As for C3, they are widely used as tools to study Rho functions [2] [4]. 

Fig. 1. Structure of class I and class II PI3Ks and their substrate specificity. PRR, proline-rich regions PX, phox homology domain.

The specificity determinants surrounding the tyrosine phospho-acceptor sites have been determined by various procedures. In PTK assays using various substrates, it was determined that glutamic residues of the N-terminal or C-terminal side of the acceptor are often preferred. The substrate specificity of PTK catalytic domains has been analyzed by peptide library screening for prediction of the optimal peptide substrates. Finally, bioinformatics has been applied to identify phospho-acceptor sites in proteins of PTKs by application of a neural network algorithm. 

Reeves, C.D., Murli, S., Ashley, G.W. et al. (2001) Alteration of the substrate specificity of a modular polyketide synthase acyltransferase domain through site-specific mutations. Biochemistry, 40, 15464. 

In another study, the carrier protein was replaced by an enzyme compatible solid-phase resin (PEGA), and enzyme-catalyzed cyclization was used to probe substrate specificity. This study demonstrated also that oxo-esters are tolerated as substrates for TE domains, and then-preparation in library format served as an excellent tool for substrate specificity studies, as well as for preparation of cyclized peptides. Figure 13.11 shows how the TycA TE showed selectivity for only residues 1 and 9 (colored in red), and changes at all other residues were tolerated [42]. Hydrogen bonding interactions are shown in green. Several compounds made from this series were shown to demonstrate improved therapeutic indices (with respect to hemolysis) while retaining antimicrobial activity. 

Under physiological conditions, NRPTKs are highly specific in directing tyrosine phosphorylation toward appropriate substrates. This specificity relies on the intrinsic predilection of the catalytic domain towards specific amino acid sequences within protein substrates. In addition, noncatalytic domains, e.g., SH2, SH3 and PH domains of NRPTKs, distribute these kinases to the subcellular region where appropriate substrates are in proximity or abundance, thus favoring phosphorylation of these proteins rather than other substrates. 

In addition to containing protein-protein interaction motifs, E3-substrate specificity may be affected by post-translational modifications. In particular, phosphorylation can alter E3-substrate interactions. One example is p53 where certain phosphorylations inhibit its direct binding to Mdm2, while others indirectly enhance their association by promoting nuclear localization of p53 [104-106]. Phosphorylation also directly enhances substrate interactions, as exemplified by the Cbls, which include phospho-tyrosine binding domains (see below) [107]. 

Li, S. j. and Hochstrasser, M. The Ulpl SUMO isopeptidase distinct domains required for viability, nuclear envelope localization, and substrate specificity, J Cdl Biol, 2003, 160, 1069— 81. 

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