The A domain

As shown in Fig. 1, the function of E-III is to transfer the phosphoryl group from P-HPr to E-II. P-phosphorylated peptides have been isolated from a number of E-III species and A domains of S. carnosus and S. aureus IIl [27], E. coli II [28], 

Ilioic 1 29] and [30], In each case the phosphoryl group is carried on a histi- 

In contrast to E. coli, B. subtilis II is a class III enzyme with a covalently attached A domain [19] but its sucrose PTS still lacks its own E-III. Sutrina et al. [18] have recently shown that II° complements B. subtilis II in sucrose phosphorylation in 

The function of the B domain has been confirmed by subcloning and preliminary kinetic measurements. We subcloned the AB domain of E. coli II, residues 348-637, after inserting a restriction site at a position corresponding to residue 348. The purified protein restored mannitol phosphorylation activity when measured with the A domain assay in Fig. 4A, and the B domain assay in Fig. 4B [42]. The B domain 

coli is the only case in which the A and B domains are linked together 

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Once numerical estimates of the weight of a trajectory and its variance (2cr ) are known we are able to use sampled trajectories to compute observables of interest. One such quantity on which this section is focused is the rate of transitions between two states in the system. We examine the transition between a domain A and a domain B, where the A domain is characterized by an inverse temperature - (3. The weight of an individual trajectory which is initiated at the A domain and of a total time length - NAt is therefore... 

The third class, represented by E. coli consists again of a single hydrophobic domain of approximately 360 residues but with two covalently attached hydrophilic domains, equal, together, in size to the hydrophobic domain [17], The A domain is proposed to function as a covalently attached E-III. Other representatives of this class include B. subtilis II° [18,19], 5. mutans 11 [20], E. coli II [21,22] and [23,24],... 

The complementation experiments in which the A domain of a class 111 E-II is used as the phosphoryl group donor to the B domain of a second E-II molecule with either the same or different sugar specificity, while both are fixed in a membrane matrix, raises some intriguing issues about the association state of these proteins and the kinetics of their interactions. Do E-IIs form stable homologous complexes in the membranes If so, is it necessary to postulate the formation of stable heterologous complexes to explain, for example, the phosphorylation of the B domain of E. coli 11° by the A domain of ll , or can the data be explained by assuming a... 

The a- and P-domains have been isolated in order to study their role in the two reaction phases. The slow reactions occur predominately with the P-domain while the fast reaction is associated entirely with the a-domain [106]. This pattern follows other circumstances where the a-domain is more reactive than the P-domain. However, the P Site has Cd ions that are thermodynamically less tightly bound and more labile to inter-site exchange. 

Fig. 5. The NMR-refmed structure of the A-domain of high-mobility group protein 1 (PDBID 1AAB). Adapted from (78).

Mapping of the tumor-derived VHL mutations on the VHL structure revealed two patches of solvent-exposed residues (Figure 7.6). One patch is located on the portion of the a-domain involved in ElonginC binding, confirming the role ElonginC binding plays in the tumor suppressor function of VHL. The second patch. 

Classical bacterial exotoxins, such as diphtheria toxin, cholera toxin, clostridial neurotoxins, and the anthrax toxins are enzymes that modify their substrates within the cytosol of mammalian cells. To reach the cytosol, these toxins must first bind to different cell-surface receptors and become subsequently internalized by the cells. To this end, many bacterial exotoxins contain two functionally different domains. The binding (B-) domain binds to a cellular receptor and mediates uptake of the enzymatically active (A-) domain into the cytosol, where the A-domain modifies its specific substrate (see Figure 1). Thus, three important properties characterize the mode of action for any AB-type toxin selectivity, specificity, and potency. Because of their selectivity toward certain cell types and their specificity for cellular substrate molecules, most of the individual exotoxins are associated with a distinct disease. Because of their enzymatic nature, placement of very few A-domain molecules in the cytosol will normally cause a cytopathic effect. Therefore, bacterial AB-type exotoxins which include the potent neurotoxins from Clostridium tetani and C. botulinum are the most toxic substances known today. However, the individual AB-type toxins can greatly vary in terms of subunit composition and enzyme activity (see Table 2). 

Figure 1 The mode of action for bacterial AB-type exotoxins. AB-toxins are enzymes that modify specific substrate molecules in the cytosol of eukaryotic cells. Besides the enzyme domain (A-domain), AB-toxins have a binding/translocation domain (B-domain) that specifically interacts with a cell-surface receptor and facilitates internalization of the toxin into cellular transport vesicles, such as endosomes. In many cases, the B-domain mediates translocation of the A-domain into the cytosol by pore formation in cellular membranes. By following receptor-mediated endocytosis, AB-type toxins exploit normal vesicle traffic pathways into cells. One type of toxin escapes from early acidified endosomes (EE) into the cytosol, thus they are referred to as short-trip-toxins . In contrast, the long-trip-toxins take a retrograde route from early endosomes (EE) through late endosomes (LE), trans-Golgi network (TGN), and Golgi apparatus into the endoplasmic reticulum (ER) from where the A-domains translocate into the cytosol to modify specific substrates.

Figure 8 Crystal structure of the C-A-T-TE SrfA-C termination module from Bacillus subtilis. The C domain is shown in gray, the A domain in yellow and blue, the T domain can be seen in red, the TE domain in green, and the C-terminal tag is shown in orange.

The structurally related myxochromides Aj.j are cyclic hexapeptides produced by several Myxococcus species. These examples contain a proline residue, which is not present in myxochromides Si 3, as the fourth amino acid in their peptide core. The NRPSs responsible for myxochromides A and S biosynthesis have exacdy the same module and domain organization thus, the fourth module of the myxochromide S synthetase must be skipped to account for the natural product. Biochemical experiments revealed that the A domain of this module activates L-proline, but the adjacent PCP domain cannot be phosphopantetheinylated by a PPTase. These results suggest that the C domain of module 5 reacts directly with the tripeptide intermediate bound to the PCP domain of module 3 in myxochromide S biosynthesis. A similar example of domain skipping has been noted in the biosynthesis of the mannopeptimycins. ... 

Many NRPs such as cyclosporin, complestatin, actinomycin, and chondramide contain N-methyl amides. M-Methyl transferase (N-MT) domains utilize S-adenosylmethionine (SAM) as a cofactor to catalyze the transfer of the methyl group from SAM to the a-amine of an aminoacyl-S-PCP substrate. The presence of M-methylamides in NRPs is believed to protect the peptide from proteolysis. Interestingly, N-MT domains are incorporated into the A domains of C-A-MT-PCP modules, between two of the core motifs (A8 and A9). MT domains contain three sequence motifs important for catalysis. ° 0-Methyl transferase domains are also found in NRPSs and likewise use the SAM cofactor. For instance, cryptophycin and anabaenopeptilide synthetases contain 0-MT domains for the methylation of tyrosine side chains. These 0-MT domains lack one of the three core motifs described for N-MT domains. ... 

Recently, bacterial NRPS modules with the organization of A-KR-PCP have been discovered in the valino-mycin and cereulide synthetases. The A domains of these modules selectively activate a-keto acids. After the resulting adenylate is transferred to the PCP domain, the a-ketoacyl- -PCP intermediate is reduced to a PCP-bound, a-hydroxythioester by the KR domain. These domains use NAD(P)H as a cofactor and are inserted into A domains between two conserved core motifs analogous to MT domains. Their substrate specificity differs from that of polyketide synthase KR domains, which reduce /3-ketoacyl substrates. Similar fungal NRPSs, such as beauvericin synthetase, utilize A domains that selectively activate a-hydroxy acids. These molecules are thought to be obtained using an in trans KR domain, which directly reduces the necessary, soluble a-keto acid. 

The termination module of surfactin synthetase is a 144 kDa four-domain enzyme responsible for the incorporation of the final amino acid (L-Leu) into the surfactin peptide and subsequent cyclization of the resulting product. The structure of the TE domain of this construct was previously solved. In the recently determined 2.6 A X-ray structure of the C-A-PCP-TE construct, the entire protein chain is evident in the electron density maps. " " The structural folds of the individual domains in this module are similar to structures of monomeric domains (Figure 13). The deviations observed in this multidomain structure include a slight difference in the hinge region of C domain subdomains and an orientation of the subdomains of the A domain that is not consistent with the open or closed conformations previously described. The A domain contains... 

Figure 13 X-ray structure of the four-domain termination moduie of surfactin synthetase (PDB code, 2VSQ). The coioring and representation of the domains is the same as in Figures 11 and 12. A cartoon diagram of the reiative domain structure is iiiustrated at the right of the two views. Ac and An signify the C-terminai and N-terminai subdomains of the A domain.

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