Multidomain enzymes

Fig. 9.9. Exon shuffling combinatorial libraries of multidomain enzymes can be created through recombination of individual modules or domains (I - IV) from multiple parents (A - D) in an ordered fashion, maintaining the parental size (left diagram) or by random reassembly. This allows...

An analysis of 40 genomes representing all three kingdoms of life identified 1307 pairwise combinations of 783 domains. Only a small proportion of domains (1%) has been used extensively in combinations with other domains. In multidomain enzymes, the P-loop nucleotide triphosphate hydrolase domain and the Rossman fold (which binds NAD /NADH) are the most commonly found. The P-loop hydrolase domain is associated with 47 and 21 different domain partners in bacteria and Archaea, respectively, and the Rossman fold with 29 and 18 domain partners in bacteria and Archaea, respectively. The repeated use of these domains reflects the utility of the reactions they facilitate - phosphoryl transfer and hydride transfer. By combining these domains with a domain that provides substrate specificity, a wide range of reactions can be accomplished without a need to reinvent something that already works well. 

The individual modules of polyketide synthases (PKSs) and of NRPSs represent another example of modularity. An electrophile and a nucleophile are covalently attached to two different subunits of a multidomain enzyme. The selectivity for the electrophile resides in the domain catalyzing bond formation between two substrates and the selectivity for the nucleophile is controlled by a transfer domain which attaches the nucleophile onto a carrier domain. ... 

Matrix metalloproteinase-9 (MMP-9) is a secreted multidomain enzyme, which plays an important role in the migration of immime cells. In a recent study, it is reported that fucoidan posttranslationally regulated MMP-9 secretion from U937 (Jintang et al., 2010). Fucoidan isolated from U. pinnatifida possesses immunomodulating activity to produce cytokines and chemokines from macrophages and splenocytes (Yoo et al., 2007). 

MaraKtcl M. Multidomain enzymes involved in peptide synthesis. FEBS Lett 1992 307 40-43. 

In the literature, several enzymatic (Kirstein et al., 1999 Cosnier et al., 1994) and nonenzymatic (Gamboa et al., 2009 Groot and Koper, 2004) electrochemical biosensors were reported for the N03 determination. The enzymatic determination of N03 using nitrate reductase (NaR)—modified electrode is novel and highly selective. NaR is a multidomain enzyme containing flavin adenine dinucleotide (FAD), two heme-Fe and molybdopterin, which catalyzes the two-electron reduction of N03 to NOa (Quan et al., 2005). 

All known eight-stranded a/p-barrel domains have enzymatic functions that include isomerization of small sugar molecules, oxidation by flavin coenzymes, phosphate transfer, and degradation of sugar polymers. In some of these enzymes the barrel domain comprises the whole subunit of the protein in others the polypeptide chain is longer and forms several additional domains. An enzymatic function in these multidomain subunits, however, is always associated with the barrel domain. 

It is equally important to work with full-length versions of enzymes whenever this is feasible. Some enzymes are expressed naturally as multidomain proteins in which the catalytic machinery is localized to a single, discrete protein domain. In... 

Multidomain synthetases can include specialized domains to modify the amino acids of peptide intermediates during chain elongation. The chemistry carried out by these domains introduces specific structural motifs, which are often important for biological activity, into the peptide natural product. A summary of additional NRPS enzyme chemistry is described below and illustrated in Figure 9. 

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... 

Other Class A polymerases. The Thermus aquati-cus (Taq) polymerase is best known for its widespread use in the polymerase chain reaction (PCR Fig. 5-47). Like E. coli I the enzyme is a large multidomain protein. The structure of the catalytic domains of the two enzymes are nearly identical, but the Taq polymerase has poor 3 -5 editing activity.276 The enzyme has been carefully engineered to improve its characteristics for use in the PCR reaction.277... 

The first DNA polymerase activity was identified in 1956 in E. coli (Kornberg et al, 1956 Lehman et al., 1958). The enzyme was subsequently named DNA polymerase I (pol I). E. coli pol I is a 109-kDa enzyme that supports a multidomain architecture containing a polymerase activity, a 5 -3 exonuclease activity, and a 3 -5 exonuclease activity. The C-terminal portion of E. coli pol I, called the Klenow fragment, which lacks the 5 -3 ... 

As most NRPS multienzymes are multidomain proteins with multiple activation domains, multiple sites may participate in the reactions assayed, and no clear result concerning a single specific site may result. In ACV synthetases, the nonadditivity of the initial rates has been observed in the S. clavuligerus enzyme [35] and the A. chrysogenum enzyme [1]. Two or more site activations of one substrate amino acid could be expected to depend on different binding constants, and thus be detectable by kinetic analysis. So far, however, no evidence for mixed types of concentration dependence has been found. It is thus not yet clear if nonadditivity results from misactivation or alteration of kinetic properties in the presence of multiple substrates. In the case of gramicidin S synthetase 2, evidence for misactivations has been reported [59],... 

Multidomain proteins tend to occur more frequently in eukaryotes than in prokaryotes. Often the eukaryotic counterpart to a set of individual prokaryotic enzymes that catalyze successive reactions is a single, multidomain protein. The theoretical advantages proposed for such an arrangement include (1) a geometry for the direct transfer of substrates from one active site to another, in a process known as substrate channeling, in order to increase the overall flux of the pathway, (2) the protection of intermediates that may be unstable in aqueous environments or may be acted on inappropriately by other enzymes, (3) the facilitation of interactions between domains for purposes of allosteric regulatory functions, and (4) the establishment of a fixed stoichiometric ratio of the... 

Another family of enzymes that have been functionally linked to the degradation of connective tissue and dissolution of epithelial and endothelial basement membrane are the MMPs.74 75 These enzymes are multidomain, zinc-containing, neutral endopeptidases including collagenases, stromelysin, gelatinases, and membrane-type metalloproteases. Each MMP exhibits a preferred substrate specificity toward individual matrix proteins, but there is overlapping specificity within the whole family.4 A striking feature of these... 

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