Sequential enzymes, protein-based

The /V-formylmethionine of a nascent protein synthesized in bacteria is removed by the sequential activities of PDF and a methionine aminopeptidase to generate the mature protein. The gene encoding PDF was cloned and overexpressed in E. coli by Meinnel and coworkers (1993). The PDF enzyme has an unusual metal ion (Fe2+) as its catalyst. However, the ferrous ion in this enzyme is unstable and can be quickly and irreversibly oxidized to ferric ion, rapidly inactivating the enzyme. PDF-based assay development therefore depended on the ability of nickel ion to replace ferrous ion in vitro, increasing the stability of the enzyme and maintaining its enzymatic activity (Groche et al., 1998 Clements et al., 2001 Hackbarth et al., 2002). 

In situ hybridization buffer is used for two purposes (1) probe denaturing and hybridization and (2) AP enzyme inactivation between two sequential AP-based BISH detections. As we previously reported (4), we learned that AP enzyme can be inactivated by incubating the tissue section with an in situ hybridization buffer containing formamide for 30 min at room temperature without losing probe-target hybridization. Thus, after the first AP-based BISH detection, an in situ hybridization buffer should be applied for inactivating prior to the second AP-based BISH detection. Since formamide can denature proteins, the slides must be washed well with a rinse solution before the second immunodetection step. 

Both of these processes require multiple enzymes acting either sequentially or, as indicated above, as molecular complexes. The hallmarks of both NER and BER are strand scission, removal of a segment of DNA containing the adducted base, 5 to 3 -oriented DNA patch synthesis through the action of a polymerase, using the intact strand as a template, and ligation of the free ends. The distinctions between these two mechanisms are the proteins involved and the types of adducts that are repaired. Bulky adducts, like those of aromatic compounds or... 

Since the PKS (polyketide synthase) gene cluster for actinorhodin (act), an antibiotic produced by Streptomyces coelicolor[ 109], was cloned, more than 20 different gene clusters encoding polyketide biosynthetic enzymes have been isolated from various organisms, mostly actinomycetes, and characterized [98, 100]. Bacterial PKSs are classified into two broad types based on gene organization and biosynthetic mechanisms [98, 100, 102]. In modular PKSs (or type I), discrete multifunctional enzymes control the sequential addition of thioester units and their subsequent modification to produce macrocyclic compounds (or complex polyketides). Type I PKSs are exemplified by 6-deoxyerythronolide B synthase (DEBS), which catalyzes the formation of the macrolactone portion of erythromycin A, an antibiotic produced by Saccharopolyspora erythraea. There are 7 different active-site domains in DEBS, but a given module contains only 3 to 6 active sites. Three domains, acyl carrier protein (ACP), acyltransferase (AT), and P-ketoacyl-ACP synthase (KS), constitute a minimum module. Some modules contain additional domains for reduction of p-carbons, e.g., P-ketoacyl-ACP reductase (KR), dehydratase (DH), and enoyl reductase (ER). The thioesterase-cyclase (TE) protein is present only at the end of module 6. 

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