Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:3.4.16.2 (PCP)
 3,761 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The capacity of human liver S9 and hepatocytes to metabolically activate 2-amino-3-methylimidazo (4,5-f) quinoline (IQ) in Salmonella mutagenicity assays more closely resembles that of preparations from Aroclor-induced rat than control rat. The extent to which hepatocyte conjugating enzymes contribute to activation in these assays has been studied. Omission of sulphate or addition of a 'specific' sulphotransferase inhibitor (2,6-DCNP) did not significantly reduce mutagenicity, nor did PAPS enhance S9-mediated bacterial mutagenicity. Conversely, mutagenicity was significantly inhibited by PCP (an inhibitor of both sulphotransferase and acetyltransferase) and an acetylation-deficient Salmonella (TA98/1,8DNP6) was unresponsive to the mutagenicity of IQ. These data suggest that acetylation but not sulphation is important in IQ bacterial mutagenesis. The addition of acetyl CoA, PAPS-generating system or ATP paradoxically reduces the mutagenicity of IQ in S9/Salmonella TA98 assays. Therefore, activation by esterification in hepatocytes does not contribute to the mutagenicity of IQ in Salmonella typhimurium possibly due to restricted access of conjugates into the bacterial cell.
 ...
PMID:Activation of 2-amino-3-methylimidazo (4,5-f) quinoline in rat and human hepatocyte/Salmonella mutagenicity assays: the contribution of hepatic conjugation. 333 2

The inhibitory effects of MgATP on neuronal nuclear acetyltransferase activities were studied using lyso platelet-activating factor (lyso-PAF, 1-alkyl-sn-glycero-3-phosphocholine) and lysophosphatidylcholine (lyso-PC, 1-acyl-sn-glycero-3-phosphocholine). The nuclear (N1) acetylation of lyso-PC was more profoundly inhibited by MgATP. MgATP did not alter the apparent Km for acetyl-CoA in either acetylation reaction. The inhibitory effects of MgATP were not seen for other nucleotides or MgAMP-PCP. Kinase inhibitors such as staurosporine (1 microM), chelerythrine, and R59022 (diglyceride kinase inhibitor I) did not block the MgATP inhibition of either acetylation. However, the addition of phospholipids to the assays indicated a selective inhibitory effect for PIP (25-50 microM) in the nuclear acetylation of lyso-PAF. When N1 was incubated with [gamma-33P]ATP, phosphatidic acid and PIP were the principal radioactive lipid products. While the extent of MgATP inhibition of lyso-PAF acetylation was similar at different concentrations of lyso-PAF, increasing lyso-PC concentrations greatly decreased the MgATP inhibition seen in lyso-PC acetylations. Nuclear envelopes prepared in the presence of PMSF, and fraction N1 exposed to PMSF, did not show the inhibitory effect of MgATP on lyso-PC acetylation. PMSF (an inhibitor of certain phospholipase and lysophospholipase activities) did not reduce the MgATP inhibition of lyso-PAF acetylation. Arachidonoyl trifluoromethylketone, an inhibitor of cytosolic phospholipases A2 and of lysophospholipase activity associated with cPLA2, also blocked the inhibitory effect of MgATP on lyso-PC acetylation. Using radioactive lyso-PC substrate, fraction N1 produced labeled free fatty acid and phosphatidylcholine. In the presence of acetyl-CoA, the production of radioactive phosphatidylcholine increased almost 6-fold when MgATP was also included in these incubations. In the presence of MgATP and acetyl-CoA, PMSF reduced the levels of radioactive free fatty acid and phosphatidylcholine derived from lyso-PC, while Triacsin C, an inhibitor of acyl CoA synthetase, decreased phosphatidylcholine labeling. These findings suggest that MgATP inhibition of lyso-PC acetylation results from a loss of lyso-PC substrate that is largely mediated by nuclear lysophospholipase, acyl-CoA synthetase and lyso-PC acylation. Thus the neuronal nuclear production of Acyl PAF may be regulated by paths that compete for the lyso-PC substrate. In contrast, the acetylation of lyso-PAF is inhibited by PIP, a product of nuclear PI kinase reactions.
 ...
PMID:MgATP has different inhibitory effects on the use of 1-acyl-lysophosphatidylcholine and lyso platelet-activating factor acceptors by neuronal nuclear acetyltransferase activities. 963 Jul 21

A key step in fungal biosynthesis of lysine, enzymatic reduction of alpha-aminoadipate at C6 to the semialdehyde, requires two gene products in Saccharomyces cerevisiae, Lys2 and Lys5. Here, we show that the 31-kDa Lys5 is a specific posttranslational modification catalyst, using coenzyme A (CoASH) as a cosubstrate to phosphopantetheinylate Ser880 of the 155-kDa Lys2 and activate it for catalysis. Lys2 was subcloned from S. cerevisiae and expressed in and purified from Escherichia coli as a full-length 155-kDa enzyme, as a 105-kDa adenylation/peptidyl carrier protein (A/PCP) fragment (residues 1-924), and as a 14-kDa PCP fragment (residues 809-924). The apo-PCP fragment was covalently modified to phosphopantetheinylated holo-PCP by pure Lys5 and CoASH with a Km of 1 microM and kcat of 3 min-1 for both the PCP and CoASH substrates. The adenylation domain of the A/PCP fragment activated S-carboxymethyl-L-cysteine (kcat/Km = 840 mM-1 min-1) at 16% the efficiency of L-alpha-aminoadipate in [32P]PPi/ATP exchange assays. The holo form of the A/PCP 105-kDa fragment of Lys2 covalently aminoacylated itself with [35S]S-carboxymethyl-L-cysteine. Addition of NADPH discharged the covalent acyl-S-PCP Lys2, consistent with a reductive cleavage of the acyl-S-enzyme intermediate. These results identify the Lys5/Lys2 pair as a two-component system in which Lys5 covalently primes Lys2, allowing alpha-aminoadipate reductase activity by holo-Lys2 with catalytic cycles of autoaminoacylation and reductive cleavage. This is a novel mechanism for a fungal enzyme essential for amino acid metabolism.
 ...
PMID:Lysine biosynthesis in Saccharomyces cerevisiae: mechanism of alpha-aminoadipate reductase (Lys2) involves posttranslational phosphopantetheinylation by Lys5. 1032 Mar 45

Yersiniabactin (Ybt) synthetase is a three-subunit, 17-domain [7 domains in high molecular weight protein (HMWP)2, 9 in HMWP1, and 1 in YbtE] enzyme producing the virulence-conferring siderophore yersiniabactin in Yersinia pestis. The 350-kDa HMWP1 subunit contains a polyketide synthase module (KS-AT-MT(2)-KR-ACP) and a nonribosomal peptide synthetase module (Cy(3)-MT(3)-PCP(3)-TE). The full-length HMWP1 was heterologously overexpressed in Escherichia coli and purified to near homogeneity. The purified HMWP1 showed thioesterase activity toward acyl-CoAs, such as acetyl-CoA, benzoyl-CoA, and malonyl-CoA, with saturation kinetics and relative catalytic efficiencies of 172:50:1. A chain-releasing thioesterase (TE) activity is ascribed to the C-terminal TE domain, and this was substantiated by the fact that acyl-N-acetylcysteamines were hydrolyzed by the didomain PCP(3)-TE fragment of HMWP1. However, PCP(3)-TE failed to hydrolyze any of the acyl-CoAs, suggesting the TE domain does not recognize CoA moiety, thus the acyl-CoA hydrolysis by HMWP1 must involve other domains. Ser-to-Ala mutants in each of the AT, ACP, PCP(3), and TE domains reduced hydrolysis rates of the two fastest substrates, acetyl-CoA and benzoyl-CoA, by more than two orders of magnitude. Thus, the acyl-CoA hydrolysis activity requires 4 of the 9 domains of HMWP1, and it is consistent with autoacylation of the AT domain active site serine and subsequent passage of the itinerant acyl chain from AT to ACP to PCP(3) to the TE domain, a cascade of four sequential acyl-enzyme intermediates, for hydrolytic turnover. This could represent an editing pathway for this polyketide synthase/nonribosomal peptide synthetase assembly line.
 ...
PMID:Acyl-CoA hydrolysis by the high molecular weight protein 1 subunit of yersiniabactin synthetase: mutational evidence for a cascade of four acyl-enzyme intermediates during hydrolytic editing. 1110 85

In fungi, nonribosomal peptide synthetases (NRP synthetases) are large multi-functional enzymes containing adenylation, thiolation (or peptidyl carrier protein, PCP) and condensation domains. These enzymes are often encoded within gene clusters. Multiple NRP synthetase ORFs have also been identified in fungi (14 in Aspergillus fumigatus). LeaA, a methyltransferase, is involved in secondary metabolite gene cluster regulation in Aspergillus spp. The NRP synthetases GliP and FtmA respectively direct the biosynthesis of the toxic metabolites gliotoxin and brevianamide F, a precursor of bioactive prenylated alkaloids. The NRP synthetase Pes1 has been shown to mediate resistance to oxidative stress, and in plant-pathogenic ascomycetes (e.g. Cochliobolus heterostrophus) an NRP synthetase, encoded by the NPS6 gene, significantly contributes to virulence and resistance to oxidative stress. Adenylation (A) domains within NRP synthetases govern the specificity of amino acid incorporation into nonribosomally synthesized peptides. To date there have only been limited demonstrations of A domain specificity (e.g. A. fumigatus GliP and in Beauveria bassiana) in fungi. Indeed, only in silico prediction data are available on A domain specificity of NRP synthetases from most fungi. NRP synthetases are activated by 4'-phosphopantetheinylation of serine residues within PCP domains by 4'-phosphopantetheinyl transferases (4'-PPTases). Coenzyme A acts as the 4'-phosphopantetheine donor, and labelled coenzyme A can be used to affinity-label apo-NRP synthetases. Emerging fungal gene disruption and gene cluster expression strategies, allied to proteomic strategies, are poised to facilitate a greater understanding of the coding potential of NRP synthetases in fungi.
 ...
PMID:Nonribosomal peptide synthesis in Aspergillus fumigatus and other fungi. 1746 44

The biological properties of the calcium-dependent antibiotics (CDAs), daptomycin and related nonribosomal lipopeptides, depend to a large extent on the nature of the N-terminal fatty acid moiety. It is suggested that the chain length of the unusually short (C6) 2,3-epoxyhexanoyl fatty acid moiety of CDA is determined by the specificity of the KAS-II enzyme encoded by fabF3 in the CDA biosynthetic gene cluster. Indeed, deletion of the downstream gene hxcO results in three new lipopeptides, all of which possess hexanoyl side chains (hCDAs). This confirms that HxcO functions as a hexanoyl-CoA or -ACP oxidase. The absence of additional CDA products with longer fatty acid groups further suggests that the CDA lipid chain is biosynthesized on a single ACP and is then transferred directly from this ACP to the first CDA peptide synthetase (CdaPS1). Interestingly, the hexanoyl-containing CDAs retain antibiotic activity. To further modulate the biological properties of CDA by introducing alternative fatty acid groups, a mutasynthesis approach was developed. This involved mutating the key active site Ser residue of the CdaPS1, module 1 PCP domain to Ala, which prevents subsequent phosphopantetheinylation. In the absence of the natural module 1 PCP tethered intermediate, it is possible to effect incorporation of different N-acyl-L-serinyl N-acetylcysteamine (NAC) thioester analogues, leading to CDA products with pentanoyl as well as hexanoyl side chains.
 ...
PMID:Engineered biosynthesis of nonribosomal lipopeptides with modified fatty acid side chains. 1802 Mar 33