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Toxicity Caused by Hydroxycinnamoyl-Coenzyme A Thioester Accumulation in Mutants of Acinetobacter sp . Strain ADP1. Donna Parke, 2004.Hydroxycinnamates, aromatic compounds that play diverse roles in plants, are dissimilated by enzymes encoded by the hca genes in the nutritionally versatile, naturally transformable bacterium Acinetobacter sp . strain ADP1 . A key step in the hca-encoded pathway is activation of the natural substrates caffeate, p-coumarate, and ferulate by an acyl:coenzyme A (acyl:CoA) ligase encoded by hcaC . As described in this paper, Acinetobacter cells with a knockout of the next enzyme in the pathway, hydroxycinnamoyl-CoA hydratase/lyase (HcaA), are extremely sensitive to the presence of the three natural hydroxycinnamate substrates; Escherichia coli cells carrying a subclone with the hcaC gene are hydroxycinnamate sensitive as well . When the hcaA mutation was combined with a mutation in the repressor HcaR, exposure of the doubly mutated Acinetobacter cells to caffeate, p-coumarate, or ferulate at 10–6 M totally inhibited the growth of cells . The toxicity of p-coumarate and ferulate to a  hcaA strain was found to be a bacteriostatic effect . Although not toxic to wild-type cells initially, the diphenolic caffeate was itself converted to a toxin over time in the absence of cells; the converted toxin was bactericidal . In an Acinetobacter strain blocked in hcaA, a secondary mutation in the ligase (HcaC) suppresses the toxic effect . Analysis of suppression due to the mutation of hcaC led to the development of a positive-selection strategy that targets mutations blocking HcaC . An hcaC mutation from one isolate was characterized and was found to result in the substitution of an amino acid that is conserved in a functionally characterized homolog of HcaC .
Molecular Analysis of the Gene Encoding a Novel Chitin-Binding Protease from Alteromonas sp . Strain O-7 and Its Role in the Chitinolytic System. Katsushiro Miyamoto, 2002.Alteromonas sp . strain O-7 secretes several proteins in response to chitin induction . We have found that one of these proteins, designated AprIV, is a novel chitin-binding protease involved in chitinolytic activity . The gene encoding AprIV (aprIV) was cloned in Escherichia coli . DNA sequencing analysis revealed that the open reading frame of aprIV encoded a protein of 547 amino acids with a calculated molecular mass of 57,104 Da . AprIV is a modular enzyme consisting of five domains: the signal sequence, the N-terminal proregion, the family A subtilase region, the polycystic kidney disease domain (PkdD), and the chitin-binding domain type 3 (ChtBD3) . Expression plasmids coding for PkdD or both PkdD and ChtBD (PkdD-ChtBD) were constructed . The PkdD-ChtBD but not PkdD exhibited strong binding to  -chitin and ß-chitin . Western and Northern analyses demonstrated that aprIV was induced in the presence of N-acetylglucosamine, N-acetylchitobiose, or chitin . Native AprIV was purified to homogeneity from Alteromonas sp . strain O-7 and characterized . The molecular mass of mature AprIV was estimated to be 44 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis . The optimum pH and temperature of AprIV were pH 11.5 and 35°C, respectively, and even at 10°C the enzyme showed 25% of the maximum activity . Pretreatment of native chitin with AprIV significantly promoted chitinase activity .
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