Substrate specificity of the phenolic acid decarboxylase from Lactobacillus plantarum and related bacteria analyzed by molecular dynamics and docking

ABSTRACT Bacillus subtilis displays a substrate-inducible decarboxylating activity with the following three phenolic acids: ferulic, p-coumaric, and caffeic acids. Based on DNA sequence homologies between the Bacillus pumilus ferulate decarboxylase gene (fdc) (A. Zago, G. Degrassi, and C. V. Bruschi, Appl. Environ. Microbiol. 61:4484–4486, 1995) and theLactobacillus plantarum p-coumarate decarboxylase gene (pdc) (J.-F. Cavin, L. Barthelmebs, and C. Diviès, Appl. Environ. Microbiol. 63:1939–1944, 1997), a DNA probe of about 300 nucleotides for the L. plantarum pdcgene was used to screen a B. subtilis genomic library in order to clone the corresponding gene in this bacterium. One clone was detected with this heterologous probe, and this clone exhibited phenolic acid decarboxylase (PAD) activity. The corresponding 5-kb insertion was partially sequenced and was found to contain a 528-bp open reading frame coding for a 161-amino-acid protein exhibiting 71 and 84% identity with the pdc- and fdc-encoded enzymes, respectively. The PAD gene (pad) is transcriptionally regulated by p-coumaric, ferulic, or caffeic acid; these three acids are the three substrates of PAD. Thepad gene was overexpressed constitutively inEscherichia coli, and the stable purified enzyme was characterized. The difference in substrate specificity between this PAD and other PADs seems to be related to a few differences in the amino acid sequence. Therefore, this novel enzyme should facilitate identification of regions involved in catalysis and substrate specificity.

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Cloning, Deletion, and Characterization of PadR, the Transcriptional Repressor of the Phenolic Acid Decarboxylase-Encoding padA Gene of Lactobacillus plantarum

Applied and Environmental Microbiology ◽

10.1128/aem.70.4.2146-2153.2004 ◽

2004 ◽

Vol 70 (4) ◽

pp. 2146-2153 ◽

Cited By ~ 76

Author(s):

Jérôme Gury ◽

Lise Barthelmebs ◽

Ngoc Phuong Tran ◽

Charles Diviès ◽

Jean-François Cavin

Keyword(s):

Lactobacillus Plantarum ◽

Phenolic Acid ◽

Stress Proteins ◽

Transcriptional Repressor ◽

Specific Protein ◽

Acid Decarboxylase ◽

Mobility Shift ◽

Specific Chemical ◽

Phenolic Acid Decarboxylase ◽

Gel Mobility Shift

ABSTRACT Lactobacillus plantarum displays a substrate-inducible padA gene encoding a phenolic acid decarboxylase enzyme (PadA) that is considered a specific chemical stress response to the inducing substrate. The putative regulator of padA was located in the padA locus based on its 52% identity with PadR, the padA gene transcriptional regulator of Pediococcus pentosaceus (L. Barthelmebs, B. Lecomte, C. Diviès, and J.-F. Cavin, J. Bacteriol. 182:6724-6731, 2000). Deletion of the L. plantarum padR gene clearly demonstrates that the protein it encodes is the transcriptional repressor of divergently oriented padA. The padR gene is cotranscribed with a downstream open reading frame (ORF1), the product of which may belong to a group of universal stress proteins (Usp). The padR deletion mutant overexpressed padA constitutively, and the padA promoter appears to be tightly regulated in this bacterium. Gel mobility shift assays using the padA gene promoter region and purified PadR expressed in Escherichia coli indicated that operator DNA binding by PadR was not eliminated by addition of p-coumarate. Gel mobility shift assays using partially purified extracts of native PadR protein from both phenolic acid-induced and noninduced L. plantarum cells demonstrate that inactivation of PadR by phenolic acids requires the integrity of L. plantarum and mediation by a specific protein absent in E. coli.

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