scholarly journals Novel 3,6-Dihydroxypicolinic Acid Decarboxylase Mediated Picolinic Acid Catabolism inAlcaligenes faecalisJQ135

2018 ◽  
Author(s):  
Qiu Jiguo ◽  
Zhang Yanting ◽  
Yao Shigang ◽  
Ren Hao ◽  
Qian Meng ◽  
...  
Keyword(s):  
Aromatic Compounds ◽  
Picolinic Acid ◽  
Degradation Pathway ◽  
Site Directed Mutagenesis ◽  
Acid Decarboxylase ◽  
Directed Mutagenesis ◽  
Deficient Mutant ◽  
Carbon And Nitrogen ◽  
Genetic Deletion ◽  
Related Proteins

AbstractAlcaligenesfaecalisstrain JQ135 utilizes picolinic acid (PA) as sole carbon and nitrogen source for growth. In this study, we screened a 6-hydroxypicolinic acid (6HPA) degradation-deficient mutant through random transposon mutagenesis. The mutant hydroxylated 6HPA into an intermediate, identified as 3,6-dihydroxypicolinic acid (3,6DHPA) with no further degradation. A novel decarboxylase PicC was identified that was found to be responsible for the decarboxylation of 3,6DHPA to 2.5-dihydroxypyridine. Although, PicC belonged to amidohydrolase_2 family, it shows low similarity (<45%) when compared to other reported amidohydrolase_2 family decarboxylases. Moreover, PicC was found to form a monophyletic group in the phylogenetic tree constructed using PicC and related proteins. Further, the genetic deletion and complementation results demonstrated thatpicCwas essential for PA degradation. The PicC was Zn2+-dependent non-oxidative decarboxylase that can specifically catalyze the irreversible decarboxylation of 3,6DHPA to 2.5-dihydroxypyridine. TheKmandkcattowards 3,6DHPA were observed to be 13.44 μM and 4.77 s-1, respectively. Site-directed mutagenesis showed that His163 and His216 were essential for PicC activity.ImportancePicolinic acid is a natural toxic pyridine derived from L-tryptophan metabolism and some aromatic compounds in mammalian and microbial cells. Microorganisms can degrade and utilize picolinic acid for their growth, and thus, a microbial degradation pathway of picolinic acid has been proposed. Picolinic acid is converted into 6-hydroxypicolinic acid, 3,6-dihydroxypicolinic acid, and 2,5-dihydroxypyridine in turn. However, there was no physiological and genetic validation for this pathway. This study demonstrated that 3,6DHPA was an intermediate in PA catabolism process and further identified and characterized a novel amidohydrolase_2 family decarboxylase PicC. It was also shown that PicC could catalyze the decarboxylation process of 3,6-dihydroxypicolinic acid into 2,5-dihydroxypyridine. This study provides a basis for understanding PA degradation pathway and the underlying molecular mechanism.

scholarly journals Novel 3,6-Dihydroxypicolinic Acid Decarboxylase-Mediated Picolinic Acid Catabolism inAlcaligenes faecalisJQ135

2019 ◽  
Vol 201 (7) ◽  
Author(s):  
Jiguo Qiu ◽  
Yanting Zhang ◽  
Shigang Yao ◽  
Hao Ren ◽  
Meng Qian ◽  
...  
Keyword(s):  
Aromatic Compounds ◽  
Picolinic Acid ◽  
Alcaligenes Faecalis ◽  
Degradation Pathway ◽  
Site Directed Mutagenesis ◽  
Pyridine Derivative ◽  
Acid Decarboxylase ◽  
Microbial Cells ◽  
Content Type ◽  
Related Proteins

ABSTRACTPicolinic acid (PA), a typical C-2-carboxylated pyridine derivative, is a metabolite ofl-tryptophan and many other aromatic compounds in mammalian and microbial cells. Microorganisms can degrade and utilize PA for growth. However, the precise mechanism of PA metabolism remains unknown.Alcaligenes faecalisstrain JQ135 utilizes PA as its carbon and nitrogen source for growth. In this study, we screened a 6-hydroxypicolinic acid (6HPA) degradation-deficient mutant through random transposon mutagenesis. The mutant hydroxylated 6HPA into an intermediate, identified as 3,6-dihydroxypicolinic acid (3,6DHPA), with no further degradation. A novel decarboxylase, PicC, was identified to be responsible for the decarboxylation of 3,6DHPA to 2,5-dihydroxypyridine. Although, PicC belonged to the amidohydrolase 2 family, it shows low similarity (<45%) compared to other reported amidohydrolase 2 family decarboxylases. Moreover, PicC was found to form a monophyletic group in the phylogenetic tree constructed using PicC and related proteins. Further, the genetic deletion and complementation results demonstrated thatpicCwas essential for PA degradation. The PicC was Zn2+-dependent nonoxidative decarboxylase that can specifically catalyze the irreversible decarboxylation of 3,6DHPA to 2,5-dihydroxypyridine. TheKmandkcattoward 3,6DHPA were observed to be 13.44 μM and 4.77 s−1, respectively. Site-directed mutagenesis showed that His163 and His216 were essential for PicC activity. This study provides new insights into the microbial metabolism of PA at molecular level.IMPORTANCEPicolinic acid is a natural toxic pyridine derived froml-tryptophan metabolism and other aromatic compounds in mammalian and microbial cells. Microorganisms can degrade and utilize picolinic acid for their growth, and thus a microbial degradation pathway of picolinic acid has been proposed. Picolinic acid is converted into 6-hydroxypicolinic acid, 3,6-dihydroxypicolinic acid, and 2,5-dihydroxypyridine in turn. However, there was no physiological and genetic validation for this pathway. This study demonstrated that 3,6-dihydroxypicolinic acid was an intermediate in picolinic acid catabolism and further identified and characterized a novel amidohydrolase 2 family decarboxylase PicC. PicC was also shown to catalyze the decarboxylation of 3,6-dihydroxypicolinic acid into 2,5-dihydroxypyridine. This study provides a basis for understanding picolinic acid degradation and its underlying molecular mechanism.

Dissection of functional domains in Bcl-2α by site-directed mutagenesis

1994 ◽  
Vol 72 (11-12) ◽  
pp. 463-469 ◽  
Author(s):  
Christoph Borner ◽  
Reynald Olivier ◽  
Isabelle Martinou ◽  
Chantal Mattmann ◽  
Jurg Tschopp ◽  
...  
Keyword(s):  
Cell Survival ◽  
Protein Structures ◽  
Cell Types ◽  
Site Directed Mutagenesis ◽  
Functional Domains ◽  
Directed Mutagenesis ◽  
Membrane Attachment ◽  
Alternatively Spliced ◽  
Membrane Bound ◽  
Related Proteins

Bcl-2α is a mitochondrial or perinuclear-associated oncoprotein that prolongs the life span of a variety of cell types by interfering with programmed cell death. How Bcl-2 confers cell survival is unknown, although antioxidant and antiprotease functions have been proposed. In addition, protein structures of Bcl-2 that are crucial for its survival activity are still ill-defined. Bcl-2 can occur as Bcl-2α or Bcl-2β, two alternatively spliced forms which solely differ in their carboxyl termini. The finding that Bcl-2α is active and membrane bound, but Bcl-2β is inactive and cytosolic, indicates that the carboxyl terminus contributes to the survival activity of Bcl-2. This region contains two subdomains, a domain X with unknown function and a hydrophobic stretch reported to mediate membrane assocation of Bcl-2α. Recently Bcl-2-related proteins have been identified. These include Bax that heterodimerizes with Bcl-2 and, when overxpressed, counteracts Bcl-2. Bax contains two highly conserved regions of sequence homology with Bcl-2, referred to as Bcl-2 homology 1 and 2 (BH1 and BH2) domains. Site-directed mutagenesis studies have revealed that both domains are not only novel dimerization motifs for the interaction of Bax with Bcl-2 but also crucial for the survival activity of Bcl-2. Interestingly, the C-terminal end of BH2 encompasses the Bcl-2α/β splice site, as well as part of domain X in Bcl-2α. To better define the role of domain X and the hydrophobic C-terminal stretch of Bcl-2α for its survival activity, we created various deletion and truncation mutations in these regions by site-directed mutagenesis. We show here that membrane attachment and therefore the hydrophobic stretch is not required for the survival activity of Bcl-2, but part of domain X appears to be indispensable.Key words: apoptosis, Bcl-2, mutagenesis, cell survival, functional domains.

scholarly journals Phosphorylation Controls Autoinhibition of Cytoplasmic Linker Protein-170

2010 ◽  
Vol 21 (15) ◽  
pp. 2661-2673 ◽  
Author(s):  
Ho-Sup Lee ◽  
Yulia A. Komarova ◽  
Elena S. Nadezhdina ◽  
Rana Anjum ◽  
John G. Peloquin ◽  
...  
Keyword(s):  
Binding Affinity ◽  
Conformational Changes ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Phosphorylation Sites ◽  
Deficient Mutant ◽  
Rich Region ◽  
The Third ◽  
Open Conformation ◽  
Intramolecular Association

Cytoplasmic linker protein (CLIP)-170 is a microtubule (MT) plus-end-tracking protein that regulates MT dynamics and links MT plus ends to different intracellular structures. We have shown previously that intramolecular association between the N and C termini results in autoinhibition of CLIP-170, thus altering its binding to MTs and the dynactin subunit p150Glued (J. Cell Biol. 2004: 166, 1003–1014). In this study, we demonstrate that conformational changes in CLIP-170 are regulated by phosphorylation that enhances the affinity between the N- and C-terminal domains. By using site-directed mutagenesis and phosphoproteomic analysis, we mapped the phosphorylation sites in the third serine-rich region of CLIP-170. A phosphorylation-deficient mutant of CLIP-170 displays an “open” conformation and a higher binding affinity for growing MT ends and p150Glued as compared with nonmutated protein, whereas a phosphomimetic mutant confined to the “folded back” conformation shows decreased MT association and does not interact with p150Glued. We conclude that phosphorylation regulates CLIP-170 conformational changes resulting in its autoinhibition.

scholarly journals Site-directed mutagenesis of K396R of the 65 kDa glutamic acid decarboxylase active site obliterates enzyme activity but not antibody binding

FEBS Letters ◽  
2001 ◽  
Vol 488 (3) ◽  
pp. 185-189 ◽  
Author(s):  
Christiane S. Hampe ◽  
Lisa P. Hammerle ◽  
Alberto Falorni ◽  
John Robertson ◽  
Åke Lernmark
Keyword(s):  
Enzyme Activity ◽  
Glutamic Acid ◽  
Glutamic Acid Decarboxylase ◽  
Active Site ◽  
Antibody Binding ◽  
Site Directed Mutagenesis ◽  
Acid Decarboxylase ◽  
Directed Mutagenesis

scholarly journals The 2-Aminoethylphosphonate-Specific Transaminase of the 2-Aminoethylphosphonate Degradation Pathway

2002 ◽  
Vol 184 (15) ◽  
pp. 4134-4140 ◽  
Author(s):  
Alexander D. Kim ◽  
Angela S. Baker ◽  
Debra Dunaway-Mariano ◽  
W. W. Metcalf ◽  
B. L. Wanner ◽  
...  
Keyword(s):  
Amino Acid ◽  
Physiological Role ◽  
Degradation Pathway ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Purification And Characterization ◽  
Ph Optimum ◽  
Serovar Typhimurium ◽  
High Degree

ABSTRACT The 2-aminoethylphosphonate transaminase (AEPT; the phnW gene product) of the Salmonella enterica serovar Typhimurium 2-aminoethylphosphonate (AEP) degradation pathway catalyzes the reversible reaction of AEP and pyruvate to form phosphonoacetaldehyde (P-Ald) and l-alanine (l-Ala). Here, we describe the purification and characterization of recombinant AEPT. pH rate profiles (log Vm and log Vm /Km versus pH) revealed a pH optimum of 8.5. At pH 8.5, K eq is equal to 0.5 and the k cat values of the forward and reverse reactions are 7 and 9 s−1, respectively. The Km for AEP is 1.11 ± 0.03 mM; for pyruvate it is 0.15 ± 0.02 mM, for P-Ald it is 0.09 ± 0.01 mM, and for l-Ala it is 1.4 ± 0.03 mM. Substrate specificity tests revealed a high degree of discrimination, indicating a singular physiological role for the transaminase in AEP degradation. The 40-kDa subunit of the homodimeric enzyme is homologous to other members of the pyridoxalphosphate-dependent amino acid transaminase superfamily. Catalytic residues conserved within well-characterized members are also conserved within the seven known AEPT sequences. Site-directed mutagenesis demonstrated the importance of three selected residues (Asp168, Lys194, and Arg340) in AEPT catalysis.

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