scholarly journals Crystal Structures of Wild-Type and F448A Mutant Citrobacter freundii Tyrosine Phenol-Lyase Complexed with a Substrate and Inhibitors: Implications for the Reaction Mechanism

Biochemistry ◽  
2018 ◽  
Vol 57 (43) ◽  
pp. 6166-6179 ◽  
Author(s):  
Robert S. Phillips ◽  
Steven Craig
Keyword(s):  
Reaction Mechanism ◽  
Crystal Structures ◽  
Citrobacter Freundii ◽  
Wild Type ◽  
Tyrosine Phenol Lyase

Threonine-124 and phenylalanine-448 in Citrobacter freundii tyrosine phenol-lyase are necessary for activity with l-tyrosine

2002 ◽  
Vol 363 (3) ◽  
pp. 745-752 ◽  
Author(s):  
Tatyana V. DEMIDKINA ◽  
Maria V. BARBOLINA ◽  
Nicolai G. FALEEV ◽  
Bakthavatsalam SUNDARARAJU ◽  
Paul D. GOLLNICK ◽  
...  
Keyword(s):  
Citrobacter Freundii ◽  
Wild Type ◽  
Proteus Vulgaris ◽  
Closed Conformation ◽  
Reaction Specificity ◽  
Phenolic Group ◽  
Tyrosine Phenol Lyase ◽  
Flow Experiments ◽  
Phenol Ring

Thr-124 and Phe-448 are located in the active site of Citrobacter freundii tyrosine phenol-lyase (TPL) near the phenol ring of a bound substrate analogue, 3-(4′-hydroxyphenyl)propionic acid [Sundararaju, Antson, Phillips, Demidkina, Barbolina, Gollnick, Dodson and Wilson (1997) Biochemistry 36, 6502–6510]. Thr-124 is replaced by Asp and Phe-448 is replaced by His in the crystal structure of a structurally similar enzyme, Proteus vulgaris tryptophan indole-lyase, which has 50% identical residues. Hence, Thr-124 and Phe-448 in TPL were mutated to Ala or Asp, and His, respectively, in order to probe the role of these residues in the reaction specificity for l-Tyr. These mutant enzymes have little or no β-elimination activity with l-Tyr or 3-fluoro-l-Tyr as a substrate, but retain significant elimination activity with S-(o-nitrophenyl)-l-cysteine, S-alkyl-l-cysteines and β-chloroalanine. Furthermore, the binding of l-Tyr and other non-substrate amino acids is not significantly affected by the mutations. The mutant TPLs form intermediates in rapid-scanning stopped-flow experiments with l-Phe, l-Tyr and l-Trp, similar to those seen with wild-type TPL. These results demonstrate that Thr-124 and Phe-448 are necessary for the reaction specificity of TPL for l-Tyr, and probably play a role in the elimination stage of the reaction mechanism. Thr-124 is within hydrogen-bonding distance of the phenolic group of the bound substrate, and may help to orientate the ring for β-elimination to occur. Phe-448 may be important to allow the formation of the closed conformation during the reaction.

Mechanisms for auto-inhibition and forced product release in glycine N-methyltransferase: crystal structures of wild-type, mutant R175K and S-adenosylhomocysteine-bound R175K enzymes

2000 ◽  
Vol 298 (1) ◽  
pp. 149-162 ◽  
Author(s):  
Yafei Huang ◽  
Junichi Komoto ◽  
Kiyoshi Konishi ◽  
Yoshimi Takata ◽  
Hirofumi Ogawa ◽  
...  
Keyword(s):  
Crystal Structures ◽  
Wild Type ◽  
Product Release ◽  
Type Mutant

scholarly journals The Type VI Secretion System Modulates Flagellar Gene Expression and Secretion in Citrobacter freundii and Contributes to Adhesion and Cytotoxicity to Host Cells

2015 ◽  
Vol 83 (7) ◽  
pp. 2596-2604 ◽  
Author(s):  
Liyun Liu ◽  
Shuai Hao ◽  
Ruiting Lan ◽  
Guangxia Wang ◽  
Di Xiao ◽  
...  
Keyword(s):  
Secretion System ◽  
Host Cells ◽  
Target Cells ◽  
Deletion Mutants ◽  
Citrobacter Freundii ◽  
Wild Type ◽  
Type Vi Secretion System ◽  
Content Type ◽  
Type Vi Secretion ◽  
Mutant Strains

The type VI secretion system (T6SS) as a virulence factor-releasing system contributes to virulence development of various pathogens and is often activated upon contact with target cells.Citrobacter freundiistrain CF74 has a complete T6SS genomic island (GI) that containsclpV,hcp-2, andvgrT6SS genes. We constructedclpV,hcp-2,vgr, and T6SS GI deletion mutants in CF74 and analyzed their effects on the transcriptome overall and, specifically, on the flagellar system at the levels of transcription and translation. Deletion of the T6SS GI affected the transcription of 84 genes, with 15 and 69 genes exhibiting higher and lower levels of transcription, respectively. Members of the cell motility class of downregulated genes of the CF74ΔT6SS mutant were mainly flagellar genes, including effector proteins, chaperones, and regulators. Moreover, the production and secretion of FliC were also decreased inclpV,hcp-2,vgr, or T6SS GI deletion mutants in CF74 and were restored upon complementation. In swimming motility assays, the mutant strains were found to be less motile than the wild type, and motility was restored by complementation. The mutant strains were defective in adhesion to HEp-2 cells and were restored partially upon complementation. Further, the CF74ΔT6SS, CF74ΔclpV, and CF74Δhcp-2mutants induced lower cytotoxicity to HEp-2 cells than the wild type. These results suggested that the T6SS GI in CF74 regulates the flagellar system, enhances motility, is involved in adherence to host cells, and induces cytotoxicity to host cells. Thus, the T6SS plays a wide-ranging role inC. freundii.

Crystal structures of the disease-causing D444V mutant and the relevant wild type human dihydrolipoamide dehydrogenase

2018 ◽  
Vol 124 ◽  
pp. 214-220 ◽  
Author(s):  
Eszter Szabo ◽  
Reka Mizsei ◽  
Piotr Wilk ◽  
Zsofia Zambo ◽  
Beata Torocsik ◽  
...  
Keyword(s):  
Crystal Structures ◽  
Wild Type ◽  
Dihydrolipoamide Dehydrogenase

Crystal structures and snapshots along the reaction pathway of human phosphoserine phosphatase

2019 ◽  
Vol 75 (6) ◽  
pp. 592-604 ◽  
Author(s):  
Marie Haufroid ◽  
Manon Mirgaux ◽  
Laurence Leherte ◽  
Johan Wouters
Keyword(s):  
Molecular Dynamics ◽  
Reaction Mechanism ◽  
Crystal Structures ◽  
Reaction Pathway ◽  
Living Cells ◽  
Homocysteic Acid ◽  
Phosphoserine Phosphatase ◽  
Key Enzyme ◽  
The Subject ◽  
Dynamics Simulations

The equilibrium between phosphorylation and dephosphorylation is one of the most important processes that takes place in living cells. Human phosphoserine phosphatase (hPSP) is a key enzyme in the production of serine by the dephosphorylation of phospho-L-serine. It is directly involved in the biosynthesis of other important metabolites such as glycine and D-serine (a neuromodulator). hPSP is involved in the survival mechanism of cancer cells and has recently been found to be an essential biomarker. Here, three new high-resolution crystal structures of hPSP (1.5–2.0 Å) in complexes with phosphoserine and with serine, which are the substrate and the product of the reaction, respectively, and in complex with a noncleavable substrate analogue (homocysteic acid) are presented. New types of interactions take place between the enzyme and its ligands. Moreover, the loop involved in the open/closed state of the enzyme is fully refined in a totally unfolded conformation. This loop is further studied through molecular-dynamics simulations. Finally, all of these analyses allow a more complete reaction mechanism for this enzyme to be proposed which is consistent with previous publications on the subject.

A special halide-stabilizing pair and enantioselectivity mechanism of DatA

2014 ◽  
Vol 70 (a1) ◽  
pp. C450-C450
Author(s):  
Lijun Guan ◽  
Hideya Yabuki ◽  
Masahiko Okai ◽  
Jun Ohtsuka ◽  
Masaru Tanokura
Keyword(s):  
Reaction Mechanism ◽  
Crystal Structures ◽  
Structural Information ◽  
Docking Simulation ◽  
Competitive Inhibitor ◽  
Residue Pair ◽  
Structural Basis ◽  
Chiral Compounds ◽  
In The Wild ◽  
Agrobacterium Tumefaciens C58

A novel haloalkane dehalogenase DatA from Agrobacterium tumefaciens C58 belongs to the HLD-II subfamily and hydrolyzes brominated and iodinated compounds, leading to the generation of the corresponding alcohol, a halide ion, and a proton. DatA possesses a unique Asn-Tyr residue pair instead of the Asn-Trp residue pair conserved among the subfamily members, thus the structural basis for its reaction mechanism merits elucidation. In addition, DatA is potentially useful for pharmaceutical and environmental applications, though several crystal structures of HLD-II dehalogenases have been reported so far, the determination of the DatA structure will provide an important contribution to those fields. This work provided insight into the reaction mechanism of DatA via a combination of X-ray crystallographic and computational analysis. The crystal structures of DatA and the Y109W mutant were determined at 1.70 Å [1] and 1.95 Å, respectively. The location of the active site was confirmed by using its novel competitive inhibitor, CHES. The structural information from these two crystal structures and the docking simulation with 1,3-dibromopropane revealed that the replacement of the Asn-Tyr pair with the Asn-Trp pair increases the binding affinity for 1,3-dibromopropane, due to the extra hydrogen bond between Trp109 and halogenated compounds; and that the key residue to bind halogenated substrate is only Asn43 in the wild-type DatA, while those in the Y109W mutant are the Asn-Trp pair. Furthermore, docking simulation using the crystal structures of DatA and some chiral compounds indicated that enantioselectivity of DatA toward brominated alkanes is determined by the large and small spaces around the halogen binding site.

scholarly journals Cloning and Random Mutagenesis of the Erwinia herbicola tyrR Gene for High-Level Expression of Tyrosine Phenol-Lyase

2000 ◽  
Vol 66 (11) ◽  
pp. 4764-4771 ◽  
Author(s):  
Takane Katayama ◽  
Hideyuki Suzuki ◽  
Takashi Koyanagi ◽  
Hidehiko Kumagai
Keyword(s):  
Random Mutagenesis ◽  
Wild Type ◽  
Reporter System ◽  
Erwinia Herbicola ◽  
E Coli ◽  
Repressive Effect ◽  
Tyrosine Phenol Lyase ◽  
High Level ◽  
Regulatory Properties

ABSTRACT Tyrosine phenol-lyase (Tpl), which can synthesize 3,4-dihydroxyphenylalanine from pyruvate, ammonia, and catechol, is a tyrosine-inducible enzyme. Previous studies demonstrated that thetpl promoter of Erwinia herbicola is activated by the TyrR protein of Escherichia coli. In an attempt to create a high-Tpl-expressing strain, we cloned the tyrRgene of E. herbicola and then randomly mutagenized it. Mutant TyrR proteins with enhanced ability to activate tplwere screened for by use of the lac reporter system inE. coli. The most increased transcription oftpl was observed for the strain with the mutanttyrR allele involving amino acid substitutions of alanine, cysteine, and glycine for valine-67, tyrosine-72, and glutamate-201, respectively. A tyrR-deficient derivative of E. herbicola was constructed and transformed with a plasmid carrying the mutant tyrR allele (V67A Y72C E201G substitutions). The resultant strain expressed Tpl without the addition of tyrosine to the medium and produced as much of it as was produced by the wild-type strain grown under tyrosine-induced conditions. The regulatory properties of the mutant TyrRV67A, TyrRY72C, TyrRE201G, and TyrRV67A Y72C E201G proteins were examined in vivo. Interestingly, as opposed to the wild-type TyrR protein, the mutant TyrRV67A protein had a repressive effect on the tyrP promoter in the presence of phenylalanine as the coeffector.

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