Mapping of catalytically important residues in the rat l-histidine decarboxylase enzyme using bioinformatic and site-directed mutagenesis approaches

HDC (l-histidine decarboxylase), the enzyme responsible for the catalytic production of histamine from l-histidine, belongs to an evolutionarily conserved family of vitamin B6-dependent enzymes known as the group II decarboxylases. Yet despite the obvious importance of histamine, mammalian HDC enzymes remain poorly characterized at both the biochemical and structural levels. By comparison with the recently described crystal structure of the homologous enzyme l-DOPA decarboxylase, we have been able to identify a number of conserved domains and motifs that are important also for HDC catalysis. This includes residues that were proposed to mediate events within the active site, and HDC proteins carrying mutations in these residues were inactive when expressed in reticulocyte cell lysates reactions. Our studies also suggest that a significant change in quartenary structure occurs during catalysis. This involves a protease sensitive loop, and incubating recombinant HDC with an l-histidine substrate analogue altered enzyme structure so that the loop was no longer exposed for tryptic proteolysis. In total, 27 mutant proteins were used to test the proposed importance of 34 different amino acid residues. This is the most extensive mutagenesis study yet to identify catalytically important residues in a mammalian HDC protein sequence and it provides a number of novel insights into the mechanism of histamine biosynthesis.

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Basic amino acid residue cluster within nuclear targeting sequence motif is essential for cytoplasmic plectin-vimentin network junctions.

The Journal of Cell Biology ◽

10.1083/jcb.134.6.1455 ◽

1996 ◽

Vol 134 (6) ◽

pp. 1455-1467 ◽

Cited By ~ 116

Author(s):

B Nikolic ◽

E Mac Nulty ◽

B Mir ◽

G Wiche

Keyword(s):

Amino Acid ◽

Intermediate Filaments ◽

Basic Amino Acid ◽

Site Directed Mutagenesis ◽

Sequence Motif ◽

Amino Acid Residues ◽

Nuclear Targeting ◽

Mutant Proteins ◽

Carboxy Terminal

We have generated a series of plectin deletion and mutagenized cDNA constructs to dissect the functional sequences that mediate plectin's interaction with intermediate filament (IF) networks, and scored their ability to coalign or disrupt intermediate filaments when ectopically expressed in rat kangaroo PtK2 cells. We show that a stretch of approximately 50 amino acid residues within plectin's carboxy-terminal repeat 5 domain serves as a unique binding site for both vimentin and cytokeratin IF networks of PtK2 cells. Part of the IF-binding domain was found to constitute a functional nuclear localization signal (NLS) motif, as demonstrated by nuclear import of cytoplasmic proteins linked to this sequence. Site directed mutagenesis revealed a specific cluster of four basic amino acid residues (arg4277-arg4280) residing within the NLS sequence motif to be essential for IF binding. When mutant proteins corresponding to those expressed in PtK2 cells were expressed in bacteria and purified proteins subjected to a sensitive quantitative overlay binding assay using Eu3+-labeled vimentin, the relative binding capacities of mutant proteins measured were fully consistent with the mutant's phenotypes observed in living cells. Using recombinant proteins we also show by negative staining and rotary shadowing electron microscopy that in vitro assembled vimentin intermediate filaments become packed into dense aggregates upon incubation with plectin repeat 5 domain, in contrast to repeat 4 domain or a mutated repeat 5 domain.

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Sites of Interaction between the FecA and FecR Signal Transduction Proteins of Ferric Citrate Transport in Escherichia coli K-12

Journal of Bacteriology ◽

10.1128/jb.185.13.3745-3752.2003 ◽

2003 ◽

Vol 185 (13) ◽

pp. 3745-3752 ◽

Cited By ~ 26

Author(s):

Sabine Enz ◽

Heidi Brand ◽

Claudia Orellana ◽

Susanne Mahren ◽

Volkmar Braun

Keyword(s):

Escherichia Coli ◽

Signal Transduction ◽

Transcription Initiation ◽

Ferric Citrate ◽

Site Directed Mutagenesis ◽

Amino Acid Residues ◽

Mutant Proteins ◽

Citrate Transport ◽

Signal Transduction Proteins ◽

K 12

ABSTRACT Transcription of the fecABCDE ferric citrate transport genes of Escherichia coli K-12 is initiated by a signaling cascade from the cell surface into the cytoplasm. FecR receives the signal in the periplasm from the outer membrane protein FecA loaded with ferric citrate, transmits the signal across the cytoplasmic membrane, and converts FecI in the cytoplasm to an active sigma factor. In this study, it was shown through the use of a bacterial two-hybrid system that, in the periplasm, the C-terminal FecR237-317 fragment interacts with the N-terminal FecA1-79 fragment. In the same C-terminal region, amino acid residues important for the interaction of FecR with FecA were identified by random and site-directed mutagenesis. They were preferentially located in and around a leucine motif (residues 247 to 268) which was found to be highly conserved in FecR-like proteins. The degree of residual binding of FecR mutant proteins to FecA was correlated with the degree of transcription initiation in response to ferric citrate in the culture medium. Three randomly generated inactive FecR mutants, FecR(L254E), FecR(L269G), and FecR(F284L), were suppressed to different degrees by the mutants FecA(G39R) and FecR(D43E). One FecR mutant, FecR (D138E, V197A), induced fecA promoter-directed transcription constitutively in the absence of ferric citrate and bound more strongly than wild-type FecR to FecA. The data showed that FecR interacts in the periplasm with FecA to confer ferric citrate-induced transcription of the fec transport genes and identified sites in FecR and FecA that are important for signal transduction.

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Identification of Essential Amino Acid Residues of the NorM Na+/Multidrug Antiporter in Vibrio parahaemolyticus

Journal of Bacteriology ◽

10.1128/jb.187.5.1552-1558.2005 ◽

2005 ◽

Vol 187 (5) ◽

pp. 1552-1558 ◽

Cited By ~ 69

Author(s):

Masato Otsuka ◽

Makoto Yasuda ◽

Yuji Morita ◽

Chie Otsuka ◽

Tomofusa Tsuchiya ◽

...

Keyword(s):

Amino Acid ◽

Ethidium Bromide ◽

Vibrio Parahaemolyticus ◽

Drug Transport ◽

Site Directed Mutagenesis ◽

Amino Acid Residues ◽

Acidic Amino Acid ◽

Wild Type ◽

Transmembrane Region ◽

Mutant Proteins

ABSTRACT NorM is a member of the multidrug and toxic compound extrusion (MATE) family and functions as a Na+/multidrug antiporter in Vibrio parahaemolyticus, although the underlying mechanism of the Na+/multidrug antiport is unknown. Acidic amino acid residues Asp32, Glu251, and Asp367 in the transmembrane region of NorM are conserved in one of the clusters of the MATE family. In this study, we investigated the role(s) of acidic amino acid residues Asp32, Glu251, and Asp367 in the transmembrane region of NorM by site-directed mutagenesis. Wild-type NorM and mutant proteins with amino acid replacements D32E (D32 to E), D32N, D32K, E251D, E251Q, D367A, D367E, D367N, and D367K were expressed and localized in the inner membrane of Escherichia coli KAM32 cells, while the mutant proteins with D32A, E251A, and E251K were not. Compared to cells with wild-type NorM, cells with the mutant NorM protein exhibited reduced resistance to kanamycin, norfloxacin, and ethidium bromide, but the NorM D367E mutant was more resistant to ethidium bromide. The NorM mutant D32E, D32N, D32K, D367A, and D367K cells lost the ability to extrude ethidium ions, which was Na+ dependent, and the ability to move Na+, which was evoked by ethidium bromide. Both E251D and D367N mutants decreased Na+-dependent extrusion of ethidium ions, but ethidium bromide-evoked movement of Na+ was retained. In contrast, D367E caused increased transport of ethidium ions and Na+. These results suggest that Asp32, Glu251, and Asp367 are involved in the Na+-dependent drug transport process.

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MhbT Is a Specific Transporter for 3-Hydroxybenzoate Uptake by Gram-Negative Bacteria

Applied and Environmental Microbiology ◽

10.1128/aem.01511-12 ◽

2012 ◽

Vol 78 (17) ◽

pp. 6113-6120 ◽

Cited By ~ 18

Author(s):

Ying Xu ◽

Xiaoli Gao ◽

Song-He Wang ◽

Hong Liu ◽

Peter A. Williams ◽

...

Keyword(s):

Fluorescent Protein ◽

Hypothetical Protein ◽

Site Directed Mutagenesis ◽

Amino Acid Residues ◽

Metabolic Intermediate ◽

Content Type ◽

Mutant Proteins ◽

Green Fluorescent ◽

Cytoplasmic Loops ◽

Conserved Residue

ABSTRACTKlebsiella pneumoniaeM5a1 is capable of utilizing 3-hydroxybenzoate via gentisate, and the 6.3-kb gene clustermhbRTDHIMconferred the ability to grow on 3-hydroxybenzoate toEscherichia coliandPseudomonas putidaPaW340. Four of the six genes (mhbDHIM) encode enzymes converting 3-hydroxybenzoate to pyruvate and fumarate via gentisate. MhbR is a gene activator, and MhbT is a hypothetical protein belonging to the transporter of the aromatic acid/H+symporter family. Since a transporter for 3-hydrxybenzoate uptake has not been characterized to date, we investigated whether MhbT is responsible for the uptake of 3-hydroxybenzoate, its metabolic intermediate gentisate, or both. The MhbT-green fluorescent protein (GFP) fusion protein was located on the cytoplasmic membrane.P. putidaPaW340 containingmhbRΔTDHIMcould not grow on 3-hydroxybenzoate; however, supplyingmhbTintransallowed the bacterium to grow on the substrate.K. pneumoniaeM5a1 andP. putidaPaW340 containing recombinant MhbT transported14C-labeled 3-hydroxybenzoate but not14C-labeled gentisate and benzoate into the cells. Site-directed mutagenesis of two conserved amino acid residues (Asp-82 and Asp-314) and a less-conserved residue (Val-311) among the members of the symporter family in the hydrophilic cytoplasmic loops resulted in the loss of 3-hydroxybenzoate uptake byP. putidaPaW340 carrying the mutant proteins. Hence, we demonstrated that MhbT is a specific 3-hydroxybenzoate transporter.

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A site-directed mutagenesis study to identify amino acid residues involved in the catalytic function of the restriction endonuclease EcoRV

Biochemistry ◽

10.1021/bi00135a010 ◽

1992 ◽

Vol 31 (20) ◽

pp. 4808-4815 ◽

Cited By ~ 92

Author(s):

Ursel Selent ◽

Thomas Rueter ◽

Eleonore Koehler ◽

Michaela Liedtke ◽

Vera Thielking ◽

...

Keyword(s):

Amino Acid ◽

Restriction Endonuclease ◽

Site Directed Mutagenesis ◽

Directed Mutagenesis ◽

Amino Acid Residues ◽

Catalytic Function ◽

Identify Amino Acid ◽

A Site ◽

Mutagenesis Study

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Catalytic residues and a predicted structure of tetrahydrobiopterin-dependent alkylglycerol mono-oxygenase

Biochemical Journal ◽

10.1042/bj20111509 ◽

2012 ◽

Vol 443 (1) ◽

pp. 279-286 ◽

Cited By ~ 10

Author(s):

Katrin Watschinger ◽

Julian E. Fuchs ◽

Vladimir Yarov-Yarovoy ◽

Markus A. Keller ◽

Georg Golderer ◽

...

Keyword(s):

Amino Acid ◽

Chinese Hamster Ovary Cells ◽

Active Form ◽

Chinese Hamster ◽

Fold Increase ◽

Site Directed Mutagenesis ◽

Fatty Aldehyde ◽

Amino Acid Residues ◽

Ovary Cells ◽

Mutant Proteins

Alkylglycerol mono-oxygenase (EC 1.14.16.5) forms a third, distinct, class among tetrahydrobiopterin-dependent enzymes in addition to aromatic amino acid hydroxylases and nitric oxide synthases. Its protein sequence contains the fatty acid hydroxylase motif, a signature indicative of a di-iron centre, which contains eight conserved histidine residues. Membrane enzymes containing this motif, including alkylglycerol mono-oxygenase, are especially labile and so far have not been purified to homogeneity in active form. To obtain a first insight into structure–function relationships of this enzyme, we performed site-directed mutagenesis of 26 selected amino acid residues and expressed wild-type and mutant proteins containing a C-terminal Myc tag together with fatty aldehyde dehydrogenase in Chinese-hamster ovary cells. Among all of the acidic residues within the eight-histidine motif, only mutation of Glu137 to alanine led to an 18-fold increase in the Michaelis–Menten constant for tetrahydrobiopterin, suggesting a role in tetrahydrobiopterin interaction. A ninth additional histidine residue essential for activity was also identified. Nine membrane domains were predicted by four programs: ESKW, TMHMM, MEMSAT and Phobius. Prediction of a part of the structure using the Rosetta membrane ab initio method led to a plausible suggestion for a structure of the catalytic site of alkylglycerol mono-oxygenase.

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Identification of amino acid residues in the C-terminal domain of the natriuretic peptide clearance receptor (NPR-C) that determine G protein coupling by site-directed mutagenesis

Gastroenterology ◽

10.1016/s0016-5085(01)82530-0 ◽

2001 ◽

Vol 120 (5) ◽

pp. A510-A510

Author(s):

H ZHOU ◽

K MURTHY

Keyword(s):

Amino Acid ◽

G Protein ◽

Natriuretic Peptide ◽

Site Directed Mutagenesis ◽

Directed Mutagenesis ◽

Amino Acid Residues ◽

G Protein Coupling ◽

Protein Coupling ◽

Terminal Domain ◽

Clearance Receptor

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A site-directed mutagenesis study of human placental aromatase.

Journal of Biological Chemistry ◽

10.1016/s0021-9258(18)48349-4 ◽

1992 ◽

Vol 267 (2) ◽

pp. 762-768

Author(s):

D J Zhou ◽

K R Korzekwa ◽

T Poulos ◽

S A Chen

Keyword(s):

Site Directed Mutagenesis ◽

Directed Mutagenesis ◽

A Site ◽

Mutagenesis Study

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Chitosanase from Streptomyces sp. strain N174: a comparative review of its structure and function

Biochemistry and Cell Biology ◽

10.1139/o97-079 ◽

1997 ◽

Vol 75 (6) ◽

pp. 687-696 ◽

Cited By ~ 20

Author(s):

Tamo Fukamizo ◽

Ryszard Brzezinski

Keyword(s):

Amino Acid ◽

Amino Acid Sequence ◽

Substrate Binding ◽

Structure And Function ◽

Site Directed Mutagenesis ◽

Directed Mutagenesis ◽

Amino Acid Residues ◽

Streptomyces Sp ◽

Binding Cleft ◽

And Function

Novel information on the structure and function of chitosanase, which hydrolyzes the beta -1,4-glycosidic linkage of chitosan, has accumulated in recent years. The cloning of the chitosanase gene from Streptomyces sp. strain N174 and the establishment of an efficient expression system using Streptomyces lividans TK24 have contributed to these advances. Amino acid sequence comparisons of the chitosanases that have been sequenced to date revealed a significant homology in the N-terminal module. From energy minimization based on the X-ray crystal structure of Streptomyces sp. strain N174 chitosanase, the substrate binding cleft of this enzyme was estimated to be composed of six monosaccharide binding subsites. The hydrolytic reaction takes place at the center of the binding cleft with an inverting mechanism. Site-directed mutagenesis of the carboxylic amino acid residues that are conserved revealed that Glu-22 and Asp-40 are the catalytic residues. The tryptophan residues in the chitosanase do not participate directly in the substrate binding but stabilize the protein structure by interacting with hydrophobic and carboxylic side chains of the other amino acid residues. Structural and functional similarities were found between chitosanase, barley chitinase, bacteriophage T4 lysozyme, and goose egg white lysozyme, even though these proteins share no sequence similarities. This information can be helpful for the design of new chitinolytic enzymes that can be applied to carbohydrate engineering, biological control of phytopathogens, and other fields including chitinous polysaccharide degradation. Key words: chitosanase, amino acid sequence, overexpression system, reaction mechanism, site-directed mutagenesis.

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