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1.
Sci Rep ; 13(1): 2248, 2023 02 08.
Article in English | MEDLINE | ID: mdl-36755087

ABSTRACT

Astaxanthin (AST), a super antioxidant with coloring and medical properties, renders it a beneficial feed additive for shrimp. This study conducted a white shrimp feeding trial of 3S, 3'S isoform AST, which was derived from metabolic-engineered Kluyveromyces marxianus fermented broth (TB) and its extract (TE) compared to sources from two chemically synthetic ASTs (Carophyll Pink [CP] and Lucantin Pink [LP]), which contain 3S, 3'S, 3R, 3'S (3S, 3'R) and 3R, 3'R isoforms ratio of 1:2:1. The effects on red coloration, immune parameters and resistance to Vibrio infection were evaluated. Four AST sources were incorporated into the diets at concentrations of 0 (control), 100 mg kg-1 (TB100, TE100, CP100, and LP100), and 200 mg kg-1 (TB200, TE200, CP200, and LP200). Results revealed that in week 4, shrimps that received AST-supplemented feeds, especially TB100, TB200, and TE200, significantly increased redness (a*) values. Immune responses including phagocytosis activity, superoxide-anion production, phenoloxidase activity, and immune-related genes were examined on days 0, 1, 2, 4, 7, 14, 21, and 28. Generally, shrimps that received AST-supplemented feeds exhibited higher immune responses on days 7 and 14 than the control feed. Gene expression levels of superoxide dismutase and glutathione peroxidase were significantly upregulated on days 7 and 14 in shrimps that received AST-supplemented feeds, while genes of penaeidins, antilipopolysaccharide factor, and lysozyme were upregulated on days 4, 7, and 14, especially received TB200 and TE200. Furthermore, shrimps that received TB100, TE100, CP100, and LP100 7 days were then challenged with Vibrio parahaemolyticus and the result demonstrated higher survival rates especially TB100 at 168 h than the control feed. In conclusion, incorporating AST into the diets enhanced shrimp red coloration, immune parameters, and resistance against V. parahaemolyticus infection. The K. marxianus-derived AST exhibited higher performance than did chemical AST to be a potential feed additive in shrimp aquaculture.


Subject(s)
Penaeidae , Vibrio parahaemolyticus , Animals , Vibrio parahaemolyticus/physiology , Immunity, Innate , Dietary Supplements , Diet
2.
J Mol Biol ; 433(13): 166960, 2021 06 25.
Article in English | MEDLINE | ID: mdl-33774034

ABSTRACT

Proteins with sequence or structure similar to those of di-Zn exopeptidases are usually classified as the M28-family enzymes, including the mammalian-type glutaminyl cyclases (QCs). QC catalyzes protein N-terminal pyroglutamate formation, a posttranslational modification important under many physiological and pathological conditions, and is a drug target for treating neurodegenerative diseases, cancers and inflammatory disorders. Without functional characterization, mammalian QCs and their orthologs remain indistinguishable at the sequence and structure levels from other M28-family proteins, leading to few reported QCs. Here, we show that a low-barrier carboxylic-acid hydrogen-bond network (CAHBN) is required for QC activity and discriminates QCs from M28-family peptidases. We demonstrate that the CAHBN-containing M28 peptidases deposited in the PDB are indeed QCs. Our analyses identify several thousands of QCs from the three domains of life, and we enzymatically and structurally characterize several. For the first time, the interplay between a CAHBN and the binuclear metal-binding center of mammalian QCs is made clear. We found that the presence or absence of CAHBN is a key discriminator for the formation of either the mono-Zn QCs or the di-Zn exopeptidases. Our study helps explain the possible roles of QCs in life.


Subject(s)
Aminoacyltransferases/metabolism , Carboxylic Acids/metabolism , Multigene Family , Animals , Archaeal Proteins/metabolism , Databases, Protein , Humans , Hydrogen Bonding , Ions , Kinetics , Mammals/metabolism , Metals/pharmacology , Phylogeny
3.
Protein Expr Purif ; 133: 121-131, 2017 05.
Article in English | MEDLINE | ID: mdl-28302513

ABSTRACT

Undecaprenyl pyrophosphate phosphatase (UppP), a cell membrane integral enzyme, catalyzes the dephosphorylation of undecaprenyl pyrophosphate to undecaprenyl phosphate, which is an essential carrier lipid in bacterial cell wall synthesis. We previously purified E. coli UppP and concluded that its catalytic site is likely located in the periplasm. To search for additional natural UppP homologs to elucidate what constitutes a common catalytic mechanism and to gain a better chance of obtaining high-resolution crystal structural information, we expressed and purified recombinant Vibrio vulnificus UppP using E. coli as a host. Mutagenesis analysis demonstrates that the proposed catalytic residues Gln-13, Glu-17, His-26 and Arg-166 are directly involved in enzyme catalysis. Additionally, mutations of most of the conserved serine and glycine residues within the proposed catalytic site (S22A, G163A and S165A) lead to complete inactivity, very low activity (<1.3% of the wild type) or no protein expression at all (G163R and G168A), whereas S23A and S167A retain enzyme activity (65% and 34%). Kinetic analysis indicates that S23A and S167A result in 1.4- and 5-fold decreases in kcat, whereas the substrate Km value exhibits only minor changes compared with wild-type UppP, implying that they are involved in enzyme catalysis. The structural modeling and molecular dynamics simulation analyses also provide a plausible structure of the catalytic core, centered on a conserved histidine (His-26) that initiates the hydrolysis of phosphate esters, rationalizing the mutagenesis data. This conclusion can be applied generally to all bacterial UppP enzymes.


Subject(s)
Bacterial Proteins , Gene Expression , Molecular Dynamics Simulation , Pyrophosphatases , Vibrio vulnificus , Bacterial Proteins/biosynthesis , Bacterial Proteins/chemistry , Bacterial Proteins/genetics , Bacterial Proteins/isolation & purification , Crystallography, X-Ray , Protein Domains , Pyrophosphatases/biosynthesis , Pyrophosphatases/chemistry , Pyrophosphatases/genetics , Pyrophosphatases/isolation & purification , Recombinant Proteins/biosynthesis , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/isolation & purification , Vibrio vulnificus/enzymology , Vibrio vulnificus/genetics
4.
Nucleic Acids Res ; 42(2): 1354-64, 2014 Jan.
Article in English | MEDLINE | ID: mdl-24150946

ABSTRACT

DNA mimic proteins are unique factors that control the DNA binding activity of target proteins by directly occupying their DNA binding sites. The extremely divergent amino acid sequences of the DNA mimics make these proteins hard to predict, and although they are likely to be ubiquitous, to date, only a few have been reported and functionally analyzed. Here we used a bioinformatic approach to look for potential DNA mimic proteins among previously reported protein structures. From ∼14 candidates, we selected the Staphylococcus conserved hypothetical protein SSP0047, and used proteomic and structural approaches to show that it is a novel DNA mimic protein. In Staphylococcus aureus, we found that this protein acts as a uracil-DNA glycosylase inhibitor, and therefore named it S. aureus uracil-DNA glycosylase inhibitor (SAUGI). We also determined and analyzed the complex structure of SAUGI and S. aureus uracil-DNA glycosylase (SAUDG). Subsequent BIAcore studies further showed that SAUGI has a high binding affinity to both S. aureus and human UDG. The two uracil-DNA glycosylase inhibitors (UGI and p56) previously known to science were both found in Bacillus phages, and this is the first report of a bacterial DNA mimic that may regulate SAUDG's functional roles in DNA repair and host defense.


Subject(s)
Bacterial Proteins/chemistry , Enzyme Inhibitors/chemistry , Staphylococcus aureus , Uracil-DNA Glycosidase/chemistry , Bacterial Proteins/metabolism , DNA/chemistry , Models, Molecular , Molecular Mimicry , Protein Conformation , Staphylococcus aureus/enzymology , Uracil-DNA Glycosidase/antagonists & inhibitors , Uracil-DNA Glycosidase/metabolism
5.
Nucleic Acids Res ; 41(9): 5127-38, 2013 May.
Article in English | MEDLINE | ID: mdl-23531546

ABSTRACT

DNA mimic proteins are unique factors that control the DNA-binding activity of target proteins by directly occupying their DNA-binding sites. To date, only a few DNA mimic proteins have been reported and their functions analyzed. Here, we present evidence that the Neisseria conserved hypothetical protein DMP12 should be added to this list. Our gel filtration and analytical ultracentrifugation results showed that the DMP12 monomer interacts with the dimeric form of the bacterial histone-like protein HU. Subsequent structural analysis of DMP12 showed that the shape and electrostatic surface of the DMP12 monomer are similar to those of the straight portion of the bent HU-bound DNA and complementary to those of HU protein dimer. DMP12 also protects HU protein from limited digestion by trypsin and enhances the growth rate Escherichia coli. Functionally, HU proteins participate in bacterial nucleoid formation, as well as recombination, gene regulation and DNA replication. The interaction between DMP12 and HU protein might, therefore, play important roles in these DNA-related mechanisms.


Subject(s)
Bacterial Proteins/chemistry , Bacterial Proteins/metabolism , DNA-Binding Proteins/metabolism , Neisseria , Models, Molecular , Molecular Mimicry , Trypsin/metabolism
6.
Nucleic Acids Res ; 40(12): 5718-30, 2012 Jul.
Article in English | MEDLINE | ID: mdl-22373915

ABSTRACT

DNA mimic proteins occupy the DNA binding sites of DNA-binding proteins, and prevent these sites from being accessed by DNA. We show here that the Neisseria conserved hypothetical protein DMP19 acts as a DNA mimic. The crystal structure of DMP19 shows a dsDNA-like negative charge distribution on the surface, suggesting that this protein should be added to the short list of known DNA mimic proteins. The crystal structure of another related protein, NHTF (Neisseria hypothetical transcription factor), provides evidence that it is a member of the xenobiotic-response element (XRE) family of transcriptional factors. NHTF binds to a palindromic DNA sequence containing a 5'-TGTNAN(11)TNACA-3' recognition box that controls the expression of an NHTF-related operon in which the conserved nitrogen-response protein [i.e. (Protein-PII) uridylyltransferase] is encoded. The complementary surface charges between DMP19 and NHTF suggest specific charge-charge interaction. In a DNA-binding assay, we found that DMP19 can prevent NHTF from binding to its DNA-binding sites. Finally, we used an in situ gene regulation assay to provide evidence that NHTF is a repressor of its down-stream genes and that DMP19 can neutralize this effect. We therefore conclude that the interaction of DMP19 and NHTF provides a novel gene regulation mechanism in Neisseria spps.


Subject(s)
Bacterial Proteins/chemistry , Bacterial Proteins/metabolism , Neisseria meningitidis/genetics , Transcription Factors/chemistry , Transcription Factors/metabolism , Amino Acid Sequence , Crystallography, X-Ray , DNA, Bacterial/metabolism , Gene Expression Regulation, Bacterial , Molecular Mimicry , Molecular Sequence Data , Neisseria meningitidis/metabolism
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