A Chimeric Variant of Two Gentisate 1,2‐Dioxygenases with Improved Catalytic Activity, Altered Structural Conformation and Substrate Specificity

A melanogenic enzyme catalysing the rearrangement of dopachrome has been identified in the ejected ink of the cuttlefish Sepia officinalis. This enzyme occurs as a heat-labile protein which co-migrates with tyrosinase under a variety of chromatographic and electrophoretic conditions. On SDS/PAGE it shows like a single band with an approx. molecular mass of 85 kDa. The enzyme possesses high substrate specificity, acting on L-dopachrome (Km = 1 mM at pH 6.8) and on L-alpha-methyl-dopachrome, but not on D-dopachrome, L-dopachrome methyl ester, dopaminochrome and adrenochrome. Significant inhibition of the catalytic activity was observed with tropolone and L-mimosine. H.p.1.c. analysis of the enzyme-catalysed rearrangement of L-dopachrome revealed the quantitative formation of the decarboxylated product, 5,6-dihydroxyindole. These results point to marked differences between melanogenesis in cephalopod pigment cells and in melanocytes, which may have important implications in relation to the use of sepiomelanin as a model for studies of mammalian melanins.

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Structure-guided rational design of the substrate specificity and catalytic activity of an enzyme

Enzyme Engineering and Evolution: General Methods - Methods in Enzymology ◽

10.1016/bs.mie.2020.04.050 ◽

2020 ◽

pp. 181-202

Author(s):

Jung Min Choi ◽

Hak-Sung Kim

Keyword(s):

Catalytic Activity ◽

Substrate Specificity ◽

Rational Design

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One functional subunit is sufficient for catalytic activity and substrate specificity of Escherichia coli endoribonuclease III artificial heterodimers

FEBS Letters ◽

10.1016/s0014-5793(02)02653-4 ◽

2002 ◽

Vol 518 (1-3) ◽

pp. 93-96 ◽

Cited By ~ 13

Author(s):

Christian Conrad ◽

Jens Guido Schmitt ◽

Elena Evguenieva-Hackenberg ◽

Gabriele Klug

Keyword(s):

Escherichia Coli ◽

Catalytic Activity ◽

Substrate Specificity

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Arabidopsis thaliana β1,2-xylosyltransferase: an unusual glycosyltransferase with the potential to act at multiple stages of the plant N-glycosylation pathway

Biochemical Journal ◽

10.1042/bj20042091 ◽

2005 ◽

Vol 388 (2) ◽

pp. 515-525 ◽

Cited By ~ 42

Author(s):

Peter BENCÚR ◽

Herta STEINKELLNER ◽

Barbara SVOBODA ◽

Jan MUCHA ◽

Richard STRASSER ◽

...

Keyword(s):

Catalytic Activity ◽

Substrate Specificity ◽

Metal Ion ◽

Catalytic Domain ◽

Protein Characterization ◽

Reaction Products ◽

Glycosylation Sites ◽

Glycosylation Pathway ◽

Multiple Stages

XylT (β1,2-xylosyltransferase) is a unique Golgi-bound glycosyltransferase that is involved in the biosynthesis of glycoprotein-bound N-glycans in plants. To delineate the catalytic domain of XylT, a series of N-terminal deletion mutants was heterologously expressed in insect cells. Whereas the first 54 residues could be deleted without affecting the catalytic activity of the enzyme, removal of an additional five amino acids led to the formation of an inactive protein. Characterization of the N-glycosylation status of recombinant XylT revealed that all three potential N-glycosylation sites of the protein are occupied by N-linked oligosaccharides. However, an unglycosylated version of the enzyme displayed substantial catalytic activity, demonstrating that N-glycosylation is not essential for proper folding of XylT. In contrast with most other glycosyltransferases, XylT is enzymatically active in the absence of added metal ions. This feature is not due to any metal ion directly associated with the enzyme. The precise acceptor substrate specificity of XylT was assessed with several physiologically relevant compounds and the xylosylated reaction products were subsequently tested as substrates of other Golgi-resident glycosyltransferases. These experiments revealed that the substrate specificity of XylT permits the enzyme to act at multiple stages of the plant N-glycosylation pathway.

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A capillary electrophoresis method to assay catalytic activity of botulinum neurotoxin serotypes: Implications for substrate specificity

Analytical Biochemistry ◽

10.1016/j.ab.2007.04.048 ◽

2007 ◽

Vol 366 (2) ◽

pp. 207-217 ◽

Cited By ~ 14

Author(s):

Angela L. Purcell ◽

Heidi M. Hoard-Fruchey

Keyword(s):

Capillary Electrophoresis ◽

Catalytic Activity ◽

Substrate Specificity ◽

Botulinum Neurotoxin ◽

Capillary Electrophoresis Method ◽

Electrophoresis Method

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Substitution of glutamic acid 109 by aspartic acid alters the substrate specificity and catalytic activity of the .beta.-subunit in the tryptophan synthase bienzyme complex from Salmonella typhimurium

Biochemistry ◽

10.1021/bi00119a030 ◽

1992 ◽

Vol 31 (4) ◽

pp. 1180-1190 ◽

Cited By ~ 38

Author(s):

Peter S. Brzovic ◽

Arvind M. Kayastha ◽

Edith Wilson Miles ◽

Michael F. Dunn

Keyword(s):

Catalytic Activity ◽

Substrate Specificity ◽

Glutamic Acid ◽

Salmonella Typhimurium ◽

Aspartic Acid ◽

Beta Subunit ◽

Tryptophan Synthase

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Substrate specificity and catalytic efficiency of aldo-keto reductases with phospholipid aldehydes

Biochemical Journal ◽

10.1042/bj20061743 ◽

2007 ◽

Vol 405 (1) ◽

pp. 95-105 ◽

Cited By ~ 64

Author(s):

Matthew Spite ◽

Shahid P. Baba ◽

Yonis Ahmed ◽

Oleg A. Barski ◽

Kanchan Nijhawan ◽

...

Keyword(s):

Catalytic Activity ◽

Substrate Specificity ◽

Reductase Activity ◽

Catalytic Efficiency ◽

Scavenger Receptors ◽

Significant Activity ◽

Oxidized Lipoproteins ◽

Phosphatidic Acids ◽

Chemotactic Factors ◽

Small Intestinal

Phospholipid oxidation generates several bioactive aldehydes that remain esterified to the glycerol backbone (‘core’ aldehydes). These aldehydes induce endothelial cells to produce monocyte chemotactic factors and enhance monocyte–endothelium adhesion. They also serve as ligands of scavenger receptors for the uptake of oxidized lipoproteins or apoptotic cells. The biochemical pathways involved in phospholipid aldehyde metabolism, however, remain largely unknown. In the present study, we have examined the efficacy of the three mammalian AKR (aldo-keto reductase) families in catalysing the reduction of phospholipid aldehydes. The model phospholipid aldehyde POVPC [1-palmitoyl-2-(5-oxovaleroyl)-sn-glycero-3-phosphocholine] was efficiently reduced by members of the AKR1, but not by the AKR6 or the ARK7 family. In the AKR1 family, POVPC reductase activity was limited to AKR1A and B. No significant activity was observed with AKR1C enzymes. Among the active proteins, human AR (aldose reductase) (AKR1B1) showed the highest catalytic activity. The catalytic efficiency of human small intestinal AR (AKR1B10) was comparable with the murine AKR1B proteins 1B3 and 1B8. Among the murine proteins AKR1A4 and AKR1B7 showed appreciably lower catalytic activity as compared with 1B3 and 1B8. The human AKRs, 1B1 and 1B10, and the murine proteins, 1B3 and 1B8, also reduced C-7 and C-9 sn-2 aldehydes as well as POVPE [1-palmitoyl-2-(5-oxovaleroyl)-sn-glycero-3-phosphoethanolamine]. AKR1A4, B1, B7 and B8 catalysed the reduction of aldehydes generated in oxidized C16:0-20:4 phosphatidylcholine with acyl, plasmenyl or alkyl linkage at the sn-1 position or C16:0-20:4 phosphatidylglycerol or phosphatidic acid. AKR1B1 displayed the highest activity with phosphatidic acids; AKR1A4 was more efficient with long-chain aldehydes such as 5-hydroxy-8-oxo-6-octenoyl derivatives, whereas AKR1B8 preferred phosphatidylglycerol. These results suggest that proteins of the AKR1A and B families are efficient phospholipid aldehyde reductases, with non-overlapping substrate specificity, and may be involved in tissue-specific metabolism of endogenous or dietary phospholipid aldehydes.

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Facile Anomer-oriented Syntheses of 4-Methylumbelliferyl Sialic Acid Glycosides

10.26434/chemrxiv.14402576 ◽

2021 ◽

Author(s):

Abdullah Hassan ◽

Stefan Oscarson

Keyword(s):

Catalytic Activity ◽

Sialic Acid ◽

Substrate Specificity ◽

Enzymatic Activity ◽

Addition Reaction ◽

Neuraminic Acid ◽

Acetylneuraminic Acid ◽

Diastereoselective Addition ◽

High Yields ◽

Stereoselective Syntheses

As part of a program to find new sialidases and determine their enzymatic specificity and catalytic activity, a library of 4-methylumbelliferyl sialic acid glycosides derivatised at the C-5 position were prepared from N-acetylneuraminic acid. Both α- and β-4-methylumbelliferyl sialic acid glycosides were prepared in high yields and excellent stereoselectivity. Alpha anomers were accessed via reagent control by utilising additive CH3CN and TBAI, whereas the beta anomers were synthesised through a diastereoselective addition reaction of iodine and the aglycone to the corresponding glycal followed by reduction of the resulting 3-iodo compounds. Both anomer-oriented synthetic pathways allow for gram-scale stereoselective syntheses of the desired C-5 modified neuraminic acid derivatives for use as tools to quantify the enzymatic activity and substrate specificity of known sialidases, and potential detection and investigation of novel sialidases.

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Functional analysis of Clostridium difficile sortase B reveals key residues for catalytic activity and substrate specificity

Journal of Biological Chemistry ◽

10.1074/jbc.ra119.011322 ◽

2020 ◽

Vol 295 (11) ◽

pp. 3734-3745

Author(s):

Chia-Yu Kang ◽

I-Hsiu Huang ◽

Chi-Chi Chou ◽

Tsai-Yu Wu ◽

Jyun-Cyuan Chang ◽

...

Keyword(s):

Cell Wall ◽

Catalytic Activity ◽

Clostridium Difficile ◽

Substrate Specificity ◽

Surface Proteins ◽

Site Directed Mutagenesis ◽

Serine Residue ◽

Peptidoglycan Cell Wall ◽

Attractive Therapeutic Target ◽

Key Residues

Most of Gram-positive bacteria anchor surface proteins to the peptidoglycan cell wall by sortase, a cysteine transpeptidase that targets proteins displaying a cell wall sorting signal. Unlike other bacteria, Clostridium difficile, the major human pathogen responsible for antibiotic-associated diarrhea, has only a single functional sortase (SrtB). Sortase's vital importance in bacterial virulence has been long recognized, and C. difficile sortase B (Cd-SrtB) has become an attractive therapeutic target for managing C. difficile infection. A better understanding of the molecular activity of Cd-SrtB may help spur the development of effective agents against C. difficile infection. In this study, using site-directed mutagenesis, biochemical and biophysical tools, LC-MS/MS, and crystallographic analyses, we identified key residues essential for Cd-SrtB catalysis and substrate recognition. To the best of our knowledge, we report the first evidence that a conserved serine residue near the active site participates in the catalytic activity of Cd-SrtB and also SrtB from Staphylococcus aureus. The serine residue indispensable for SrtB activity may be involved in stabilizing a thioacyl-enzyme intermediate because it is neither a nucleophilic residue nor a substrate-interacting residue, based on the LC-MS/MS data and available structural models of SrtB–substrate complexes. Furthermore, we also demonstrated that residues 163–168 located on the β6/β7 loop of Cd-SrtB dominate specific recognition of the peptide substrate PPKTG. The results of this work reveal key residues with roles in catalysis and substrate specificity of Cd-SrtB.

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