scholarly journals VvLAR1 and VvLAR2 Are Bifunctional Enzymes for Proanthocyanidin Biosynthesis in Grapevine

2019 ◽  
Vol 180 (3) ◽  
pp. 1362-1374 ◽  
Author(s):  
Keji Yu ◽  
Ji Hyung Jun ◽  
Changqing Duan ◽  
Richard A. Dixon
Keyword(s):  
Bifunctional Enzymes

scholarly journals Mechanistic Dissection of the Enzyme Complexes Involved in Biosynthesis of Lacticin 3147 and Nisin

2008 ◽  
Vol 74 (21) ◽  
pp. 6591-6597 ◽  
Author(s):  
Anneke Kuipers ◽  
Jenny Meijer-Wierenga ◽  
Rick Rink ◽  
Leon D. Kluskens ◽  
Gert N. Moll
Keyword(s):  
Lactococcus Lactis ◽  
Bifunctional Enzyme ◽  
Bifunctional Enzymes ◽  
Lacticin 3147 ◽  
Two Component ◽  
Modified Peptides ◽  
Enzyme Complexes ◽  
Insight Into ◽  
Design And Production

ABSTRACT The thioether rings in the lantibiotics lacticin 3147 and nisin are posttranslationally introduced by dehydration of serines and threonines, followed by coupling of these dehydrated residues to cysteines. The prepeptides of the two-component lantibiotic lacticin 3147, LtnA1 and LtnA2, are dehydrated and cyclized by two corresponding bifunctional enzymes, LtnM1 and LtnM2, and are subsequently processed and exported via one bifunctional enzyme, LtnT. In the nisin synthetase complex, the enzymes NisB, NisC, NisT, and NisP dehydrate, cyclize, export, and process prenisin, respectively. Here, we demonstrate that the combination of LtnM2 and LtnT can modify, process, and transport peptides entirely different from LtnA2 and that LtnT can process and transport unmodified LtnA2 and unrelated peptides. Furthermore, we demonstrate a higher extent of NisB-mediated dehydration in the absence of thioether rings. Thioether rings apparently inhibited dehydration, which implies alternating actions of NisB and NisC. Furthermore, certain (but not all) NisC-cyclized peptides were exported with higher efficiency as a result of their conformation. Taken together, these data provide further insight into the applicability of Lactococcus lactis strains containing lantibiotic enzymes for the design and production of modified peptides.

scholarly journals Engineering of Bifunctional Enzymes with Uricase and Peroxidase Activities for Simple and Rapid Quantification of Uric Acid in Biological Samples

Catalysts ◽  
2020 ◽  
Vol 10 (4) ◽  
pp. 428
Author(s):  
Thanawat Phuadraksa ◽  
Jurairat Chittrakanwong ◽  
Kittitouch Tullayaprayouch ◽  
Naruthai Onsirisakul ◽  
Sineewanlaya Wichit ◽  
...  
Keyword(s):  
Uric Acid ◽  
Low Cost ◽  
Colorimetric Detection ◽  
Amplex Red ◽  
Bifunctional Enzymes ◽  
Bifunctional Proteins ◽  
Frame Fusion ◽  
The Cost ◽  
Optimized Conditions ◽  
Catalyzed Oxidation

Serum uric acid (SUA) is an important biomarker for prognosis and management of gout and other diseases. The development of a low-cost, simple, rapid and reliable assay for SUA detection is of great importance. In the present study, to save the cost of enzyme production and to shorten the reaction time for uric acid quantification, bifunctional proteins with uricase and peroxidase activities were engineered. In-frame fusion of Candida utilis uricase (CUOX) and Vitreoscilla hemoglobin (VHb) resulted in two versions of the bifunctional protein, CUOX-VHb (CV) and VHb-CUOX (VC). To our knowledge, this is the first report to describe the production of proteins with uricase and peroxidase activities. Based on the measurement of the initial rates of the coupled reaction (between uricase and peroxidase), CV was proven to be the most efficient enzyme followed by VC and native enzymes (CUOX+VHb), respectively. CV was further applied for the development of an assay for colorimetric detection of SUA, which was based on VHb-catalyzed oxidation of Amplex Red in the presence of hydrogen peroxide (H2O2). Under the optimized conditions, the assay exhibited a linear relationship between the absorbance and UA concentration over the range of 2.5 to 50 μM, with a detection limit of 1 μM. In addition, the assay can be performed at a single pH (8.0) so adjustment of the pH for peroxidase activity was not required. This advantage helped to further reduce costs and time. The developed assay was also successfully applied to detect UA in pooled human serum with the recoveries over 94.8%. These results suggest that the proposed assay holds great potential for clinical application.

scholarly journals Structure of an ancestral ADP-dependent kinase with fructose-6P reveals key residues for binding, catalysis, and ligand-induced conformational changes

2020 ◽  
pp. jbc.RA120.015376
Author(s):  
Sebastián M. Muñoz ◽  
Victor Castro-Fernandez ◽  
Victoria Guixe
Keyword(s):  
Conformational Changes ◽  
Active Site ◽  
Substrate Specificities ◽  
Closed Conformation ◽  
Sugar Binding ◽  
Bifunctional Enzymes ◽  
Glucose Binding ◽  
Critical Residue ◽  
Key Residues ◽  
Human And Mouse

ADP-dependent kinases were first described in archaea, although their presence has also been reported in bacteria and eukaryotes (human and mouse). This enzyme family comprises three substrate specificities; specific phosphofructokinases (ADP-PFK), specific glucokinases (ADP-GK), and bifunctional enzymes (ADP-PFK/GK). Although many structures are available for members of this family, no one exhibits fructose-6P at the active site. Employing an ancestral enzyme, we obtain the first structure of an ADP-dependent kinase (AncMsPFK) with fructose-6P at its active site. Key residues for sugar-binding and catalysis were identified by alanine scanning, being D36 a critical residue for F6P binding and catalysis. However, this residue hinders glucose binding since its mutation to alanine converts the AncMsPFK enzyme into a specific ADP-GK. Residue K179 is critical for F6P binding, while residues N181 and R212 are also important for this sugar-binding, but to a lesser extent. This structure also provides evidence for the requirement of both substrates (sugar and nucleotide) to accomplish the conformational change leading to a closed conformation. This suggests that AncMsPFK mainly populates two states (open and closed) during the catalytic cycle, as reported for specific ADP-PFK. This situation differs from that described for specific ADP-GK enzymes, where each substrate independently causes a sequential domain closure, resulting in three conformational states (open, semi-closed and closed).

Interrupted adenylation domains: unique bifunctional enzymes involved in nonribosomal peptide biosynthesis

2015 ◽  
Vol 32 (5) ◽  
pp. 641-653 ◽  
Author(s):  
Kristin J. Labby ◽  
Stoyan G. Watsula ◽  
Sylvie Garneau-Tsodikova
Keyword(s):  
Amino Acid ◽  
Adenylation Domain ◽  
Nonribosomal Peptide ◽  
Bifunctional Enzymes ◽  
Peptide Biosynthesis ◽  
Single Enzyme

This highlight focuses on one of Nature's key strategies to doubly modify an amino acid during nonribosomal peptide biosynthesis by using a single enzyme, an interrupted adenylation domain.

Enzyme redesign and interactions of substrate analogues with sterol methyltransferase to understand phytosterol diversity, reaction mechanism and the nature of the active site

2005 ◽  
Vol 33 (5) ◽  
pp. 1189-1196 ◽  
Author(s):  
W.D. Nes
Keyword(s):  
Active Site ◽  
Functional Divergence ◽  
Product Distribution ◽  
Site Directed Mutagenesis ◽  
Site Model ◽  
Bifunctional Enzymes ◽  
And Function ◽  
The Relationship ◽  
Sterol Methyltransferase

Several STM (sterol methyltransferase) genes have been cloned, sequenced and expressed in bacteria recently, making it possible to address questions of the relationship between sterol structure and function. The active site and mechanism of action of a set of phylogenetically diverse SMTs have been probed by site-directed mutagenesis as well as by using substrate and related analogues of the SMT-catalysed reaction. An active-site model has been developed that is in accord with the results presented, which is consistent with the hypothesis that SMTs are bifunctional enzymes kinetically responsible to bind Δ24-acceptor sterols of specific steric and electronic character and rigid orientation imposed by multiple hydrophobic active site contacts exacted from a common waxy core. Functional divergence influenced by the architectural role of sterols in membranes is considered to govern the evolution of product distribution and specificity of individual SMTs as discussed.

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