Production and Characterization of Bifunctional Enzymes. Substrate Channeling in the Aspartate Pathway†

Biochemistry ◽  
2002 ◽  
Vol 41 (11) ◽  
pp. 3726-3731 ◽  
Author(s):  
Cindy L. James ◽  
Ronald E. Viola
Keyword(s):  
Substrate Channeling ◽  
Bifunctional Enzymes ◽  
Aspartate Pathway

scholarly journals Protein complex formation in methionine chain-elongation and leucine biosynthesis

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Li-Qun Chen ◽  
Shweta Chhajed ◽  
Tong Zhang ◽  
Joseph M. Collins ◽  
Qiuying Pang ◽  
...  
Keyword(s):  
Protein Complexes ◽  
Complete Characterization ◽  
Bimolecular Fluorescence Complementation ◽  
Chain Elongation ◽  
Substrate Channeling ◽  
Leucine Biosynthesis ◽  
Protein Complex Formation ◽  
Genes Encoding ◽  
Isopropylmalate Dehydrogenase

AbstractDuring the past two decades, glucosinolate (GLS) metabolic pathways have been under extensive studies because of the importance of the specialized metabolites in plant defense against herbivores and pathogens. The studies have led to a nearly complete characterization of biosynthetic genes in the reference plant Arabidopsis thaliana. Before methionine incorporation into the core structure of aliphatic GLS, it undergoes chain-elongation through an iterative three-step process recruited from leucine biosynthesis. Although enzymes catalyzing each step of the reaction have been characterized, the regulatory mode is largely unknown. In this study, using three independent approaches, yeast two-hybrid (Y2H), coimmunoprecipitation (Co-IP) and bimolecular fluorescence complementation (BiFC), we uncovered the presence of protein complexes consisting of isopropylmalate isomerase (IPMI) and isopropylmalate dehydrogenase (IPMDH). In addition, simultaneous decreases in both IPMI and IPMDH activities in a leuc:ipmdh1 double mutants resulted in aggregated changes of GLS profiles compared to either leuc or ipmdh1 single mutants. Although the biological importance of the formation of IPMI and IPMDH protein complexes has not been documented in any organisms, these complexes may represent a new regulatory mechanism of substrate channeling in GLS and/or leucine biosynthesis. Since genes encoding the two enzymes are widely distributed in eukaryotic and prokaryotic genomes, such complexes may have universal significance in the regulation of leucine biosynthesis.

Characterization of an Aldolase−Dehydrogenase Complex That Exhibits Substrate Channeling in the Polychlorinated Biphenyls Degradation Pathway

Biochemistry ◽  
2009 ◽  
Vol 48 (27) ◽  
pp. 6551-6558 ◽  
Author(s):  
Perrin Baker ◽  
Dan Pan ◽  
Jason Carere ◽  
Adam Rossi ◽  
Weijun Wang ◽  
...  
Keyword(s):  
Polychlorinated Biphenyls ◽  
Degradation Pathway ◽  
Substrate Channeling ◽  
Dehydrogenase Complex

Synthesis and Characterization of Allosteric Probes of Substrate Channeling in the Tryptophan Synthase Bienzyme Complex†,‡

Biochemistry ◽  
2007 ◽  
Vol 46 (26) ◽  
pp. 7713-7727 ◽  
Author(s):  
Huu Ngo ◽  
Rodney Harris ◽  
Novelle Kimmich ◽  
Patricia Casino ◽  
Dimitri Niks ◽  
...  
Keyword(s):  
Synthesis And Characterization ◽  
Tryptophan Synthase ◽  
Substrate Channeling

scholarly journals Biochemical Characterization of a Bifunctional Enzyme Constructed by the Fusion of a Glucuronan Lyase and a Chitinase from Trichoderma sp.

Life ◽  
2020 ◽  
Vol 10 (10) ◽  
pp. 234
Author(s):  
Zeineb Baklouti ◽  
Cédric Delattre ◽  
Guillaume Pierre ◽  
Christine Gardarin ◽  
Slim Abdelkafi ◽  
...  
Keyword(s):  
Biochemical Characterization ◽  
Biochemical Properties ◽  
Cell Wall Degrading Enzymes ◽  
Promising Candidate ◽  
Structural Modifications ◽  
Chimeric Enzymes ◽  
Bifunctional Enzymes ◽  
Trichoderma Sp ◽  
Ph Variations

Bifunctional enzymes created by the fusion of a glucuronan lyase (TrGL) and a chitinase (ThCHIT42) from Trichoderma sp. have been constructed with the aim to validate a proof of concept regarding the potential of the chimera lyase/hydrolase by analyzing the functionality and the efficiency of the chimeric constructions compared to parental enzymes. All the chimeric enzymes, including or nor linker (GGGGS), were shown functional with activities equivalent or higher to native enzymes. The velocity of glucuronan lyase was considerably increased for chimeras, and may involved structural modifications at the active site. The fusion has induced a slightly decrease of the thermostability of glucuronan lyase, without modifying its catalytic activity regarding pH variations ranging from 5 to 8. The biochemical properties of chitinase seemed to be more disparate between the different fusion constructions suggesting an impact of the linkers or structural interactions with the linked glucuronan lyase. The chimeric enzymes displayed a decreased stability to temperature and pH variations, compared to parental one. Overall, TrGL-ThCHIT42 offered the better compromise in terms of biochemical stability and enhanced activity, and could be a promising candidate for further experiments in the field of fungi Cell Wall-Degrading Enzymes (CWDEs).

A cautionary tale of structure-guided inhibitor development against an essential enzyme in the aspartate-biosynthetic pathway

2014 ◽  
Vol 70 (12) ◽  
pp. 3244-3252 ◽  
Author(s):  
Alexander G. Pavlovsky ◽  
Bharani Thangavelu ◽  
Pravin Bhansali ◽  
Ronald E. Viola
Keyword(s):  
Active Site ◽  
Enzyme Inhibitor ◽  
Bacterial Survival ◽  
Cautionary Tale ◽  
Lead Compounds ◽  
Semialdehyde Dehydrogenase ◽  
Fragment Library ◽  
Aspartate Pathway ◽  
Essential Enzyme

The aspartate pathway is essential for the production of the amino acids required for protein synthesis and of the metabolites needed in bacterial development. This pathway also leads to the production of several classes of quorum-sensing molecules that can trigger virulence in certain microorganisms. The second enzyme in this pathway, aspartate β-semialdehyde dehydrogenase (ASADH), is absolutely required for bacterial survival and has been targeted for the design of selective inhibitors. Fragment-library screening has identified a new set of inhibitors that, while they do not resemble the substrates for this reaction, have been shown to bind at the active site of ASADH. Structure-guided development of these lead compounds has produced moderate inhibitors of the target enzyme, with some selectivity observed between the Gram-negative and Gram-positive orthologs of ASADH. However, many of these inhibitor analogs and derivatives have not yet achieved the expected enhanced affinity. Structural characterization of these enzyme–inhibitor complexes has provided detailed explanations for the barriers that interfere with optimal binding. Despite binding in the same active-site region, significant changes are observed in the orientation of these bound inhibitors that are caused by relatively modest structural alterations. Taken together, these studies present a cautionary tale for issues that can arise in the systematic approach to the modification of lead compounds that are being used to develop potent inhibitors.

A STUDY ON KINETIC CHARACTERIZATION OF CHANNEL-BLOCKING MUTANTS IN BRADYRHIZOBIUM JAPONICUM UTILIZATION A

2021 ◽  
pp. 49-52
Author(s):  
Anand Shanker Singh ◽  
G. Radhika ◽  
R. Praveen Kumar ◽  
Debarshi Jana
Keyword(s):  
Active Site ◽  
Bradyrhizobium Japonicum ◽  
Kinetic Data ◽  
Active Sites ◽  
Substrate Channeling ◽  
Site Analysis ◽  
Proline Utilization ◽  
Channel Blocking ◽  
Proline Utilization A

Proline utilization A (PutA) from Bradyrhizobium japonicum (BjPutA) is a bifunctional avoenzyme that catalyzes the oxidation of proline to glutamate using fused proline dehydrogenase (PRODH) and ∆1-pyrroline-5-carboxylate dehydrogenase (P5CDH) domains. Recent crystal structures and kinetic data suggest an intramolecular channel connects the two active sites, promoting substrate channeling of the intermediate P5C from the PRODH domain to the P5CDH domain. In this work several mutations were made along the channel in an effort to block passage of P5C to the second active site. Analysis of several site-specic mutants in the substrate channel of BjPutA revealed an important role for D779 in the channeling path. BjPutA mutants D779Y and D779W signicantly decreased the overall PRODH-P5CDH channeling reaction indicating that bulky mutations at residue D779 impede travel of P5C through the channel. Interestingly, D779Y and D779W also exhibited lower P5CDH activity, suggesting that exogenous P5C must enter the channel upstream of D779. Replacing D779 with a smaller residue (D779A) had no effect on the catalytic and channeling properties of BjPutA showing that the carboxylate group of D779 is not essential for channeling. An identical mutation at D778 (D778Y) did not impact BjPutA channeling activity. Thus, D779 is optimally orientated so that replacement with the larger side chains of Tyr/Trp blocks P5C movment through the channel. The kinetic data reveal not only that bulky mutations at residue D779 hinder passage of P5C to the second active site, but also P5C must use the channel to efciently access the P5CDH domain. Moreover, these mutants may be used to learn more about the hydrolysis event that is thought to take place within the channel

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