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16 results on '"Chatukuta, Patience"'

7. An Arabidopsis Pentatricopeptide Repeat Is A Moonlighting Protein With Cross-Talking In Vitro Adenylyl Cyclase And Kinase Activities

Author

Dikobe, Tshegofatso, Sehlabane, Katlego, Bobo, Enetia, Sibanda‑Makuvise, Angela, Chatukuta, Patience, Kawadza, David, Ruzvidzo, Oziniel, Dikobe, Tshegofatso, Sehlabane, Katlego, Bobo, Enetia, Sibanda‑Makuvise, Angela, Chatukuta, Patience, Kawadza, David, and Ruzvidzo, Oziniel

Abstract

Downstream signalling involving adenylyl cyclases (ACs) and kinases is a key component of several processes in plants including cell division, growth, and response to stress. ACs are enzymes that generate the second messenger molecule, 3′,5′-cyclic adenosine monophosphate (cAMP) from 5′-adenosine triphosphate (ATP) while kinases are enzymes that catalyze the addition of a phosphate group to other molecules (trans-phosphorylation) or themselves (auto-phosphorylation). Apparently, while there has been an expanded record of various ACs and kinases identified in plants, no plant molecule to date has been shown to possess both the AC and kinase activities/functions and with such activities/functions having the characteristic of cross-talking interactions. Therefore, in an endeavor to find such a molecule, we searched the amino acid sequence of a known Arabidopsis AC, pentatricopeptide repeat (AtPPR) protein, and found a kinase-specific sequence signature (KSSS), which we speculated to be working in synergy with the AC center in this protein during downstream signalling. So, in order to test if this additional center is catalytically active and perhaps also having some cross-talking interactions with the AC center, we cloned, expressed, and affinity purified a truncated version of AtPPR, harboring both the AC and KSSS centers (AtPPR-AC/K). When tested in vitro, the recombinant AtPPR-AC/K showed a Mn2+-dependent AC activity that is positively enhanced by Ca2+ and HCO3 − and a trans-/auto-phosphorylation kinase activity capable of utilizing both ATP and GTP as substrates and specific to the serine, threonine, and tyrosine amino acids as target residues. In addition, the kinase activity of AtPPR-AC/K was found to be reduced by cAMP while at the same time, it was totally shut down by Ca2+. This thus qualified both cAMP and Ca2+ as molecular switches or modulators, capable of regulating AtPPR functions through cross-talking interactions between the activities of its two domain

Published
2023

15. Recombinant expression and functional characterization of a novel toll interleukin receptor nucleotide-binding site leucine-rich repeat protein from arabidopsis thaliana

Author

Chatukuta, Patience, Ruzvidzo, O., and 22714839 - Ruzvidzo, Oziniel (Supervisor)

Subjects
Primers, Reverse transcription polymerase chain reaction (RT-PCR), Adenylate cyclases (ACs), fungi, Second messenger, food and beverages, Guanylate cyclases (GCs), Enzyme immunoassay, Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE)
Abstract

MSc (Biology), North-West University, Mafikeng Campus The presence of adenylate cyclases in higher plants has generally been questioned. A BLAST search of the Arabidopsis genome using a 14-amino acid motif with specificity for ATP binding bioinformatically identified the toll interleukin-like receptor nucleotide binding site leucine rich repeat (TIR-NBS-LRR) protein encoded by the At3g04220 gene from Arabidopsis thaliana to have a putative adenylate cyclase catalytic centre. To test whether TIR-NBS-LRR possesses adenylate cyclase activity, total mRNA was extracted from the leaf material of 6-week old A. thaliana plants and used as template for complementary DNA synthesis and amplification of a 114 amino acid-long adenylate cyclase catalytic domain fragment of the At3g04220 gene using Reverse Transcriptase Polymerase Chain Reaction in conjunction with sequence-specific primers. The amplified fragment was then cloned into a pCR®T7/NT-TOPO® vector and the recombinant vector was transformed into the expression host, Escherichia coli BL21 (DE3) pLysS. Positive transformants were determined using Sodium Dodecyl Sulphate Polyacrylamide Gel Electrophoresis; and confirmed by sequence-specific colony PCR and restriction double digestion. The His-tagged recombinant protein was over-expressed following induction with isopropyl-β-D-1-thiogalactopyranoside and purified using a nickel affinity gel system. The endogenous and in vitro adenylate cyclase activities of the recombinant TIR-NBS-LRR were then tested via a cAMP-specific enzyme immunoassaying system and the in vivo adenylate cyclase activity was tested through a complementation test using the Escherichia coli cyaA SP850 mutant. The results of all these three assays indicated that the TIR-NBS-LRR protein encoded by the At3g04220 gene from A. thaliana possessed endogenous, in vitro and in vivo adenylate cyclase activities, and thus, confirmed TIR-NBS-LRR as a higher plant adenylate cyclase with a possible cAMP-mediated signaling system. Masters

Published
2012

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