ORCID Profile
0000-0002-5472-7956
Current Organisations
CSIRO
,
CSIRO Black Mountain Laboratories
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Publisher: Frontiers Media SA
Date: 12-11-2019
Publisher: Frontiers Media SA
Date: 22-08-2018
Publisher: Cold Spring Harbor Laboratory
Date: 03-09-2019
DOI: 10.1101/755116
Abstract: To engineer Mo dependent nitrogenase function in plants expression of proteins NifD and NifK will be an absolute requirement. Although mitochondria have been established as a suitable eukaryotic environment for biosynthesis of oxygen-sensitive enzymes such as NifH, expression of NifD in this organelle has proven difficult due to cryptic NifD degradation. Here we describe a solution to this problem. Using molecular and proteomic methods, we found NifD degradation to be a consequence of mitochondrial endoprotease activity at a specific motif within NifD. Focusing on this functionally sensitive region, we designed NifD variants comprising between one and three amino acid substitutions and distinguished several that were resistant to degradation when expressed in both plant and yeast mitochondria. Nitrogenase activity assays of these resistant variants in E. coli identified a subset that retained function, including a single amino acid (Y100Q) variant. The Y100Q variant also enabled expression of a NifD(Y100Q)-linker-NifK translational polyprotein in plant mitochondria, confirmed by identification of the polyprotein in the soluble fraction of plant extracts. The NifD(Y100Q)-linker-NifK retained function in E. coli based nitrogenase assays, demonstrating this polyprotein permits expression of NifD and NifK in a defined stoichiometry supportive of activity. Our results exemplify how protein design can overcome impediments encountered when expressing synthetic proteins in novel environments. Specifically, these findings outline our progress toward the assembly of the catalytic unit of nitrogenase within mitochondria.
Publisher: Oxford University Press (OUP)
Date: 20-04-2004
DOI: 10.1105/TPC.020487
Publisher: Wiley
Date: 15-05-2003
DOI: 10.1046/J.1469-8137.2003.00785.X
Abstract: Arabidopsis is remarkable for having eight members of the type 1 B heavy metal‐transporting P‐type ATPase subfamily. Sequence analyses indicate that four, two of which may be targeted to plastids, are related to known Cu(I) transporters and contain N‐terminal metal‐binding site (MBS) motifs similar to those identified in other organisms. The remaining four are more closely related to known alent cation transporters of prokaryotes. Three of these form a closely related group and are believed to be Zn(II) transporters. These contain a predicted N‐terminal MBS that is a variant of those found in Cu transporters in addition to extended C‐terminal regions that contain likely metal‐binding sequences. Our current limited knowledge of the physiological roles of these transporters is reviewed and their evolutionary relationships are explored, including an hypothesis that some, particularly the putative alent cation transporters, are derived from horizontal gene transfer events.
Publisher: Springer Berlin Heidelberg
Date: 2010
Publisher: Wiley
Date: 13-07-2019
DOI: 10.1111/PBI.12959
Publisher: Frontiers Media SA
Date: 06-03-2020
Publisher: Cold Spring Harbor Laboratory
Date: 27-12-2019
DOI: 10.1101/2019.12.23.887703
Abstract: Industrial nitrogen fertilizer is intrinsic to modern agriculture yet expensive and environmentally harmful. We aim to reconstitute bacterial nitrogenase function within plant mitochondria to reduce nitrogen fertilizer usage. Many nitrogen fixation (Nif) proteins are required for biosynthesis and function of the mature nitrogenase enzyme, and these will need to be correctly processed and soluble within mitochondria as a pre-requisite for function. Here we present our workflow that assessed processing, solubility and relative abundance of 16 Klebsiella oxytoca Nif proteins targeted to the plant mitochondrial matrix using an Arabidopsis mitochondrial targeting peptide (MTP). The functional consequence of the N-terminal modifications required for mitochondrial targeting of Nif proteins was tested using bacterial nitrogenase assays. We found that despite the use of the same constitutive promoter and MTP, MTP::Nif processing and relative abundance in plant leaf varied considerably. Assessment of solubility for all MTP::Nif proteins found NifF, M, N, S, U, W, X, Y and Z were soluble, while NifB, E, H, J, K, Q and V were mostly insoluble. Although most Nif proteins tolerated the N-terminal extension as a consequence of mitochondrial processing, this extension in NifM reduced nitrogenase activity to 10% of controls. Using proteomics, we detected a ∼50-fold increase in the abundance of NifM when it contained the N-terminal MTP extension, which may account for this reduction seen in nitrogenase activity. Based on plant mitochondrial processing and solubility, and retention of function in a bacterial assay, our workflow has identified that NifF, N, S, U, W, Y and Z satisfied all these criteria. Future work can now focus on improving these parameters for the remaining Nif components to assemble a complete set of plant-ready Nif proteins for reconstituting nitrogen fixation in plant mitochondria.
Publisher: Elsevier BV
Date: 04-2008
DOI: 10.1016/J.BBRC.2007.11.098
Abstract: A robust cross-link between Gln(23) in phospholamban (PLN) and Lys(328) in the sarco(endo)plasmic reticulum Ca(2+) ATPase (SERCA1a) is formed in the presence or absence of oxidant and is susceptible to both PLN phosphorylation and SERCA1a Ca(2+) binding. This cross-link provides precisely the evidence needed to support our earlier proposal that collision of the PLN transmembrane helix at Asn(27) with the cytosolic extension of M4 at Leu(321) leads to unwinding of the helix. In a study of site-specific interactions among PLN, sarcolipin (SLN), and SERCA1a, we determined that mutations of some specific amino acids in PLN or SLN diminish either the super-inhibition imposed on SERCA1a function by the PLN-SLN binary complex or the physical interactions between PLN and SLN or both. These results have led to a revision of our earlier model for the PLN-SLN-SERCA1a complex.
Publisher: Wiley
Date: 23-11-2005
Publisher: Proceedings of the National Academy of Sciences
Date: 31-08-2020
Abstract: Engineering nitrogenase in plants may help alleviate economic and environmental issues due to the use of nitrogen fertilizer. Mitochondria have shown promise in supporting the function of nitrogenase, including electron donation and metallocluster assembly. Despite these successes, formation of the catalytic unit, NifDK, has proven difficult. Here, we find that when relocated to plant mitochondria, NifD is subject to errant peptidase-based cleavage and is insoluble. Guided by NifD sequence variation amongst bacteria and structural modeling, we designed NifD variants that avoided cleavage and retained function in bacterial assays. Fusion of NifK to degradation-resistant NifD also improved solubility, and the polyprotein retained function in bacterial assays. This work advances efforts to produce crops less reliant on nitrogen fertilizer.
Publisher: Springer Science and Business Media LLC
Date: 10-2000
Publisher: Frontiers Media SA
Date: 10-09-2020
Location: Australia
No related grants have been discovered for Dawar Hussain.