Development of a System for Directed Evolution of \u3cem\u3eArabidopsis\u3c/em\u3e Formate Dehydrogenase to Utilize NADP as a Cofactor

Formate dehydrogenase (FDH) is a NAD-dependent enzyme found in methylotrophic bacteria, yeast and plants. This enzyme catalyzes the reversible oxidation of formate to carbon dioxide. The goal of this research was to determine the feasibility of using a directed evolution approach to generate an altered Arabidopsis FDH with a high affinity for NADP as a cofactor. A PCR procedure that induced approximately 1.5 mutations in the wild-type Arabidopsis FDH sequence per thousand base pairs was developed and the amplified products were transformed into E. coli cells. Approximately 1300 cell lines were assayed in 96-well microplates for activity with NADP+ and 100 putative mutants were selected for further study. One particular mutant line, pFDH-18, possessed reproducible NADP+-FDH activity. Sequence analysis showed that a single T in the wild-type DNA sequence had been changed to a G. The result of this mutation was that an isoleucine (Ile) residue at position 188 in the wild-type enzyme was converted to a methionine. This particular Ile residue is conserved in the known FDH sequences from higher plants and is located in the region of the enzyme that contains the binding domain for the NAD cofactor

Phylloquinone (vitamin K 1 ) biosynthesis in plants: two peroxisomal thioesterases of lactobacillales origin hydrolyze 1,4‐dihydroxy‐2‐naphthoyl‐coa

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/92396/1/TPJ_4972_sm_FigS3.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/92396/2/TPJ_4972_sm_TableS1.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/92396/3/TPJ_4972_sm_FigS2.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/92396/4/TPJ_4972_sm_TableS4.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/92396/5/TPJ_4972_sm_FigS6.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/92396/6/j.1365-313X.2012.04972.x.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/92396/7/TPJ_4972_sm_FigS1.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/92396/8/TPJ_4972_sm_TableS3.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/92396/9/TPJ_4972_sm_FigS5.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/92396/10/TPJ_4972_sm_TableS2.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/92396/11/TPJ_4972_sm_FigS4.pd

The Origin and Biosynthesis of the Benzenoid Moiety of Ubiquinone (Coenzyme Q) in \u3ci\u3eArabidopsis\u3c/i\u3e

It is not known how plants make the benzenoid ring of ubiquinone, a vital respiratory cofactor. Here, we demonstrate that Arabidopsis thaliana uses for that purpose two separate biosynthetic branches stemming from phenylalanine and tyrosine. Gene network modeling and characterization of T-DNA mutants indicated that acyl-activating enzyme encoded by At4g19010 contributes to the biosynthesis of ubiquinone specifically from phenylalanine. CoA ligase assays verified that At4g19010 prefers para-coumarate, ferulate, and caffeate as substrates. Feeding experiments demonstrated that the at4g19010 knockout cannot use para-coumarate for ubiquinone biosynthesis and that the supply of 4-hydroxybenzoate, the side-chain shortened version of para-coumarate, can bypass this blockage. Furthermore, a trans-cinnamate 4-hydroxylase mutant, which is impaired in the conversion of trans-cinnamate into para-coumarate, displayed similar defects in ubiquinone biosynthesis to that of the at4g19010 knockout. Green fluorescent protein fusion experiments demonstrated that At4g19010 occurs in peroxisomes, resulting in an elaborate biosynthetic architecture where phenylpropanoid intermediates have to be transported from the cytosol to peroxisomes and then to mitochondria where ubiquinone is assembled. Collectively, these results demonstrate that At4g19010 activates the propyl side chain of para-coumarate for its subsequent β-oxidative shortening. Evidence is shown that the peroxisomal ABCD transporter (PXA1) plays a critical role in this branch. Includes supplementary files

Remarkable Features of the Hotdog-fold Thioesterases Involved in Phylloquinone (vitamin K1) Biosynthesis

Phylloquinone (vitamin K1) is a bipartite molecule, consisting of a naphthoquinone ring attached to a phytyl side chain, that is synthesized by plants and certain cyanobacteria to serve as an electron carrier in photosystem I. The coupling of the ring and isoprenyl moieties relies on the cleavage of the CoA-thioester linkage with 1,4-dihydroxy-2- naphthoate (DHNA). It has long been a mystery if this hydrolysis is an enzymatic or chemical process. Using comparative genomics, protein biochemistry, genetics and metabolic profiling, we identified a cyanobacterial thioesterase responsible for the in vivo hydrolysis of DHNA-CoA. This enzyme bears a signature domain of the 4- hydroxybenzoyl-CoA thioesterase (4HBT) family of Hotdog-fold proteins. Surprisingly, plants, which obtained most of their phylloquinone biosynthetic genes with the acquisition of the plastid, do not contain orthologs of cyanobacterial DHNA-CoA thioesterase. We tested all of the predicted 4HBT Hotdog-fold proteins in Arabidopsis by functional complementation of the cyanobacterial mutant. We found two genes encoding functional DHNA-CoA thioesterases that display low percentages of identity and dissimilar catalytic motifs from their cyanobacterial counterparts. It appears that plant DHNA-CoA thioesterases originated from a horizontal gene transfer with a species of the Lactobacillales order. The cognate T-DNA knockout lines exhibit reduced DHNA-CoA thioesterase activity and phylloquinone content. Fluorescently tagging the Arabidopsis enzymes revealed that they are localized to the peroxisome. Subcellular fractionation assays confirmed this providing the first biochemical evidence for the involvement of peroxisomes in phylloquinone biosynthesis. Recent proteomics and GFP-reporter projects suggest that the two steps preceding DHNA-CoA thioesterase are also peroxisomal. Thus, the current model of phylloquinone biosynthesis reflects a split between plastids and peroxisomes, implying the movement of intermediates between the organelles. To assess the importance of the cognate transport steps, we have re-routed the peroxisomal branch of the pathway to plastids in Camelina sativa. Here we report the findings of our metabolic engineering strategy on the pool of phylloquinone

Detection and quantification of vitamin K\u3csub\u3e1\u3c/sub\u3e quinol in leaf tissues

Phylloquinone (2-methyl-3-phytyl-1,4-naphthoquinone; vitamin K1) is vital to plants. It is responsible for the oneelectron transfer at the A1 site of photosystem I, a process that involves turnover between the quinone and semi-quinone forms of phylloquinone. Using HPLC coupled with fluorometric detection to analyze Arabidopsis leaf extracts, we detected a third redox form of phylloquinone corresponding to its fully reduced – quinol–naphthoquinone ring (PhQH2). A method was developed to quantify PhQH2 and its corresponding oxidized quinone (PhQ) counterpart in a single HPLC run. PhQH2 was found in leaves of all dicotyledonous and monocotyledonous species tested, but not in fruits or in tubers. Its level correlated with that of PhQ, and represented 5–10% of total leaf phylloquinone. Analysis of purified pea chloroplasts showed that these organelles accounted for the bulk of PhQH2. The respective pool sizes of PhQH2 and PhQ were remarkably stable throughout the development of Arabidopsis green leaves. On the other hand, in Arabidopsis and tomato senescing leaves, PhQH2 was found to increase at the expense of PhQ, and represented 25–35% of the total pool of phylloquinone. Arabidopsis leaves exposed to light contained lower level of PhQH2 than those kept in the dark. These data indicate that PhQH2 does not originate from the photochemical reduction of PhQ, and point to a hitherto unsuspected function of phylloquinone in plants. The putative origin of PhQH2 and its recycling into PhQ are discussed

Specialized naphthoquinones present in Impatiens glandulifera nectaries inhibit the growth of fungal nectar microbes

Abstract The invasion success of Impatiens glandulifera (Himalayan balsam) in certain parts of Europe and North America has been partially attributed to its ability to compete for bee pollinators with its rich nectar and due to its capacity to produce and release allelopathic 1,4‐naphthoquinones (1,4‐NQs) from its roots and leaves. Given that other 1,4‐NQs present in the digestive fluids of certain carnivorous plants are proposed to control microbial colonization, we investigated the potential for the 1,4‐NQs, 2‐methoxy‐1,4‐naphthoquinone (2‐MNQ) and lawsone, to fulfill an analogous role in the nectaries of I. glandulifera. Both 2‐MNQ and lawsone were detected in the floral nectaries of I. glandulifera at levels comparable to leaves and roots, but were discovered to be at significantly higher levels in its extra‐floral nectaries (EFNs) and to be present in EFN nectar itself. Nectar microbe inhibition assays revealed that the common nectar bacteria Gluconobacter oxydans and Asaia prunellae are not inhibited by 2‐MNQ or lawsone, although both compounds were found to inhibit the growth of the common fungal nectar microbes Metschnikowia reukaufii and Aureobasidium pullulans. Taken together, these findings suggest that 2‐MNQ and lawsone could serve to protect the rich nectar of I. glandulifera against fungal growth. The high abundance of 2‐MNQ and lawsone in I. glandulifera EFNs may also point to an unsuspected mechanism for how allelopathic 1,4‐NQs are leached into the soil where they exhibit their known allelopathic effects

Agricultural Uses of Juglone: Opportunities and Challenges

Application of conventional synthetic pesticides and agrochemicals has boosted the yield and productivity of crops by reducing pest infestation and promoting crop growth yet increasing reliance on many of these products poses serious environmental threats. This has led to growing interest in obtaining more environmentally friendly alternatives to conventional pesticides and agrochemicals. Allelochemicals produced by plants, fungi, and microbes offer options for developing novel natural product-based pesticides and agrochemicals that are effective but with lower environmental half-lives. Here, we review the current state of knowledge about the potential use of juglone (5-hydroxy-1,4-naphthoquinone), the allelochemical produced by black walnut trees (Juglans nigra), which has been investigated for applications across a range of different agricultural purposes. We then offer our perspective on what opportunities and challenges exist for harnessing juglone as a component of sustainable agriculture

Functional convergence of structurally distinct thioesterases from cyanobacteria and plants involved in phylloquinone biosynthesis

The synthesis of phylloquinone (vitamin K1) in photosynthetic organisms requires a thioesterase that hydrolyzes 1,4-dihydroxy- 2-naphthoyl-CoA (DHNA-CoA) to release 1,4- dihydroxy-2-naphthoate (DHNA). Cyanobacteria and plants contain distantly related hotdog-fold thioesterases that catalyze this reaction, although the structural basis of these convergent enzymatic activities is unknown. To investigate this, the crystal structures of hotdog-fold DHNA-CoA thioesterases from the cyanobacterium Synechocystis (Slr0204) and the flowering plant Arabidopsis thaliana (AtDHNAT1) were determined. These enzymes form distinct homotetramers and use different active sites to catalyze hydrolysis of DHNACoA, similar to the 4-hydroxybenzoyl-CoA (4-HBA-CoA) thioesterases from Pseudomonas and Arthrobacter. Like the 4-HBA-CoA thioesterases, the DHNA-CoA thioesterases contain either an active-site aspartate (Slr0204) or glutamate (AtDHNAT1) that are predicted to be catalytically important. Computational modeling of the substrate-bound forms of both enzymes indicates the residues that are likely to be involved in substrate binding and catalysis. Both enzymes are selective for DHNA-CoA as a substrate, but this selectivity is achieved using divergent predicted binding strategies. The Slr0204 binding pocket is predominantly hydrophobic and closely conforms to DHNA, while that of AtDHNAT1 is more polar and solvent-exposed. Considered in light of the related 4-HBA-CoA thioesterases, these structures indicate that hotdog-fold thioesterases using either an active-site aspartate or glutamate diverged into distinct clades prior to the evolution of strong substrate specificity in these enzymes

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Phylloquinone (vitamin K1) is a bipartite molecule that consists of a naphthoquinone ring attached to a phytyl side chain. The coupling of these 2 moieties depends on the hydrolysis of the CoA thioester of 1,4-dihydroxy-2-naphthoate (DHNA), which forms the naphthalenoid backbone. It is not known whether such a hydrolysis is enzymatic or chemical. In this study, comparative genomic analyses identified orthologous genes of unknown function that in most species of cyanobacteria cluster with predicted phylloquinone biosynthetic genes. The encoded approximately 16-kDa proteins display homology with some Hotdog domain-containing CoA thioesterases that are involved in the catabolism of 4-hydroxybenzoyl-CoA and gentisyl-CoA (2,5-dihydroxybenzoyl-CoA) in certain soil-dwelling bacteria. The Synechocystis ortholog, encoded by gene slr0204, was expressed as a recombinant protein and was found to form DHNA as reaction product. Unlike its homologs in the Hotdog domain family, Slr0204 showed strict substrate specificity. The Synechocystis slr0204 knockout was devoid of DHNA-CoA thioesterease activity and accumulated DHNA-CoA. As a result, knockout cells contained 13-fold less phylloquinone than their wild-type counterparts and displayed the typical photosensitivity to high light associated to phylloquinone deficiency in cyanobacteria