Membrane Association, Mechanism of Action, and Structure of Arabidopsis Embryonic Factor 1 (FAC1)

Abstract

Embryonic factor 1 (FAC1) is one of the earliest expressed plant genes and encodes an AMP deaminase (AMPD), which is also an identified herbicide target. This report identifies an N-terminal transmembrane domain in Arabidopsis FAC1, explores subcellular fractionation, and presents a 3.3-Å globular catalytic domain x-ray crystal structure with a bound herbicide-based transition state inhibitor that provides the first glimpse of a complete AMPD active site. FAC1 contains an (␣/␤) 8 -barrel characterized by loops in place of strands 5 and 6 that places it in a small subset of the amidohydrolase superfamily with imperfect folds. Unlike tetrameric animal orthologs, FAC1 is a dimer and each subunit contains an exposed Walker A motif that may be involved in the dramatic combined K m (25-80-fold lower) and V max (5-6-fold higher) activation by ATP. Normal mode analysis predicts a hinge motion that flattens basic surfaces on each monomer that flank the dimer interface, which suggests a reversible association between the FAC1 globular catalytic domain and intracellular membranes, with N-terminal transmembrane and disordered linker regions serving as the anchor and attachment to the globular catalytic domain, respectively.Embryonic factor 1 (FAC1) 5 was recently identified as one of the earliest expressed plant genes and is essential for the zygote to embryo transition in Arabidopsis thaliana (1). The zygote-lethal phenotype is characterized by developmental arrest at the 8 -16-cell stage and mutant embryo shriveling 2-3 days after fertilization. The Arabidopsis FAC1 locus encodes an AMP deaminase (AMPD; EC 3.5.4.6), which is a eukaryotic enzyme that catalyzes the hydrolytic deamination of AMP to IMP. AMPD has also been identified as the intracellular target for a class of herbicides that are produced by fungal pathogens. Carbocyclic coformycin was initially discovered in Saccharothrix (2), and plant cells can take up this diffusible nucleoside and 5Ј-phosphorylate it to produce a potent transition state inhibitor of AMPD (3). Exposure to carbocyclic coformycin results in cessation of seedling growth, followed by paling and necrosis at the apical meristem (3). Coformycin, a structurally related compound produced by a number of microbes (4, 5), also has herbicidal properties (5). Although the intracellular metabolism of this compound in plants has not been examined, its mode of action is presumably similar because coformycin 5Ј-phosphate is a transition state inhibitor of rabbit muscle AMPD (6). Both herbicides are inhibitors of mammalian adenosine deaminase (ADA) (3, 6), but the lack of this enzyme in plants (3, 7-9) supports the argument that AMPD is the intracellular target for their nucleotide derivatives. Taken together, these observations suggest that AMPD is essential throughout the plant life cycle. However, the underlying basis for lethality of a FAC1 null phenotype and for herbicidal toxicity related to the catalytic inhibition of this enzyme is not known.AMPD catalyzes the initial step in adenine to guanine ribon...

Multiple Lines of Evidence Localize Signaling, Morphology, and Lipid Biosynthesis Machinery to the Mitochondrial Outer Membrane of Arabidopsis

“…Nevertheless, the identification of this protein implies a conserved role for it, both on bacterial membranes and evidently on the mitochondrial outer membrane in plants. Another well-conserved protein is Embryonic Factor1 (FAC1; At2g38280.1), for which the subcellular location has long been discussed (Han et al, 2006), but in this study we report an outer mitochondrial membrane localization for this protein, on the basis of both proteomic and GFP evidence. Interestingly, like PECT1 and MIRO1, it has been observed that plants have a seed-lethal phenotype when FAC1 is knocked out (Xu et al, 2005;Meinke et al, 2008), indicating a vital role for these outer mitochondrial membrane proteins in Arabidopsis development.…”

Adenine Nucleotide Pool Perturbation Is a Metabolic Trigger for AMP Deaminase Inhibitor-Based Herbicide Toxicity

“…Although the intracellular metabolism of this compound in plants has not been examined, its mode of action is presumably similar because coformycin 5#-phosphate is a potent inhibitor of rabbit muscle AMPD (Frieden et al, 1980). The recently solved x-ray crystal structure of FAC1 complexed with coformycin 5#-phosphate confirmed this mode of inhibition and also provided the first glimpse of a complete AMPD active site in plants (Han et al, 2006). Coformycin and carbocyclic coformycin are also inhibitors of mammalian adenosine deaminase (Frieden et al, 1980;Dancer et al, 1997), but the lack of this enzyme in plants ( Le Floc'h et al, 1982;Yabuki and Ashihara, 1991;Dancer et al, 1997) supports the argument that AMPD is the primary intracellular target once these compounds are converted to their respective nucleotide derivatives.…”

“…These combined observations demonstrate that a functional AMPD enzyme effectively prevents the detrimental expansion of the adenine nucleotide pool in response to increased salvage synthesis of adenine. This functional significance can be attributed to a dramatic K m and V max ATP activation, as reported for the endogenous Catharanthus roseus (Yabuki and Ashihara, 1992) and recombinant Arabidopsis (Han et al, 2006) enzymes. This combined regulatory effect is not seen with orthologs in the animal kingdom, which suggests that plant AMPD may have evolved this property to compensate for the lack of an alternate AMP catabolic pathway.…”

“…K i Determination of DF 5#-Monophosphate Using a Purified Arabidopsis AMPD Recombinant Enzyme A 3.3 Å x-ray crystal structure of an Arabidopsis AMPD N-truncated (DI139M) recombinant enzyme complexed with coformycin 5#-phosphate (Han et al, 2006) has provided direct evidence that 5#-phosphorylated forms of these herbicides bind to the active site. The DI139M recombinant enzyme (K m [2ATP] 5 12 6 3 mM [Han et al, 2006]) was assayed at a constant substrate concentration (2.4 mM AMP) in the presence of variable concentrations of DF 5#-phosphate (none, and 100 nM to 4.5 mM).…”

“…The DI139M recombinant enzyme (K m [2ATP] 5 12 6 3 mM [Han et al, 2006]) was assayed at a constant substrate concentration (2.4 mM AMP) in the presence of variable concentrations of DF 5#-phosphate (none, and 100 nM to 4.5 mM). Inhibitor plots ([V max /V o ] 21 versus inhibitor concentration) were generated using three independent enzyme preparations to reveal a K i of 601 6 340 nM (data not shown).…”