Structure-function analysis of plant G-protein regulatory mechanisms identifies key Gα-RGS protein interactions.

Heterotrimeric GTP-binding protein alpha subunit (Gα) and its cognate regulator of G-protein signaling (RGS) protein transduce signals in eukaryotes spanning protists, amoeba, animals, fungi, and plants. The core catalytic mechanisms of the GTPase activity of Gα and the interaction interface with RGS for the acceleration of GTP hydrolysis seem to be conserved across these groups; however, the RGS gene is under low selective pressure in plants, resulting in its frequent loss. Our current understanding of the structural basis of Gα:RGS regulation in plants has been shaped by Arabidopsis Gα, (AtGPA1), which has a cognate RGS protein. To gain a comprehensive understanding of this regulation beyond Arabidopsis, we obtained the x-ray crystal structures of Oryza sativa Gα, which has no RGS, and Selaginella moellendorffi (a lycophyte) Gα that has low sequence similarity with AtGPA1 but has an RGS. We show that the three-dimensional structure, protein-protein interaction with RGS, and the dynamic features of these Gα are similar to AtGPA1 and metazoan Gα. Molecular dynamic simulation of the Gα-RGS interaction identifies the contacts established by specific residues of the switch regions of GTP-bound Gα, crucial for this interaction, but finds no significant difference due to specific amino acid substitutions. Together, our data provide valuable insights into the regulatory mechanisms of plant G-proteins but do not support the hypothesis of adaptive co-evolution of Gα:RGS proteins in plants.

Potent, specific MEPicides for treatment of zoonotic staphylococci.

Coagulase-positive staphylococci, which frequently colonize the mucosal surfaces of animals, also cause a spectrum of opportunistic infections including skin and soft tissue infections, urinary tract infections, pneumonia, and bacteremia. However, recent advances in bacterial identification have revealed that these common veterinary pathogens are in fact zoonoses that cause serious infections in human patients. The global spread of multidrug-resistant zoonotic staphylococci, in particular the emergence of methicillin-resistant organisms, is now a serious threat to both animal and human welfare. Accordingly, new therapeutic targets that can be exploited to combat staphylococcal infections are urgently needed. Enzymes of the methylerythritol phosphate pathway (MEP) of isoprenoid biosynthesis represent potential targets for treating zoonotic staphylococci. Here we demonstrate that fosmidomycin (FSM) inhibits the first step of the isoprenoid biosynthetic pathway catalyzed by deoxyxylulose phosphate reductoisomerase (DXR) in staphylococci. In addition, we have both enzymatically and structurally determined the mechanism by which FSM elicits its effect. Using a forward genetic screen, the glycerol-3-phosphate transporter GlpT that facilitates FSM uptake was identified in two zoonotic staphylococci, Staphylococcus schleiferi and Staphylococcus pseudintermedius. A series of lipophilic ester prodrugs (termed MEPicides) structurally related to FSM were synthesized, and data indicate that the presence of the prodrug moiety not only substantially increased potency of the inhibitors against staphylococci but also bypassed the need for GlpT-mediated cellular transport. Collectively, our data indicate that the prodrug MEPicides selectively and robustly inhibit DXR in zoonotic staphylococci, and further, that DXR represents a promising, druggable target for future development.

Molecular Basis of the Evolution of Methylthioalkylmalate Synthase and the Diversity of Methionine-Derived Glucosinolates.

The globally cultivated Brassica species possess diverse aliphatic glucosinolates, which are important for plant defense and animal nutrition. The committed step in the side chain elongation of methionine-derived aliphatic glucosinolates is catalyzed by methylthioalkylmalate synthase, which likely evolved from the isopropylmalate synthases of leucine biosynthesis. However, the molecular basis for the evolution of methylthioalkylmalate synthase and its generation of natural product diversity in Brassica is poorly understood. Here, we show that Brassica genomes encode multiple methylthioalkylmalate synthases that have differences in expression profiles and 2-oxo substrate preferences, which account for the diversity of aliphatic glucosinolates across Brassica accessions. Analysis of the 2.1 Å resolution x-ray crystal structure of Brassica juncea methylthioalkylmalate synthase identified key active site residues responsible for controlling the specificity for different 2-oxo substrates and the determinants of side chain length in aliphatic glucosinolates. Overall, these results provide the evolutionary and biochemical foundation for the diversification of glucosinolate profiles across globally cultivated Brassica species, which could be used with ongoing breeding strategies toward the manipulation of beneficial glucosinolate compounds for animal health and plant protection.

Biochemical and clinical studies of putative allergens to assess what distinguishes them from other non-allergenic proteins in the same family.

Many protein families have numerous members listed in databases as allergens; however, some allergen database entries, herein called "orphan allergens", are members of large families of which all other members are not allergens. These orphan allergens provide an opportunity to assess whether specific structural features render a protein allergenic. Three orphan allergens [Cladosporium herbarum aldehyde dehydrogenase (ChALDH), Alternaria alternata ALDH (AaALDH), and C. herbarum mannitol dehydrogenase (ChMDH)] were recombinantly produced and purified for structure characterization and for clinical skin prick testing (SPT) in mold allergic participants. Examination of the X-ray crystal structures of ChALDH and ChMDH and a homology structure model of AaALDH did not identify any discernable epitopes that distinguish these putative orphan allergens from their non-allergenic protein relatives. SPT results were aligned with ChMDH being an allergen, 53% of the participants were SPT (+). AaALDH did not elicit SPT reactivity above control proteins not in allergen databases (i.e., Psedomonas syringae indole-3-acetaldehyde dehydrogenase and Zea mays ALDH). Although published results showed consequential human IgE reactivity with ChALDH, no SPT reactivity was observed in this study. With only one of these three orphan allergens, ChMDH, eliciting SPT(+) reactions consistent with the protein being included in allergen databases, this underscores the complicated nature of how bioinformatics is used to assess the potential allergenicity of food proteins that could be newly added to human diets and, when needed, the subsequent clinical testing of that bioinformatic assessment.Trial registration number and date of registration AAC-2017-0467, approved as WIRB protocol #20172536 on 07DEC2017 by WIRB-Copernicus (OHRP/FDA Registration #: IRB00000533, organization #: IORG0000432).

Molecular basis for branched steviol glucoside biosynthesis.

Steviol glucosides, such as stevioside and rebaudioside A, are natural products roughly 200-fold sweeter than sugar and are used as natural, noncaloric sweeteners. Biosynthesis of rebaudioside A, and other related stevia glucosides, involves formation of the steviol diterpenoid followed by a series of glycosylations catalyzed by uridine diphosphate (UDP)-dependent glucosyltransferases. UGT76G1 from Stevia rebaudiana catalyzes the formation of the branched-chain glucoside that defines the stevia molecule and is critical for its high-intensity sweetness. Here, we report the 3D structure of the UDP-glucosyltransferase UGT76G1, including a complex of the protein with UDP and rebaudioside A bound in the active site. The X-ray crystal structure and biochemical analysis of site-directed mutants identifies a catalytic histidine and how the acceptor site of UGT76G1 achieves regioselectivity for branched-glucoside synthesis. The active site accommodates a two-glucosyl side chain and provides a site for addition of a third sugar molecule to the C3' position of the first C13 sugar group of stevioside. This structure provides insight on the glycosylation of other naturally occurring sweeteners, such as the mogrosides from monk fruit, and a possible template for engineering of steviol biosynthesis.

The plant pathogen enzyme AldC is a long-chain aliphatic aldehyde dehydrogenase.

Aldehyde dehydrogenases are versatile enzymes that serve a range of biochemical functions. Although traditionally considered metabolic housekeeping enzymes because of their ability to detoxify reactive aldehydes, like those generated from lipid peroxidation damage, the contributions of these enzymes to other biological processes are widespread. For example, the plant pathogen Pseudomonas syringae strain PtoDC3000 uses an indole-3-acetaldehyde dehydrogenase to synthesize the phytohormone indole-3-acetic acid to elude host responses. Here we investigate the biochemical function of AldC from PtoDC3000. Analysis of the substrate profile of AldC suggests that this enzyme functions as a long-chain aliphatic aldehyde dehydrogenase. The 2.5 Å resolution X-ray crystal of the AldC C291A mutant in a dead-end complex with octanal and NAD+ reveals an apolar binding site primed for aliphatic aldehyde substrate recognition. Functional characterization of site-directed mutants targeting the substrate- and NAD(H)-binding sites identifies key residues in the active site for ligand interactions, including those in the "aromatic box" that define the aldehyde-binding site. Overall, this study provides molecular insight for understanding the evolution of the prokaryotic aldehyde dehydrogenase superfamily and their diversity of function.

Indole-3-acetaldehyde dehydrogenase-dependent auxin synthesis contributes to virulence of Pseudomonas syringae strain DC3000.

The bacterial pathogen Pseudomonas syringae modulates plant hormone signaling to promote infection and disease development. P. syringae uses several strategies to manipulate auxin physiology in Arabidopsis thaliana to promote pathogenesis, including its synthesis of indole-3-acetic acid (IAA), the predominant form of auxin in plants, and production of virulence factors that alter auxin responses in the host; however, the role of pathogen-derived auxin in P. syringae pathogenesis is not well understood. Here we demonstrate that P. syringae strain DC3000 produces IAA via a previously uncharacterized pathway and identify a novel indole-3-acetaldehyde dehydrogenase, AldA, that functions in IAA biosynthesis by catalyzing the NAD-dependent formation of IAA from indole-3-acetaldehyde (IAAld). Biochemical analysis and solving of the 1.9 Å resolution x-ray crystal structure reveal key features of AldA for IAA synthesis, including the molecular basis of substrate specificity. Disruption of aldA and a close homolog, aldB, lead to reduced IAA production in culture and reduced virulence on A. thaliana. We use these mutants to explore the mechanism by which pathogen-derived auxin contributes to virulence and show that IAA produced by DC3000 suppresses salicylic acid-mediated defenses in A. thaliana. Thus, auxin is a DC3000 virulence factor that promotes pathogenicity by suppressing host defenses.

Modification of auxinic phenoxyalkanoic acid herbicides by the acyl acid amido synthetase GH3.15 from Arabidopsis.

Herbicide-resistance traits are the most widely used agriculture biotechnology products. Yet, to maintain their effectiveness and to mitigate selection of herbicide-resistant weeds, the discovery of new resistance traits that use different chemical modes of action is essential. In plants, the Gretchen Hagen 3 (GH3) acyl acid amido synthetases catalyze the conjugation of amino acids to jasmonate and auxin phytohormones. This reaction chemistry has not been explored as a possible approach for herbicide modification and inactivation. Here, we examined a set of Arabidopsis GH3 proteins that use the auxins indole-3-acetic acid (IAA) and indole-3-butyric acid (IBA) as substrates along with the corresponding auxinic phenoxyalkanoic acid herbicides 2,4-dichlorophenoxylacetic acid (2,4-D) and 4-(2,4-dichlorophenoxy)butyric acid (2,4-DB). The IBA-specific AtGH3.15 protein displayed high catalytic activity with 2,4-DB, which was comparable to its activity with IBA. Screening of phenoxyalkanoic and phenylalkyl acids indicated that side-chain length of alkanoic and alkyl acids is a key feature of AtGH3.15's substrate preference. The X-ray crystal structure of the AtGH3.15·2,4-DB complex revealed how the herbicide binds in the active site. In root elongation assays, Arabidopsis AtGH3.15-knockout and -overexpression lines grown in the presence of 2,4-DB exhibited hypersensitivity and tolerance, respectively, indicating that the AtGH3.15-catalyzed modification inactivates 2,4-DB. These findings suggest a potential use for AtGH3.15, and perhaps other GH3 proteins, as herbicide-modifying enzymes that employ a mode of action different from those of currently available herbicide-resistance traits.

Molecular basis of the evolution of alternative tyrosine biosynthetic routes in plants.

L-Tyrosine (Tyr) is essential for protein synthesis and is a precursor of numerous specialized metabolites crucial for plant and human health. Tyr can be synthesized via two alternative routes by different key regulatory TyrA family enzymes, prephenate dehydrogenase (PDH, also known as TyrAp) or arogenate dehydrogenase (ADH, also known as TyrAa), representing a unique divergence of primary metabolic pathways. The molecular foundation underlying the evolution of these alternative Tyr pathways is currently unknown. Here we characterized recently diverged plant PDH and ADH enzymes, obtained the X-ray crystal structure of soybean PDH, and identified a single amino acid residue that defines TyrA substrate specificity and regulation. Structures of mutated PDHs co-crystallized with Tyr indicate that substitutions of Asn222 confer ADH activity and Tyr sensitivity. Reciprocal mutagenesis of the corresponding residue in divergent plant ADHs further introduced PDH activity and relaxed Tyr sensitivity, highlighting the critical role of this residue in TyrA substrate specificity that underlies the evolution of alternative Tyr biosynthetic pathways in plants.

Conformational changes in the di-domain structure of Arabidopsis phosphoethanolamine methyltransferase leads to active-site formation.

Phosphocholine (pCho) is a precursor for phosphatidylcholine and osmoprotectants in plants. In plants, de novo synthesis of pCho relies on the phosphobase methylation pathway. Phosphoethanolamine methyltransferase (PMT) catalyzes the triple methylation of phosphoethanolamine (pEA) to pCho. The plant PMTs are di-domain methyltransferases that divide the methylation of pEA in one domain from subsequent methylations in the second domain. To understand the molecular basis of this architecture, we examined the biochemical properties of three Arabidopsis thaliana PMTs (AtPMT1-3) and determined the X-ray crystal structures of AtPMT1 and AtPMT2. Although each isoform synthesizes pCho from pEA, their physiological roles differ with AtPMT1 essential for normal growth and salt tolerance, whereas AtPMT2 and AtPMT3 overlap functionally. The structures of AtPMT1 and AtPMT2 reveal unique features in each methyltransferase domain, including active sites that use different chemical mechanisms for phosphobase methylation. These structures also show how rearrangements in both the active sites and the di-domain linker form catalytically competent active sites and provide insight on the evolution of the PMTs in plants, nematodes, and apicomplexans. Connecting conformational changes with catalysis in modular enzymes, like the PMT, provides new insights on interdomain communication in biosynthetic systems.

Structure and Mechanism of Isopropylmalate Dehydrogenase from Arabidopsis thaliana: INSIGHTS ON LEUCINE AND ALIPHATIC GLUCOSINOLATE BIOSYNTHESIS.

Isopropylmalate dehydrogenase (IPMDH) and 3-(2'-methylthio)ethylmalate dehydrogenase catalyze the oxidative decarboxylation of different ß-hydroxyacids in the leucine- and methionine-derived glucosinolate biosynthesis pathways, respectively, in plants. Evolution of the glucosinolate biosynthetic enzyme from IPMDH results from a single amino acid substitution that alters substrate specificity. Here, we present the x-ray crystal structures of Arabidopsis thaliana IPMDH2 (AtIPMDH2) in complex with either isopropylmalate and Mg(2+) or NAD(+) These structures reveal conformational changes that occur upon ligand binding and provide insight on the active site of the enzyme. The x-ray structures and kinetic analysis of site-directed mutants are consistent with a chemical mechanism in which Lys-232 activates a water molecule for catalysis. Structural analysis of the AtIPMDH2 K232M mutant and isothermal titration calorimetry supports a key role of Lys-232 in the reaction mechanism. This study suggests that IPMDH-like enzymes in both leucine and glucosinolate biosynthesis pathways use a common mechanism and that members of the ß-hydroxyacid reductive decarboxylase family employ different active site features for similar reactions.

Gα and regulator of G-protein signaling (RGS) protein pairs maintain functional compatibility and conserved interaction interfaces throughout evolution despite frequent loss of RGS proteins in plants.

Signaling pathways regulated by heterotrimeric G-proteins exist in all eukaryotes. The regulator of G-protein signaling (RGS) proteins are key interactors and critical modulators of the Gα protein of the heterotrimer. However, while G-proteins are widespread in plants, RGS proteins have been reported to be missing from the entire monocot lineage, with two exceptions. A single amino acid substitution-based adaptive coevolution of the Gα:RGS proteins was proposed to enable the loss of RGS in monocots. We used a combination of evolutionary and biochemical analyses and homology modeling of the Gα and RGS proteins to address their expansion and its potential effects on the G-protein cycle in plants. Our results show that RGS proteins are widely distributed in the monocot lineage, despite their frequent loss. There is no support for the adaptive coevolution of the Gα:RGS protein pair based on single amino acid substitutions. RGS proteins interact with, and affect the activity of, Gα proteins from species with or without endogenous RGS. This cross-functional compatibility expands between the metazoan and plant kingdoms, illustrating striking conservation of their interaction interface. We propose that additional proteins or alternative mechanisms may exist which compensate for the loss of RGS in certain plant species.

Structural basis for regulation of rhizobial nodulation and symbiosis gene expression by the regulatory protein NolR.

The symbiosis between rhizobial microbes and host plants involves the coordinated expression of multiple genes, which leads to nodule formation and nitrogen fixation. As part of the transcriptional machinery for nodulation and symbiosis across a range of Rhizobium, NolR serves as a global regulatory protein. Here, we present the X-ray crystal structures of NolR in the unliganded form and complexed with two different 22-base pair (bp) double-stranded operator sequences (oligos AT and AA). Structural and biochemical analysis of NolR reveals protein-DNA interactions with an asymmetric operator site and defines a mechanism for conformational switching of a key residue (Gln56) to accommodate variation in target DNA sequences from diverse rhizobial genes for nodulation and symbiosis. This conformational switching alters the energetic contributions to DNA binding without changes in affinity for the target sequence. Two possible models for the role of NolR in the regulation of different nodulation and symbiosis genes are proposed. To our knowledge, these studies provide the first structural insight on the regulation of genes involved in the agriculturally and ecologically important symbiosis of microbes and plants that leads to nodule formation and nitrogen fixation.

Molecular basis for AUXIN RESPONSE FACTOR protein interaction and the control of auxin response repression.

In plants, the AUXIN RESPONSE FACTOR (ARF) transcription factor family regulates gene expression in response to auxin. In the absence of auxin, ARF transcription factors are repressed by interaction with AUXIN/INDOLE 3-ACETIC ACID (Aux/IAA) proteins. Although the C termini of ARF and Aux/IAA proteins facilitate their homo- and heterooligomerization, the molecular basis for this interaction remained undefined. The crystal structure of the C-terminal interaction domain of Arabidopsis ARF7 reveals a Phox and Bem1p (PB1) domain that provides both positive and negative electrostatic interfaces for directional protein interaction. Mutation of interface residues in the ARF7 PB1 domain yields monomeric protein and abolishes interaction with both itself and IAA17. Expression of a stabilized Aux/IAA protein (i.e., IAA16) bearing PB1 mutations in Arabidopsis suggests a multimerization requirement for ARF protein repression, leading to a refined auxin-signaling model.

Recapitulating the Structural Evolution of Redox Regulation in Adenosine 5'-Phosphosulfate Kinase from Cyanobacteria to Plants.

In plants, adenosine 5'-phosphosulfate (APS) kinase (APSK) is required for reproductive viability and the production of 3'-phosphoadenosine 5'-phosphosulfate (PAPS) as a sulfur donor in specialized metabolism. Previous studies of the APSK from Arabidopsis thaliana (AtAPSK) identified a regulatory disulfide bond formed between the N-terminal domain (NTD) and a cysteine on the core scaffold. This thiol switch is unique to mosses, gymnosperms, and angiosperms. To understand the structural evolution of redox control of APSK, we investigated the redox-insensitive APSK from the cyanobacterium Synechocystis sp. PCC 6803 (SynAPSK). Crystallographic analysis of SynAPSK in complex with either APS and a non-hydrolyzable ATP analog or APS and sulfate revealed the overall structure of the enzyme, which lacks the NTD found in homologs from mosses and plants. A series of engineered SynAPSK variants reconstructed the structural evolution of the plant APSK. Biochemical analyses of SynAPSK, SynAPSK H23C mutant, SynAPSK fused to the AtAPSK NTD, and the fusion protein with the H23C mutation showed that the addition of the NTD and cysteines recapitulated thiol-based regulation. These results reveal the molecular basis for structural changes leading to the evolution of redox control of APSK in the green lineage from cyanobacteria to plants.

Adaptive Engineering of Phytochelatin-based Heavy Metal Tolerance.

Metabolic engineering approaches are increasingly employed for environmental applications. Because phytochelatins (PC) protect plants from heavy metal toxicity, strategies directed at manipulating the biosynthesis of these peptides hold promise for the remediation of soils and groundwaters contaminated with heavy metals. Directed evolution of Arabidopsis thaliana phytochelatin synthase (AtPCS1) yields mutants that confer levels of cadmium tolerance and accumulation greater than expression of the wild-type enzyme in Saccharomyces cerevisiae, Arabidopsis, or Brassica juncea. Surprisingly, the AtPCS1 mutants that enhance cadmium tolerance and accumulation are catalytically less efficient than wild-type enzyme. Metabolite analyses indicate that transformation with AtPCS1, but not with the mutant variants, decreases the levels of the PC precursors, glutathione and γ-glutamylcysteine, upon exposure to cadmium. Selection of AtPCS1 variants with diminished catalytic activity alleviates depletion of these metabolites, which maintains redox homeostasis while supporting PC synthesis during cadmium exposure. These results emphasize the importance of metabolic context for pathway engineering and broaden the range of tools available for environmental remediation.

Defining a two-pronged structural model for PB1 (Phox/Bem1p) domain interaction in plant auxin responses.

Phox/Bem1p (PB1) domains are universal structural modules that use surfaces of different charge for protein-protein association. In plants, PB1-mediated interactions of auxin response factors (ARF) and auxin/indole 3-acetic acid inducible proteins regulate transcriptional events modulated by the phytohormone auxin. Here we investigate the thermodynamic and structural basis for Arabidopsis thaliana ARF7 PB1 domain self-interaction. Isothermal titration calorimetry and NMR experiments indicate that key residues on both the basic and acidic faces of the PB1 domain contribute to and organize coordinately to stabilize protein-protein interactions. Calorimetric analysis of ARF7PB1 site-directed mutants defines a two-pronged electrostatic interaction. The canonical PB1 interaction between a lysine and a cluster of acidic residues provides one prong with an arginine and a second cluster of acidic residues defining the other prong. Evolutionary conservation of this core recognition feature and other co-varying interface sequences allows for versatile PB1-mediated interactions in auxin signaling.

An alternative mechanism for the methylation of phosphoethanolamine catalyzed by Plasmodium falciparum phosphoethanolamine methyltransferase.

The phosphobase methylation pathway catalyzed by the phosphoethanolamine methyltransferase in Plasmodium falciparum (PfPMT), the malaria parasite, offers an attractive target for anti-parasitic drug development. PfPMT methylates phosphoethanolamine (pEA) to phosphocholine for use in membrane biogenesis. Quantum mechanics and molecular mechanics (QM/MM) calculations tested the proposed reaction mechanism for methylation of pEA involving the previously identified Tyr-19-His-132 dyad, which indicated an energetically unfavorable mechanism. Instead, the QM/MM calculations suggested an alternative mechanism involving Asp-128. The reaction coordinate involves the stepwise transfer of a proton to Asp-128 via a bridging water molecule followed by a typical Sn2-type methyl transfer from S-adenosylmethionine to pEA. Functional analysis of the D128A, D128E, D128Q, and D128N PfPMT mutants shows a loss of activity with pEA but not with the final substrate of the methylation pathway. X-ray crystal structures of the PfPMT-D128A mutant in complex with S-adenosylhomocysteine and either pEA or phosphocholine reveal how mutation of Asp-128 disrupts a hydrogen bond network in the active site. The combined QM/MM, biochemical, and structural studies identify a key role for Asp-128 in the initial step of the phosphobase methylation pathway in Plasmodium and provide molecular insight on the evolution of multiple activities in the active site of the PMT.

Structure and mechanism of soybean ATP sulfurylase and the committed step in plant sulfur assimilation.

Enzymes of the sulfur assimilation pathway are potential targets for improving nutrient content and environmental stress responses in plants. The committed step in this pathway is catalyzed by ATP sulfurylase, which synthesizes adenosine 5'-phosphosulfate (APS) from sulfate and ATP. To better understand the molecular basis of this energetically unfavorable reaction, the x-ray crystal structure of ATP sulfurylase isoform 1 from soybean (Glycine max ATP sulfurylase) in complex with APS was determined. This structure revealed several highly conserved substrate-binding motifs in the active site and a distinct dimerization interface compared with other ATP sulfurylases but was similar to mammalian 3'-phosphoadenosine 5'-phosphosulfate synthetase. Steady-state kinetic analysis of 20 G. max ATP sulfurylase point mutants suggests a reaction mechanism in which nucleophilic attack by sulfate on the α-phosphate of ATP involves transition state stabilization by Arg-248, Asn-249, His-255, and Arg-349. The structure and kinetic analysis suggest that ATP sulfurylase overcomes the energetic barrier of APS synthesis by distorting nucleotide structure and identifies critical residues for catalysis. Mutations that alter sulfate assimilation in Arabidopsis were mapped to the structure, which provides a molecular basis for understanding their effects on the sulfur assimilation pathway.

Structure and Mechanism of Ferulic Acid Decarboxylase (FDC1) from Saccharomyces cerevisiae.

The nonoxidative decarboxylation of aromatic acids occurs in a range of microbes and is of interest for bioprocessing and metabolic engineering. Although phenolic acid decarboxylases provide useful tools for bioindustrial applications, the molecular bases for how these enzymes function are only beginning to be examined. Here we present the 2.35-Å-resolution X-ray crystal structure of the ferulic acid decarboxylase (FDC1; UbiD) from Saccharomyces cerevisiae. FDC1 shares structural similarity with the UbiD family of enzymes that are involved in ubiquinone biosynthesis. The position of 4-vinylphenol, the product of p-coumaric acid decarboxylation, in the structure identifies a large hydrophobic cavity as the active site. Differences in the ß2e-α5 loop of chains in the crystal structure suggest that the conformational flexibility of this loop allows access to the active site. The structure also implicates Glu285 as the general base in the nonoxidative decarboxylation reaction catalyzed by FDC1. Biochemical analysis showed a loss of enzymatic activity in the E285A mutant. Modeling of 3-methoxy-4-hydroxy-5-decaprenylbenzoate, a partial structure of the physiological UbiD substrate, in the binding site suggests that an ∼30-Å-long pocket adjacent to the catalytic site may accommodate the isoprenoid tail of the substrate needed for ubiquinone biosynthesis in yeast. The three-dimensional structure of yeast FDC1 provides a template for guiding protein engineering studies aimed at optimizing the efficiency of aromatic acid decarboxylation reactions in bioindustrial applications.