Phenylalanine ammonia-lyase (PAL) is the first enzyme of the general phenylpropanoid pathway catalyzing the nonoxidative elimination of ammonia from l-phenylalanine to give -cinnamate. In monocots, PAL also displays tyrosine ammonia lyase (TAL) activity, leading to the formation of-coumaric acid. The catalytic mechanism and substrate specificity of a major PAL from sorghum (; SbPAL1), a strategic plant for bioenergy production, were deduced from crystal structures, molecular docking, site-directed mutagenesis, and kinetic and thermodynamic analyses. This first crystal structure of a monocotyledonous PAL displayed a unique conformation in its flexible inner loop of the 4-methylidene-imidazole-5-one (MIO) domain compared with that of dicotyledonous plants. The side chain of histidine-123 in the MIO domain dictated the distance between the catalytic MIO prosthetic group created from Ala-Ser-Gly residues and the bound l-phenylalanine and l-tyrosine, conferring the deamination reaction through either the Friedel-Crafts or E reaction mechanism. Several recombinant mutant SbPAL1 enzymes were generated via structure-guided mutagenesis, one of which, H123F-SbPAL1, has 6.2 times greater PAL activity without significant TAL activity. Additional PAL isozymes of sorghum were characterized and categorized into three groups. Taken together, this approach identified critical residues and explained substrate preferences among PAL isozymes in sorghum and other monocots, which can serve as the basis for the engineering of plants with enhanced biomass conversion properties, disease resistance, or nutritional quality.

EDTA has become a major organic pollutant in the environment because of its extreme usage and resistance to biodegradation. Recently, two critical enzymes, EDTA monooxygenase (EmoA) and NADH:FMN oxidoreductase (EmoB), belonging to the newly established two-component flavin-diffusible monooxygenase family, were identified in the EDTA degradation pathway in Mesorhizobium sp. BNC1. EmoA is an FMNH 2 -dependent enzyme that requires EmoB to provide FMNH 2 for the conversion of EDTA to ethylenediaminediacetate. To understand the molecular basis of this FMN-mediated reaction, the crystal structures of the apo-form, FMN⅐FMN complex, and FMN⅐NADH complex of EmoB were determined at 2.5 Å resolution. The structure of EmoB is a homotetramer consisting of four ␣/␤-single-domain monomers of five parallel ␤-strands flanked by five ␣-helices, which is quite different from those of other known two-component flavin-diffusible monooxygenase family members, such as PheA2 and HpaC, in terms of both tertiary and quaternary structures. For the first time, the crystal structures of both the FMN⅐FMN and FMN⅐NADH complexes of an NADH:FMN oxidoreductase were determined. Two stacked isoalloxazine rings and nicotinamide/isoalloxazine rings were at a proper distance for hydride transfer. The structures indicated a ping-pong reaction mechanism, which was confirmed by activity assays. Thus, the structural data offer detailed mechanistic information for hydride transfer between NADH to an enzyme-bound FMN and between the bound FMNH 2 and a diffusible FMN.EDTA has quietly become a major organic pollutant, currently present in the environment at higher concentrations than any other organic pollutant (1). A high level of EDTA in natural waters is due to its extensive usage, such as in industrial cleaning to remove calcium deposits, in detergent as a sequestering agent, in phytoremediation to mobilize heavy metals, and in scientific laboratories as a chelating agent (2, 3). EDTA is recalcitrant to biodegradation and exists mainly in metal⅐EDTA complexes, many of which are toxic (4, 5). In addition, the codisposal of EDTA with radionuclides has led to the enhanced mobilization of radionuclides in groundwater, rapidly spreading radioactive contamination (3, 6 -8). Concerns over EDTA recalcitrance and the potential mobilization of heavy metals and radionuclides have led the European Union, Australia, and some parts of the United States to ban EDTA in detergent. It is now also being carefully controlled in many other products to reduce contamination of water resources.Several bacteria that can degrade EDTA and the related compound, nitrilotriacetate, and use them as a sole source of carbon and energy have been isolated (9 -12). They are phylogenetically related to Mesorhizobium and Agrobacterium species (11), likely forming a new branch within the Phyllobacteriaceae, the "Mesorhizobia" family (13). In these bacteria, reduced flavin mononucleotide (FMNH 2 )-dependent EDTA monooxygenase (EmoA) and NADH:FMN oxidoreductase (EmoB) together oxidize EDTA to e...

Cinnamyl alcohol dehydrogenase (CAD) catalyzes the final step in monolignol biosynthesis, reducing sinapaldehyde, coniferaldehyde, and -coumaraldehyde to their corresponding alcohols in an NADPH-dependent manner. Because of its terminal location in monolignol biosynthesis, the variation in substrate specificity and activity of CAD can result in significant changes in overall composition and amount of lignin. Our in-depth characterization of two major CAD isoforms, SbCAD2 (Brown midrib 6 [bmr6]) and SbCAD4, in lignifying tissues of sorghum (), a strategic plant for generating renewable chemicals and fuels, indicates their similarity in both structure and activity to Arabidopsis () CAD5 and sinapyl alcohol dehydrogenase, respectively. This first crystal structure of a monocot CAD combined with enzyme kinetic data and a catalytic model supported by site-directed mutagenesis allows full comparison with dicot CADs and elucidates the potential signature sequence for their substrate specificity and activity. The L119W/G301F-SbCAD4 double mutant displayed its substrate preference in the order coniferaldehyde> -coumaraldehyde> sinapaldehyde, with higher catalytic efficiency than that of both wild-type SbCAD4 and SbCAD2. As SbCAD4 is the only major CAD isoform in mutants, replacing SbCAD4 with L119W/G301F-SbCAD4 in plants could produce a phenotype that is more amenable to biomass processing.

Summary Ethylenediaminetetraacetate (EDTA) is currently the most abundant organic pollutant due to its recalcitrance and extensive use. Only a few bacteria can degrade it, using EDTA monooxygenase (EmoA) to initiate the degradation. EmoA is an FMNH2-dependent monooxygenase that requires an NADH:FMN oxidoreductase (EmoB) to provide FMNH2 as a cosubstrate. Although EmoA has been identified from Chelativorans (ex. Mesorhizobium) sp. BNC1, its catalytic mechanism is unknown. Crystal structures of EmoA revealed a domain-like insertion into a TIM-barrel, which might serve as a flexible lid for the active site. Docking of MgEDTA2− into EmoA identified an intricate hydrogen bond network connected to Tyr71, which should potentially lower its pKa. Tyr71, along with nearby Glu70 and a peroxy flavin, facilitates a keto-enol transition of the leaving acetyl group of EDTA. Further, for the first time, the physical interaction between EmoA and EmoB was observed by ITC, molecular docking and enzyme kinetic assay, which enhanced both EmoA and EmoB activities probably through coupled channeling of FMNH2.

In bacterial chemotaxis, chemoreceptors in signaling complexes modulate the activity of two-component histidine kinase CheA in response to chemical stimuli. CheA catalyzes phosphoryl transfer from ATP to a histidinyl residue of its P1 domain. That phosphoryl group is transferred to two response regulators. Receptor control is almost exclusively at autophosphorylation, but the aspect of enzyme action on which that control acts is unclear. We investigated this by a kinetic analysis of activated kinase in signaling complexes. We found that phosphoryl transfer from ATP to P1 is an ordered sequential reaction in which the binding of ATP to CheA is the necessary first step; the second substrate, the CheA P1 domain, binds only to an ATP-occupied enzyme; and phosphorylated P1 is released prior to the second product, namely, ADP. We confirmed the crucial features of this kinetically deduced ordered mechanism by assaying P1 binding to the enzyme. In the absence of a bound nucleotide, there was no physiologically significant binding, but the enzyme occupied with a nonhydrolyzable ATP analog bound P1. Previous structural and computational analyses indicated that ATP binding creates the P1-binding site by ordering the “ATP lid.” This process identifies the structural basis for the ordered kinetic mechanism. Recent mathematical modeling of kinetic data identified ATP binding as a focus of receptor-mediated kinase control. The ordered kinetic mechanism provides the biochemical logic of that control. We conclude that chemoreceptors modulate kinase by controlling ATP binding. Structural similarities among two-component kinases, particularly the ATP lid, suggest that ordered mechanisms and control of ATP binding are general features of two-component signaling. IMPORTANCE Our work provides important new insights into the action of the chemotaxis signaling kinase CheA by identifying the kinetic mechanism of its autophosphorylation as an ordered sequential reaction, in which the required first step is binding of ATP. These insights provide a framework for integrating previous kinetic, mathematical modeling, structural, simulation, and docking observations to conclude that chemoreceptors control the activity of the chemotaxis kinase by regulating binding of the autophosphorylation substrate ATP. Previously observed conformational changes in the ATP lid of the enzyme active site provide a structural basis for the ordered mechanism. Such lids are characteristic of two-component histidine kinases in general, suggesting that ordered sequential mechanisms and regulation by controlling ATP binding are common features of these kinases.