EMS-induced missense mutation in TaCHLI-7D affects leaf color and yield-related traits in wheat.

KEY MESSAGE: Mutations in TaCHLI impact chlorophyll levels and yield-related traits in wheat. Natural variations in TaCHLI-7A/B influence plant productivity, offering potential for molecular breeding. Chlorophyll is essential for plant growth and productivity. The CHLI subunit of the magnesium chelatase protein plays a key role inserting magnesium into protoporphyrin IX during chlorophyll biosynthesis. Here, we identify a novel wheat mutant chlorophyll (chl) that exhibits yellow-green leaves, reduced chlorophyll levels, and increased carotenoid content, leading to an overall decline in yield-related traits. Map-based cloning reveals that the chl phenotype is caused by a point mutation (Asp186Asn) in the TaCHLI-7D gene, which encodes subunit I of magnesium chelatase. Furthermore, the three TaCHLI mutants: chl-7b-1 (Pro82Ser), chl-7b-2 (Ala291Thr), and chl-7d-1 (Gly357Glu), also showed significant reductions in chlorophyll content and yield-related traits. However, TaCHLI-7D overexpression in rice significantly decreased thousand kernel weight, yield per plant, and germination. Additionally, natural variations in TaCHLI-7A/B are significantly associated with flag leaf, spike exsertion length, and yield per plant. Notably, the favorable haplotype, TaCHLI-7B-HapII, which displayed higher thousand kernel weight and yield per plant, is positively selected in wheat breeding. Our study provides insights on the regulatory molecular mechanisms underpinning leaf color and chlorophyll biosynthesis, and highlights TaCHLI functions, which provide useful molecular markers and genetic resources for wheat breeding.

Persistence and pathway of glyphosate degradation in the coastal wetland soil of central Delaware.

Glyphosate is a globally dominant herbicide. Here, we studied the degradation and microbial response to glyphosate application in a wetland soil in central Delaware for controlling invasive species (Phragmites australis). We applied a two-step solid-phase extraction method using molecularly imprinted polymers designed for the separation and enrichment of glyphosate and aminomethylphosphonic acid (AMPA) from soils before their analysis by ultra-high-performance liquid chromatography (UHPLC) and Q Exactive Orbitrap mass spectrometry methods. Our results showed that approximately 90 % of glyphosate degraded over 100 d after application, with AMPA being a minor (<10 %) product. Analysis of glyphosate-specific microbial genes to identify microbial response and function revealed that the expression of the phnJ gene, which codes C-P lyase enzyme, was consistently dominant over the gox gene, which codes glyphosate oxidoreductase enzyme, after glyphosate application. Both gene and concentration data independently suggested that C-P bond cleavage-which forms sarcosine or glycine-was the dominant degradation pathway. This is significant because AMPA, a more toxic product, is reported to be the preferred pathway of glyphosate degradation in other soil and natural environments. The degradation through a safer pathway is encouraging for minimizing the detrimental impacts of glyphosate on the environment.

Helicobacter pylori and Campylobacter jejuni bacterial holocytochrome c synthase structure-function analysis reveals conservation of heme binding.

Heme trafficking is essential for cellular function, yet mechanisms of transport and/or heme interaction are not well defined. The System I and System II bacterial cytochrome c biogenesis pathways are developing into model systems for heme trafficking due to their functions in heme transport, heme stereospecific positioning, and mediation of heme attachment to apocytochrome c. Here we focus on the System II pathway, CcsBA, that is proposed to be a bi-functional heme transporter and holocytochrome c synthase. An extensive structure-function analysis of recombinantly expressed Helicobacter pylori and Campylobacter jejuni CcsBAs revealed key residues required for heme interaction and holocytochrome c synthase activity. Homologous residues were previously identified to be required for heme interaction in Helicobacter hepaticus CcsBA. This study provides direct, biochemical evidence that mechanisms of heme interaction are conserved, leading to the proposal that the CcsBA WWD heme-handling domain represents a novel target for therapeutics.

Insights into ACO genes across Rosaceae: evolution, expression, and regulatory networks in fruit development.

BACKGROUND: ACO (1-aminocyclopropane-1-carboxylic acid) serves as a pivotal enzyme within the plant ethylene synthesis pathway, exerting influence over critical facets of plant biology such as flowering, fruit ripening, and seed development. OBJECTIVE: This study aims to identify ACO genes from representative Rosaceae genomes, reconstruct their phylogenetic relationships by integrating synteny information, and investigate their expression patterns and networks during fruit development. METHODS: we utilize a specialized Hidden Markov Model (HMM), crafted on the sequence attributes of ACO gene-encoded proteins, to systematically identify and analyze ACO gene family members across 12 representative species within the Rosaceae botanical family. Through transcriptome analysis, we delineate the expression patterns of ACO genes in six distinct Rosaceae fruits. RESULTS: Our investigation reveals the presence of 62 ACO genes distributed among the surveyed Rosaceae species, characterized by hydrophilic proteins predominantly expressed within the cytoplasm. Phylogenetic analysis categorizes these ACO genes into three discernible classes, namely Class I, Class II, and Class III. Further scrutiny via collinearity assessment indicates a lack of collinearity relationships among these classes, highlighting variations in conserved motifs and promoter types within each class. Transcriptome analysis unveils significant disparities in both expression levels and trends of ACO genes in fruits exhibiting respiratory bursts compared to those that do not. Employing Weighted Gene Co-Expression Network Analysis (WGCNA), we discern that the co-expression correlation of ACO genes within loquat fruit notably differs from that observed in apples. Our findings, derived from Gene Ontology (GO) enrichment results, signify the involvement of ACO genes and their co-expressed counterparts in biological processes linked to terpenoid metabolism and carbohydrate synthesis in loquat. Moreover, our exploration of gene regulatory networks (GRN) highlights the potential pivotal role of the GNAT transcription factor (Ejapchr1G00010380) in governing the overexpression of the ACO gene (Ejapchr10G00001110) within loquat fruits. CONCLUSION: The constructed HMM of ACO proteins offers a precise and systematic method for identifying plant ACO proteins, facilitating phylogenetic reconstruction. ACO genes from representative Rosaceae fruits exhibit diverse expression and regulative patterns, warranting further function characterizations.

Substitution of 2-oxoglutarate alters reaction outcomes of the Pseudomonas savastanoi ethylene-forming enzyme.

In seeding plants, biosynthesis of the phytohormone ethylene, which regulates processes including fruit ripening and senescence, is catalyzed by 1-aminocyclopropane-1-carboxylic acid (ACC) oxidase. The plant pathogen Pseudomonas savastanoi (previously classified as: Pseudomonas syringae) employs a different type of ethylene-forming enzyme (psEFE), though from the same structural superfamily as ACC oxidase, to catalyze ethylene formation from 2-oxoglutarate (2OG) in an arginine dependent manner. psEFE also catalyzes the more typical oxidation of arginine to give L-Δ1-pyrroline-5-carboxylate (P5C), a reaction coupled to oxidative decarboxylation of 2OG giving succinate and CO2. We report on the effects of C3 and/or C4 substituted 2OG derivatives on the reaction modes of psEFE. 1H NMR assays, including using the pure shift method, reveal that, within our limits of detection, none of the tested 2OG derivatives is converted to an alkene; some are converted to the corresponding ß-hydroxypropionate or succinate derivatives, with only the latter being coupled to arginine oxidation. The NMR results reveal that the nature of 2OG derivatization can affect the outcome of the bifurcating reaction, with some 2OG derivatives exclusively favoring the arginine oxidation pathway. Given that some of the tested 2OG derivatives are natural products, the results are of potential biological relevance. There are also opportunities for therapeutic or biocatalytic regulation of the outcomes of reactions catalyzed by 2OG-dependent oxygenases by the use of 2OG derivatives.

Identification and Characterization of Thiamine Diphosphate-Dependent Lyases with an Unusual CDG Motif.

The thiamine diphosphate (ThDP)-binding motif, characterized by the canonical GDG(X)24-27N sequence, is highly conserved among ThDP-dependent enzymes. We investigated a ThDP-dependent lyase (JanthE from Janthinobacterium sp. HH01) with an unusual cysteine (C458) replacing the first glycine of this motif. JanthE exhibits a high substrate promiscuity and accepts long aliphatic α-keto acids as donors. Sterically hindered aromatic aldehydes or non-activated ketones are acceptor substrates, giving access to a variety of secondary and tertiary alcohols as carboligation products. The crystal structure solved at a resolution of 1.9 Šreveals that C458 is not primarily involved in cofactor binding as previously thought for the canonical glycine. Instead, it coordinates methionine 406, thus ensuring the integrity of the active site and the enzyme activity. In addition, we have determined the long-sought genuine tetrahedral intermediates formed with pyruvate and 2-oxobutyrate in the pre-decarboxylation states and deciphered the atomic details for their stabilization in the active site. Collectively, we unravel an unexpected role for the first residue of the ThDP-binding motif and unlock a family of lyases that can perform valuable carboligation reactions.

Escherichia coli monothiol glutaredoxin GrxD replenishes Fe-S clusters to the essential ErpA A-type carrier under low iron stress.

Iron-sulfur (Fe-S) clusters are required for essential biological pathways, including respiration and isoprenoid biosynthesis. Complex Fe-S cluster biogenesis systems have evolved to maintain an adequate supply of this critical protein cofactor. In Escherichia coli, two Fe-S biosynthetic systems, the "housekeeping" Isc and "stress responsive" Suf pathways, interface with a network of cluster trafficking proteins, such as ErpA, IscA, SufA, and NfuA. GrxD, a Fe-S cluster-binding monothiol glutaredoxin, also participates in Fe-S protein biogenesis in both prokaryotes and eukaryotes. Previous studies in E. coli showed that the ΔgrxD mutation causes sensitivity to iron depletion, spotlighting a critical role for GrxD under conditions that disrupt Fe-S homeostasis. Here, we utilized a global chemoproteomic mass spectrometry approach to analyze the contribution of GrxD to the Fe-S proteome. Our results demonstrate that (1) GrxD is required for biogenesis of a specific subset of Fe-S proteins under iron-depleted conditions, (2) GrxD is required for cluster delivery to ErpA under iron limitation, (3) GrxD is functionally distinct from other Fe-S trafficking proteins, and (4) GrxD Fe-S cluster binding is responsive to iron limitation. All these results lead to the proposal that GrxD is required to maintain Fe-S cluster delivery to the essential trafficking protein ErpA during iron limitation conditions.

Transgenic Arabidopsis thaliana plants expressing bacterial γ-hexachlorocyclohexane dehydrochlorinase LinA.

BACKGROUND: γ-Hexachlorocyclohexane (γ-HCH), an organochlorine insecticide of anthropogenic origin, is a persistent organic pollutant (POP) that causes environmental pollution concerns worldwide. Although many γ-HCH-degrading bacterial strains are available, inoculating them directly into γ-HCH-contaminated soil is ineffective because of the low survival rate of the exogenous bacteria. Another strategy for the bioremediation of γ-HCH involves the use of transgenic plants expressing bacterial enzyme for γ-HCH degradation through phytoremediation. RESULTS: We generated transgenic Arabidopsis thaliana expressing γ-HCH dehydrochlroninase LinA from bacterium Sphingobium japonicum strain UT26. Among the transgenic Arabidopsis T2 lines, we obtained one line (A5) that expressed and accumulated LinA well. The A5-derived T3 plants showed higher tolerance to γ-HCH than the non-transformant control plants, indicating that γ-HCH is toxic for Arabidopsis thaliana and that this effect is relieved by LinA expression. The crude extract of the A5 plants showed γ-HCH degradation activity, and metabolites of γ-HCH produced by the LinA reaction were detected in the assay solution, indicating that the A5 plants accumulated the active LinA protein. In some A5 lines, the whole plant absorbed and degraded more than 99% of γ-HCH (10 ppm) in the liquid medium within 36 h. CONCLUSION: The transgenic Arabidopsis expressing active LinA absorbed and degraded γ-HCH in the liquid medium, indicating the high potential of LinA-expressing transgenic plants for the phytoremediation of environmental γ-HCH. This study marks a crucial step toward the practical use of transgenic plants for the phytoremediation of POPs.

The transcription factor WRKY22 modulates ethylene biosynthesis and root development through transactivating the transcription of ACS5 and ACO5 in Arabidopsis.

The WRKY transcription factor (TF) genes form a large family in higher plants, with 72 members in Arabidopsis (Arabidopsis thaliana). The gaseous phytohormone ethylene (ET) regulates multiple physiological processes in plants. It is known that 1-aminocyclopropane-1-carboxylic acid (ACC) synthases (ACSs, EC 4.4.1.14) limit the enzymatic reaction rate of ethylene synthesis. However, whether WRKY TFs regulate the expression of ACSs and/or ACC oxidases (ACOs, EC 1.14.17.4) remains largely elusive. Here, we demonstrated that Arabidopsis WRKY22 positively regulated the expression of a few ACS and ACO genes, thus promoting ethylene production. Inducible overexpression of WRKY22 caused shorter hypocotyls without ACC treatment. A qRT-PCR screening demonstrated that overexpression of WRKY22 activates the expression of several ACS and ACO genes. The promoter regions of ACS5, ACS11, and ACO5 were also activated by WRKY22, which was revealed by a dual luciferase reporter assay. A follow-up chromatin immunoprecipitation coupled with quantitative PCR (ChIP-qPCR) and electrophoretic mobility shift assay (EMSA) showed that the promoter regions of ACS5 and ACO5 could be bound by WRKY22 directly. Moreover, wrky22 mutants had longer primary roots and more lateral roots than wild type, while WRKY22-overexpressing lines showed the opposite phenotype. In conclusion, this study revealed that WRKY22 acts as a novel TF activating, at least, the expression of ACS5 and ACO5 to increase ethylene synthesis and modulate root development.

Nickel-chelatase activity of SirB variants mimicking the His arrangement in the naturally occurring nickel-chelatase CfbA.

Metal-tetrapyrrole cofactors are involved in multiple cellular functions, and chelatases are key enzymes for the biosynthesis of these cofactors. CfbA is an ancestral, homodimeric-type class II chelatase which is able to use not only Ni2+ as a physiological metal substrate, but also Co2+ as a nonphysiological substrate with higher activity than for Ni2+. The Ni/Co-chelatase function found in CfbA is also observed in SirB, a descendant, monomeric-type class II chelatase. This is despite the distinct active site structure of CfbA and SirB; specifically, CfbA shows a unique four His residue arrangement, unlike other monomeric class II chelatases such as SirB. Herein, we studied the Ni-chelatase activity of SirB variants R134H, L200H, and R134H/L200H, the latter of which mimics the His alignment of CfbA. Our results showed that the SirB R134H variant exhibited the highest Ni-chelatase activity among the SirB enzymes, which in turn suggests that the position of His134 could be more important for the Ni-chelatase activity than that of His200. The SirB R134H/L200H variant showed lower activity than R134H, despite the four His residues found in SirB R134H/L200H. CD spectroscopy showed secondary structure denaturation and a slight difficulty in Ni-binding of SirB R134H/L200H, which may be related to its lower activity. Finally, a docking simulation suggested that the His134 of the SirB R134H variant could function as a base catalyst for the Ni-chelatase reaction in a class II chelatase architecture.

Gene identification, expression analysis, and molecular docking of SAT and OASTL in the metabolic pathway of selenium in Cardamine hupingshanensis.

KEY MESSAGE: Identification of selenium stress-responsive expression and molecular docking of serine acetyltransferase (SAT) and O-acetyl serine (thiol) lyase (OASTL) in Cardamine hupingshanensis. A complex coupled with serine acetyltransferase (SAT) and O-acetyl serine (thiol) lyase (OASTL) is the key enzyme that catalyzes selenocysteine (Sec) synthesis in plants. The functions of SAT and OASTL genes were identified in some plants, but it is still unclear whether SAT and OASTL are involved in the selenium metabolic pathway in Cardamine hupingshanensis. In this study, genome-wide identification and comparative analysis of ChSATs and ChOASTLs were performed. The eight ChSAT genes were divided into three branches, and the thirteen ChOASTL genes were divided into four branches by phylogenetic analysis and sequence alignment, indicating the evolutionary conservation of the gene structure and its association with other plant species. qRT-PCR analysis showed that the ChSAT and ChOASTL genes were differentially expressed in different tissues under various selenium levels, suggesting their important roles in Sec synthesis. The ChSAT1;2 and ChOASTLA1;2 were silenced by the VIGS system to investigate their involvement in selenium metabolites in C. hupingshanensis. The findings contribute to understanding the gene functions of ChSATs and ChOASTLs in the selenium stress and provide a reference for further exploration of the selenium metabolic pathway in plants.

Cryoradiolysis of oxygenated cytochrome P450 17A1 with lyase substrates generates expected products.

When subjected to γ-irradiation at cryogenic temperatures the oxygenated complexes of Cytochrome P450 CYP17A1 (CYP17A1) bound with either of the lyase substrates, 17α-Hydroxypregnenolone (17-OH PREG) or 17α-Hydroxyprogesterone (17-OH PROG) are shown to generate the corresponding lyase products, dehydroepiandrosterone (DHEA) and androstenedione (AD) respectively. The current study uses gas chromatography-mass spectrometry (GC/MS) to document the presence of the initial substrates and products in extracts of the processed samples. A rapid and efficient method for the simultaneous determination of residual substrate and products by GC/MS is described without derivatization of the products. It is also shown that no lyase products were detected for similarly treated control samples containing no nanodisc associated CYP17 enzyme, demonstrating that the product is formed during the enzymatic reaction and not by GC/MS conditions, nor the conditions produced by the cryoradiolysis process.

Structural, Spectroscopic, and Computational Insights from Canavanine-Bound and Two Catalytically Compromised Variants of the Ethylene-Forming Enzyme.

The ethylene-forming enzyme (EFE) is an Fe(II), 2-oxoglutarate (2OG), and l-arginine (l-Arg)-dependent oxygenase that either forms ethylene and three CO2/bicarbonate from 2OG or couples the decarboxylation of 2OG to C5 hydroxylation of l-Arg. l-Arg binds with C5 toward the metal center, causing 2OG to change from monodentate to chelate metal interaction and OD1 to OD2 switch of D191 metal coordination. We applied anaerobic UV-visible spectroscopy, X-ray crystallography, and computational approaches to three EFE systems with high-resolution structures. The ineffective l-Arg analogue l-canavanine binds to the EFE with O5 pointing away from the metal center while promoting chelate formation by 2OG but fails to switch the D191 metal coordination from OD1 to OD2. Substituting alanine for R171 that interacts with 2OG and l-Arg inactivates the protein, prevents metal chelation by 2OG, and weakens l-Arg binding. The R171A EFE had electron density at the 2OG binding site that was identified by mass spectrometry as benzoic acid. The substitution by alanine of Y306 in the EFE, a residue 12 Å away from the catalytic metal center, generates an interior cavity that leads to multiple local and distal structural changes that reduce l-Arg binding and significantly reduce the enzyme activity. Flexibility analyses revealed correlated and anticorrelated motions in each system, with important distinctions from the wild-type enzyme. In combination, the results are congruent with the currently proposed enzyme mechanism, reinforce the importance of metal coordination by OD2 of D191, and highlight the importance of the second coordination sphere and longer range interactions in promoting EFE activity.

Identification of Key Ubiquitination Sites Involved in the Proteasomal Degradation of AtACS7 in Arabidopsis.

The gaseous hormone ethylene plays pivotal roles in plant growth and development. The rate-limiting enzyme of ethylene biosynthesis in seed plants is 1-aminocyclopropane-1-carboxylic acid (ACC) synthase (ACS). ACS proteins are encoded by a multigene family and the expression of ACS genes is highly regulated, especially at a post-translational level. AtACS7, the only type III ACS in Arabidopsis, is degraded in a 26S proteasome-dependent pathway. Here, by using liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS) analysis, two lysine residues of AtACS7, lys285 (K285) and lys366 (K366), were revealed to be ubiquitin-modified in young, light-grown Arabidopsis seedlings but not in etiolated seedlings. Deubiquitylation-mimicking mutations of these residues significantly increased the stability of the AtACS7K285RK366R mutant protein in cell-free degradation assays. All results suggest that K285 and K366 are the major ubiquitination sites on AtACS7, providing deeper insights into the post-translational regulation of AtACS7 in Arabidopsis.

Mitochondrial transcription factor A (TFAM) has 5'-deoxyribose phosphate lyase activity in vitro.

Mitochondrial DNA (mtDNA) plays a key role in mitochondrial and cellular functions. mtDNA is maintained by active DNA turnover and base excision repair (BER). In BER, one of the toxic repair intermediates is 5'-deoxyribose phosphate (5'dRp). Human mitochondrial DNA polymerase γ has weak dRp lyase activities, and another known dRp lyase in the nucleus, human DNA polymerase ß, can also localize to mitochondria in certain cell and tissue types. Nonetheless, whether additional proteins have the ability to remove 5'dRp in mitochondria remains unknown. Our prior work on the AP lyase activity of mitochondrial transcription factor A (TFAM) has prompted us to examine its ability to remove 5'dRp residues in vitro. TFAM is the primary DNA-packaging factor in human mitochondria and interacts with mitochondrial DNA extensively. Our data demonstrate that TFAM has the dRp lyase activity with different DNA substrates. Under single-turnover conditions, TFAM removes 5'dRp residues at a rate comparable to that of DNA polymerase (pol) ß, albeit slower than that of pol λ. Among the three proteins examined, pol λ shows the highest single-turnover rates in dRp lyase reactions. The catalytic effect of TFAM is facilitated by lysine residues of TFAM via Schiff base chemistry, as evidenced by the observation of dRp-lysine adducts in mass spectrometry experiments. The catalytic effect of TFAM observed here is analogous to the AP lyase activity of TFAM reported previously. Together, these results suggest a potential role of TFAM in preventing the accumulation of toxic DNA repair intermediates.

GT-11 impairs insulin signaling through modulation of sphingolipid metabolism in C2C12 myotubes.

AIMS: Sphingolipids are involved in the regulation of insulin signaling, which is linked to the development of insulin resistance, leading to diabetes mellitus. We aimed to study whether modulation of sphingolipid levels by GT-11 may regulate insulin signaling in C2C12 myotubes. MAIN METHODS: We investigated the effects of sphingolipid metabolism on Akt phosphorylation and glucose uptake using C2C12 myotubes. Either GT-11, an inhibitor of dihydroceramide desaturase 1 and S1P lyase, or siRNA targeting Sgpl1, the gene encoding the enzyme, was employed to determine the effect of sphingolipid metabolism modulation on insulin signaling. Western blotting and glucose uptake assays were used to evaluate the effect of treatments on insulin signaling. Sphingolipid metabolites were analyzed by high performance liquid chromatography (HPLC). KEY FINDINGS: Treatment with GT-11 resulted in decreased Akt phosphorylation and reduced glucose uptake. Silencing the Sgpl1 gene, which encodes S1P lyase, mimicked these findings, suggesting the potential for regulating insulin signaling through S1P lyase modulation. GT-11 modulated sphingolipid metabolism, inducing the accumulation of sphingolipids. Using PF-543 and ARN14974 to inhibit sphingosine kinases and acid ceramidase, respectively, we identified a significant interplay between sphingosine, S1P lyase, and insulin signaling. Treatment with either exogenous sphingosine or palmitic acid inhibited Akt phosphorylation, and reduced S1P lyase activity. SIGNIFICANCE: Our findings highlight the importance of close relationship between sphingolipid metabolism and insulin signaling in C2C12 myotubes, pointing to its potential therapeutic relevance for diabetes mellitus.

Review on sterilization techniques, and the application potential of phage lyase and lyase immobilization in fighting drug-resistant bacteria.

Many human health problems and property losses caused by pathogenic contamination cannot be underestimated. Bactericidal techniques have been extensively studied to address this issue of public health and economy. Bacterial resistance develops as a result of the extensive use of single or multiple but persistent usage of sterilizing drugs, and the emergence of super-resistant bacteria brings new challenges. Therefore, it is crucial to control pathogen contamination by applying innovative and effective sterilization techniques. As organisms that exist in nature and can specifically kill bacteria, phages have become the focus as an alternative to antibacterial agents. Furthermore, phage-encoded lyases are proteins that play important roles in phage sterilization. The in vitro sterilization of phage lyase has been developed as a novel biosterilization technique to reduce bacterial resistance and is more environmentally friendly than conventional sterilization treatments. For the shortcomings of enzyme applications, this review discusses the enzyme immobilization methods and the application potential of immobilized lyases for sterilization. Although some techniques provide effective solutions, immobilized lyase sterilization technology has been proven to be a more effective innovation for efficient pathogen killing and reducing bacterial resistance. We hope that this review can provide new insights for the development of sterilization techniques.

Charge neutralization and ß-elimination cleavage mechanism of family 42 L-rhamnose-α-1,4-D-glucuronate lyase revealed using neutron crystallography.

Gum arabic (GA) is widely used as an emulsion stabilizer and edible coating and consists of a complex carbohydrate moiety with a rhamnosyl-glucuronate group capping the non-reducing ends. Enzymes that can specifically cleave the glycosidic chains of GA and modify their properties are valuable for structural analysis and industrial application. Cryogenic X-ray crystal structure of GA-specific L-rhamnose-α-1,4-D-glucuronate lyase from Fusarium oxysporum (FoRham1), belonging to the polysaccharide lyase (PL) family 42, has been previously reported. To determine the specific reaction mechanism based on its hydrogen-containing enzyme structure, we performed joint X-ray/neutron crystallography of FoRham1. Large crystals were grown in the presence of L-rhamnose (a reaction product), and neutron and X-ray diffraction datasets were collected at room temperature at 1.80 and 1.25 Å resolutions, respectively. The active site contained L-rhamnose and acetate, the latter being a partial analog of glucuronate. Incomplete H/D exchange between Arg166 and acetate suggested that a strong salt-bridge interaction was maintained. Doubly deuterated His105 and deuterated Tyr150 supported the interaction between Arg166 and the acetate. The unique hydrogen-rich environment functions as a charge neutralizer for glucuronate and stabilizes the oxyanion intermediate. The NE2 atom of His85 was deprotonated and formed a hydrogen bond with the deuterated O1 hydroxy of L-rhamnose, indicating the function of His85 as the base/acid catalyst for bond cleavage via ß-elimination. Asp83 functions as a pivot between the two catalytic histidine residues by bridging them. This His-His-Asp structural motif is conserved in the PL 24, 25, and 42 families.

Systematic Analysis of the MIO-forming Residues of Aromatic Ammonia Lyases.

Aromatic ammonia lyases (AALs) and tyrosine/phenylalanine ammonia mutases (TAM/PAM) are 3,5-dihydro-5-methylidene-4H-imidazol-4-one (MIO)-dependent enzymes. Usually, the MIO moiety is autocatalytically formed from the tripeptide Ala-Ser-Gly (ASG) and acts as an electrophile during the enzymatic reaction. However, the MIO-forming residues (ASG) have some diversity in this enzyme class. In this work, a systematic investigation on the variety of MIO-forming residues was carried out using in-depth sequence analyses. Several protein clusters of AAL-like enzymes with unusual MIO-forming residues such as ACG, TSG, SSG, and CSG were identified, including two novel histidine ammonia lyases and one PAM with CSG and TSG residues, respectively, as well as three novel ergothioneine trimethylammonia lyases without MIO motif. The mutagenesis of common MIO-groups confirmed the function of these MIO variants, which provides good starting points for future functional prediction and mutagenesis research of AALs.

Steric Perturbation of the Grob-like Final Step of Ethylene-Forming Enzyme Enables 3-Hydroxypropionate and Propylene Production.

Ethylene-forming enzyme (EFE) is an iron(II)-dependent dioxygenase that fragments 2-oxoglutarate (2OG) to ethylene (from C3 and C4) and 3 equivs of carbon dioxide (from C1, C2, and C5). This major ethylene-forming pathway requires l-arginine as the effector and competes with a minor pathway that merely decarboxylates 2OG to succinate as it oxidatively fragments l-arginine. We previously proposed that ethylene forms in a polar-concerted (Grob-like) fragmentation of a (2-carboxyethyl)carbonatoiron(II) intermediate, formed by the coupling of a C3-C5-derived propion-3-yl radical to a C1-derived carbonate coordinated to the Fe(III) cofactor. Replacement of one or both C4 hydrogens of 2OG by fluorine, methyl, or hydroxyl favored the elimination products 2-(F1-2/Me/OH)-3-hydroxypropionate and CO2 over the expected olefin or carbonyl products, implying strict stereoelectronic requirements in the final step, as is known for Grob fragmentations. Here, we substituted active-site residues expected to interact sterically with the proposed Grob intermediate, aiming to disrupt or enable the antiperiplanar disposition of the carboxylate electrofuge and carbonate nucleofuge required for concerted fragmentation. The bulk-increasing A198L substitution barely affects the first partition between the major and minor pathways but then, as intended, markedly diminishes ethylene production in favor of 3-hydroxypropionate. Conversely, the bulk-diminishing L206V substitution enables propylene formation from (4R)-methyl-2OG, presumably by allowing the otherwise sterically disfavored antiperiplanar conformation of the Grob intermediate bearing the extra methyl group. The results provide additional evidence for a polar-concerted ethylene-yielding step and thus for the proposed radical-polar crossover via substrate-radical coupling to the Fe(III)-coordinated carbonate.