AflaILVB/G/I and AflaILVD are involved in mycelial production, aflatoxin biosynthesis, and fungal virulence in Aspergillus flavus.

Aflatoxins (AFs) are produced by fungi such as Aspergillus flavus and A. parasiticus and are one of the most toxic mycotoxins found in agricultural products and food. Aflatoxin contamination, which requires the control of A. flavus, remains problematic because of the lack of effective strategies and the exploration of new compounds that can inhibit A. flavus growth and mycotoxin production is urgently required to alleviate potential deleterious effects. Acetohydroxy acid synthase (AHAS) and dihydroxy acid dehydratase are important enzymes in the biosynthetic pathways of branched-chain amino acids (BCAAs), including isoleucine, leucine, and valine. Enzymes involved in BCAA biosynthesis are present in bacteria, plants, and fungi, but not in mammals, and are therefore, attractive targets for antimicrobial and herbicide development. In this study, we characterized AflaILVB/G/I and AflaILVD, which encode the catalytic and regulatory subunits of AHAS and dihydroxy acid dehydratase, from the pathogenic fungus Aspergillus flavus. The AflaILVB/G/I and AflaILVD deletion mutant grew slower and produced smaller colonies than the wild-type strain when grown on glucose minimal medium, potato dextrose agar, and yeast extract medium for three days at 28°C, and disruption of AflaILVB/G/I caused a significant reduction in conidia production when grown on all kinds of media. Cellular stress assays determined that all strains were sensitive to H2O2. Importantly, the pathogenicity and aflatoxin production were affected when AflaILVB/G/I and AflaILVD were knocked out, particularly AflaILVB/G/I. A series of genes that encoded enzymes involved in aflatoxin synthesis were downregulated, meaning that the knockout of AflaILVB/G/I influenced aflatoxin synthesis in A. flavus strain WT. Collectively, our results demonstrate the potential value of antifungals targeting AflaILVB/G/I in A. flavus.

HadBD dehydratase from Mycobacterium tuberculosis fatty acid synthase type II: A singular structure for a unique function.

Worldwide, tuberculosis is the second leading infectious killer and multidrug resistance severely hampers disease control. Mycolic acids are a unique category of lipids that are essential for viability, virulence, and persistence of the causative agent, Mycobacterium tuberculosis (Mtb). Therefore, enzymes involved in mycolic acid biosynthesis represent an important class of drug targets. We previously showed that the (3R)-hydroxyacyl-ACP dehydratase (HAD) protein HadD is dedicated mainly to the production of ketomycolic acids and plays a determinant role in Mtb biofilm formation and virulence. Here, we discovered that HAD activity requires the formation of a tight heterotetramer between HadD and HadB, a HAD unit encoded by a distinct chromosomal region. Using biochemical, structural, and cell-based analyses, we showed that HadB is the catalytic subunit, whereas HadD is involved in substrate binding. Based on HadBDMtb crystal structure and substrate-bound models, we identified determinants of the ultra-long-chain lipid substrate specificity and revealed details of structure-function relationship. HadBDMtb unique function is partly due to a wider opening and a higher flexibility of the substrate-binding crevice in HadD, as well as the drastically truncated central α-helix of HadD hotdog fold, a feature described for the first time in a HAD enzyme. Taken together, our study shows that HadBDMtb , and not HadD alone, is the biologically relevant functional unit. These results have important implications for designing innovative antivirulence molecules to fight tuberculosis, as they suggest that the target to consider is not an isolated subunit, but the whole HadBD complex.

Isolation and characterization of a bis(dithiolene)-supported tungsten-acetylenic complex as a model for acetylene hydratase.

Acetylene hydratase is currently the only known mononuclear tungstoenzyme that does not catalyze a net redox reaction. The conversion of acetylene to acetaldehyde is proposed to occur at a W(IV) active site through first-sphere coordination of the acetylene substrate. To date, a handful of tungsten complexes have been shown to bind acetylene, but many lack the bis(dithiolene) motif of the native enzyme. The model compound, [W(O)(mnt)2]2-, where mnt2- is 1,2-dicyano-1,2-dithiolate, was previously reported to bind an electrophilic acetylene substrate, dimethyl acetylenedicarboxylate, and characterized by FT-IR, UV-vis, potentiometry, and mass spectrometry (Yadav, J; Das, S. K.; Sarkar, S., J. Am. Chem. Soc., 1997, 119, 4316-4317). By slightly changing the electrophilic acetylene substrate, an acetylenic-bis(dithiolene)­tungsten(IV) complex has been isolated and characterized by FT-IR, UV-vis, NMR, X-ray diffraction, and X-ray absorption spectroscopy. Activation parameters for complex formation were also determined and suggest coordination-sphere reorganization is a limiting factor in the model complex reactivity.

Immobilization of aldoxime dehydratases on metal affinity resins and use of the immobilized catalysts for the synthesis of nitriles important in fragrance industry.

Nitriles have a wide range of uses as building blocks, solvents, and alternative fuels, but also as intermediates and components of flavors and fragrances. The enzymatic synthesis of nitriles by aldoxime dehydratase (Oxd) is an emerging process with significant advantages over conventional approaches. Here we focus on the immobilization of His-tagged Oxds on metal affinity resins, an approach that has not been used previously for these enzymes. The potential of the immobilized Oxd was demonstrated for the synthesis of phenylacetonitrile (PAN) and E-cinnamonitrile, compounds applicable in the fragrance industry. A comparison of Talon and Ni-NTA resins showed that Ni-NTA with its higher binding capacity was more suitable for the immobilization of Oxd. Immobilized Oxds were prepared from purified enzymes (OxdFv from Fusarium vanettenii and OxdBr1 from Bradyrhizobium sp.) or the corresponding cell-free extracts. The immobilization of cell-free extracts reduced time and cost of the catalyst production. The immobilized OxdBr1 was superior in terms of recyclability (22 cycles) in the synthesis of PAN from 15 mM E/Z-phenylacetaldoxime at pH 7.0 and 30 °C (100% conversion, 61% isolated yield after product purification). The volumetric and catalyst productivity was 10.5 g/L/h and 48.3 g/g of immobilized protein, respectively.

Targeted macrophage phagocytosis by Irg1/itaconate axis improves the prognosis of intracerebral hemorrhagic stroke and peritonitis.

BACKGROUND: Macrophages are innate immune cells whose phagocytosis function is critical to the prognosis of stroke and peritonitis. cis-aconitic decarboxylase immune-responsive gene 1 (Irg1) and its metabolic product itaconate inhibit bacterial infection, intracellular viral replication, and inflammation in macrophages. Here we explore whether itaconate regulates phagocytosis. METHODS: Phagocytosis of macrophages was investigated by time-lapse video recording, flow cytometry, and immunofluorescence staining in macrophage/microglia cultures isolated from mouse tissue. Unbiased RNA-sequencing and ChIP-sequencing assays were used to explore the underlying mechanisms. The effects of Irg1/itaconate axis on the prognosis of intracerebral hemorrhagic stroke (ICH) and peritonitis was observed in transgenic (Irg1flox/flox; Cx3cr1creERT/+, cKO) mice or control mice in vivo. FINDINGS: In a mouse model of ICH, depletion of Irg1 in macrophage/microglia decreased its phagocytosis of erythrocytes, thereby exacerbating outcomes (n = 10 animals/group, p < 0.05). Administration of sodium itaconate/4-octyl itaconate (4-OI) promoted macrophage phagocytosis (n = 7 animals/group, p < 0.05). In addition, in a mouse model of peritonitis, Irg1 deficiency in macrophages also inhibited phagocytosis of Staphylococcus aureus (n = 5 animals/group, p < 0.05) and aggravated outcomes (n = 9 animals/group, p < 0.05). Mechanistically, 4-OI alkylated cysteine 155 on the Kelch-like ECH-associated protein 1 (Keap1), consequent in nuclear translocation of nuclear factor erythroid 2-related factor 2 (Nrf2) and transcriptional activation of Cd36 gene. Blocking the function of CD36 completely abolished the phagocytosis-promoting effects of Irg1/itaconate axis in vitro and in vivo. INTERPRETATION: Our findings provide a potential therapeutic target for phagocytosis-deficiency disorders, supporting further development towards clinical application for the benefit of stroke and peritonitis patients. FUNDING: The National Natural Science Foundation of China (32070735, 82371321 to Q. Li, 82271240 to F. Yang) and the Beijing Natural Science Foundation Program and Scientific Research Key Program of Beijing Municipal Commission of Education (KZ202010025033 to Q. Li).

Plastid ancestors lacked a complete Entner-Doudoroff pathway, limiting plants to glycolysis and the pentose phosphate pathway.

The Entner-Doudoroff (ED) pathway provides an alternative to glycolysis. It converts 6-phosphogluconate (6-PG) to glyceraldehyde-3-phosphate and pyruvate in two steps consisting of a dehydratase (EDD) and an aldolase (EDA). Here, we investigate its distribution and significance in higher plants and determine the ED pathway is restricted to prokaryotes due to the absence of EDD genes in eukaryotes. EDDs share a common origin with dihydroxy-acid dehydratases (DHADs) of the branched chain amino acid pathway (BCAA). Each dehydratase features strict substrate specificity. E. coli EDD dehydrates 6-PG to 2-keto-3-deoxy-6-phosphogluconate, while DHAD only dehydrates substrates from the BCAA pathway. Structural modeling identifies two divergent domains which account for their non-overlapping substrate affinities. Coupled enzyme assays confirm only EDD participates in the ED pathway. Plastid ancestors lacked EDD but transferred metabolically promiscuous EDA, which explains the absence of the ED pathway from the Viridiplantae and sporadic persistence of EDA genes across the plant kingdom.

The 3-hydroxyacyl-CoA dehydratase 1/2 form complex with trans-2-enoyl-CoA reductase involved in substrates transfer in very long chain fatty acid elongation.

Very long-chain fatty acids (VLCFAs) are fatty acids with a carbon chain length greater than 18 carbons (>C18) and exhibit various functions, such as in skin barrier formation, liver homeostasis, myelin maintenance, spermatogenesis, retinal function, and anti-inflammation. VLCFAs are absorbed by dietary or elongated from endogenous hexadecanoyl acids (C16). Similar to long-chain fatty acid synthesis, VLCFAs elongation begins with acyl-CoA and malonyl-CoA as sources, and the length of the acyl chain is extended by two carbon units in each cycle. However, the VLCFAs elongation machinery is located in ER membrane and consists of four components, FA elongase (ELOVL), 3-ketoacyl-CoA reductase (KAR), 3-hydroxyacyl-CoA dehydratase (HACD), and trans-2-enoyl-CoA reductase (TECR), which is different with the long-chain fatty acid machinery fatty acid synthase (FAS) complex. Although the critical components in the elongation cycle are identified, the detailed catalytic and regulation mechanisms are still poorly understood. Here, we focused on the structural and biochemical analysis of TECR-associated VLCFA elongation reactions. Firstly, we identified a stable complex of human HACD2-TECR based on extensive in vitro characterizations. Combining computational modeling and biochemical analysis, we confirmed the critical interactions between TECR and HACD1/2. Then, we proposed the putative substrate binding sites and catalytic residues for TECR and HACD2. Besides, we revealed the structural similarities of HACD with ELOVLs and proposed the possible competition mechanism of TECR-associated complex formation.

Nitrile-synthesizing enzymes and biocatalytic synthesis of volatile nitrile compounds: A review.

Nitriles (R-CN) comprise a broad group of chemicals industrially produced and used in fine chemicals, pharmaceuticals, and bulk applications, polymer chemistry, solvents, etc. Nitriles are important starting materials for producing carboxylic acids, amides, amines, and several other compounds. In addition, some volatile nitriles have been evaluated for their potential as ingredients in fragrance and flavor formulations. However, many nitrile synthesis methods have drawbacks, such as drastic reaction conditions, limited substrate scope, lack of readily available reagents, poor yields, and long reaction times. In contrast to chemical synthesis, biocatalytic approaches using enzymes can produce nitriles without harsh conditions, such as high temperatures and pressures, or toxic compounds. In this review, we summarize the nitrile-synthesizing enzymes from microorganisms, plants, and animals. Furthermore, we introduce several examples of biocatalytic synthesis of volatile nitrile compounds, particularly those using aldoxime dehydratase.

Structure and Substrate Specificity of S-Methyl Thiourocanate Hydratase.

Nicotinamide adenine dinucleotide (NAD+) is a common cofactor in enzyme-catalyzed reactions that involve hydride transfers. In contrast, urocanase and urocanase-like enzymes use NAD+ for covalent electrophilic catalysis. Deciphering avenues by which this unusual catalytic strategy has diversified by evolution may point to approaches for the design of novel enzymes. In this report, we describe the S-methyl thiourocanate hydratase (S-Me-TUC) from Variovorax sp. RA8 as a novel member of this small family of NAD+-dependent hydratases. This enzyme catalyzes the 1,4-addition of water to S-methyl thiourocanate as the second step in the catabolism of S-methyl ergothioneine. The crystal structure of this enzyme in complex with the cofactor and a product analogue identifies critical sequence motifs that explain the narrow and nonoverlapping substrate scopes of S-methyl thiourocanate-, urocanate-, thiourocanate-, and Nτ-methyl urocanate-specific hydratases. The discovery of a S-methyl ergothioneine catabolic pathway also suggests that S-methylation or alkylation may be a significant activity in the biology of ergothioneine.

Interrogating l-fuconate dehydratase with tartronate and 3-hydroxypyruvate reveals subtle differences within the mandelate racemase-subgroup of the enolase superfamily.

Enzymes of the enolase superfamily share a conserved structure and a common partial reaction (i.e., metal-assisted, Brønsted base-catalyzed enol(ate) formation). The architectures of the enolization apparatus at the active sites of the mandelate racemase (MR)-subgroup members MR and l-fuconate dehydratase (FucD) are almost indistinguishable at the structural level. Tartronate and 3-hydroxypyruvate (3-HP) recognize the enolization apparatus and can be used to interrogate the active sites for differences that may not be apparent from structural data. We report a circular dichroism-based assay of FucD activity that monitors the change in ellipticity at 216 nm (Δ[Θ]S-P = 8985 ± 87 deg cm2 mol-1) accompanying the conversion of l-fuconate to 2-keto-3-deoxy-l-fuconate. Tartronate was a linear mixed-type inhibitor of FucD (Ki = 8.4 ± 0.7 mM, αKi = 63 ± 11 mM), binding 18-fold weaker than l-fuconate, compared with 2-fold weaker binding of tartronate by MR relative to mandelate. 3-HP irreversibly inactivated FucD (kinact/KI = 0.018 ± 0.002 M-1s-1) with an efficiency that was ∼4.6 × 103-fold less than that observed with MR. The inactivation arose predominantly from modifications at multiple sites and Tris-HCl, but not l-fuconate, afforded protection against inactivation. Similar to the reaction of 3-HP with MR, 3-HP modified the Brønsted base catalyst (Lys 220) at the active site of FucD, which was facilitated by the Brønsted acid catalyst His 351. Thus, the interactions of tartronate and 3-HP with MR and FucD revealed differences in binding affinity and reactivity that differentiated between the enzymes' enolization apparatuses.

Progressive encephalopathy after routine 4-month immunizations in a patient with NAXD genetic variant.

Metabolic pathways are known to generate byproducts-some of which have no clear metabolic function and some of which are toxic. Nicotinamide adenine dinucleotide phosphate hydrate (NAD(P)HX) is a toxic metabolite that is produced by stressors such as a fever, infection, or physical stress. Nicotinamide adenine dinucleotide phosphate hydrate dehydratase (NAXD) and nicotinamide adenine dinucleotide phosphate hydrate epimerase (NAXE) are part of the nicotinamide repair system that function to break down this toxic metabolite. Deficiency of NAXD and NAXE interrupts the critical intracellular repair of NAD(P)HX and allows for its accumulation. Clinically, deficiency of NAXE manifests as progressive, early onset encephalopathy with brain edema and/or leukoencephalopathy (PEBEL) 1, while deficiency of NAXD manifests as PEBEL2. In this report, we describe a case of probable PEBEL2 in a patient with a variant of unknown significance (c.362C>T, p.121L) in the NAXD gene who presented after routine immunizations with significant skin findings and in the absence of fevers.

Suppressor analysis links trans-translation and ribosomal protein uS7 to RluD function in Escherichia coli.

The pseudouridine (ψ) synthase, RluD is responsible for three ψ modifications in the helix 69 (H69) of bacterial 23S rRNA. While normally dispensable, rluD becomes critical for rapid cell growth in bacteria that are defective in translation-termination. In slow-growing rluD- bacteria, suppressors affecting termination factors RF2 and RF3 arise frequently and restore normal termination and rapid cell growth. Here we describe two weaker suppressors, affecting rpsG, encoding ribosomal protein uS7 and ssrA, encoding tmRNA. In K-12 strains of E. coli, rpsG terminates at a TGA codon. In the suppressor strain, alteration of an upstream CAG to a TAG stop codon results in a shortened uS7 and partial alleviation of slow growth, likely by replacing an inefficient TGA stop codon with the more efficient TAG. Inefficient termination events, such as occurs in some rluD- strains, are targeted by trans-translation. Inactivation of the ssrA gene in slow-growing, termination-defective mutants lacking RluD and RF3, also partially restores robust growth, most probably by preventing destruction of completed polypeptides on ribosomes at slow-terminating stop codons. Finally, an additional role for RluD has been proposed, independent of its pseudouridine synthase activity. This is based on the observation that plasmids expressing catalytically dead (D139N or D139T) RluD proteins could nonetheless restore robust growth to an E. coli K-12 rluD- mutant. However, newly constructed D139N and D139T rluD plasmids do not have any growth-restoring activity and the original observations were likely due to the appearance of suppressors.

Novel tetrahydrofolate-dependent d-serine dehydratase activity of serine hydroxymethyltransferases.

d-Serine plays vital physiological roles in the functional regulation of the mammalian brain, where it is produced from l-serine by serine racemase and degraded by d-amino acid oxidase. In the present study, we identified a new d-serine metabolizing activity of serine hydroxymethyltransferase (SHMT) in bacteria as well as mammals. SHMT is known to catalyze the conversion of l-serine and tetrahydrofolate (THF) to glycine and 5,10-methylenetetrahydrofolate, respectively. In addition, we found that human and Escherichia coli SHMTs have d-serine dehydratase activity, which degrades d-serine to pyruvate and ammonia. We characterized this enzymatic activity along with canonical SHMT activity. Intriguingly, SHMT required THF to catalyze d-serine dehydration and did not exhibit dehydratase activity toward l-serine. Furthermore, SHMT did not use d-serine as a substrate in the canonical hydroxymethyltransferase reaction. The d-serine dehydratase activities of two isozymes of human SHMT were inhibited in the presence of a high concentration of THF, whereas that of E. coli SHMT was increased. The pH and temperature profiles of d-serine dehydratase and serine hydroxymethyltransferase activities of these three SHMTs were partially distinct. The catalytic efficiency (kcat /Km ) of dehydratase activity was lower than that of hydroxymethyltransferase activity. Nevertheless, the d-serine dehydratase activity of SHMT was physiologically important because d-serine inhibited the growth of an SHMT deletion mutant of E. coli, ∆glyA, more than that of the wild-type strain. Collectively, these results suggest that SHMT is involved not only in l- but also in d-serine metabolism through the degradation of d-serine.

Exploration and utilization of novel aldoxime, nitrile, and nitro compounds metabolizing enzymes from plants and arthropods.

Aldoxime (R1R2C=NOH) and nitrile (R-C≡N) are nitrogen-containing compounds that are found in species representing all kingdoms of life. The enzymes discovered from the microbial "aldoxime-nitrile" pathway (aldoxime dehydratase, nitrile hydratase, amidase, and nitrilase) have been thoroughly studied because of their industrial importance. Although plants utilize cytochrome P450 monooxygenases to produce aldoxime and nitrile, many biosynthetic pathways are yet to be studied. Cyanogenic millipedes accumulate various nitrile compounds, such as mandelonitrile. However, no such aldoxime- and nitrile-metabolizing enzymes have been identified in millipedes. Here, I review the exploration of novel enzymes from plants and millipedes with characteristics distinct from those of microbial enzymes, the catalysis of industrially useful reactions, and applications of these enzymes for nitrile compound production.

Characterization of a novel L-fuconate dehydratase involved in the non-phosphorylated pathway of L-fucose metabolism from bacteria.

A sugar acid dehydratase from Paraburkholderia mimosarum, potentially involved in the non-phosphorylated L-fucose pathway, was functionally characterized. A biochemical analysis revealed its unique heterodimeric structure and higher specificity toward L-fuconate than D-arabinonate, D-altronate, and L-xylonate, which differed from homomeric homologs. This unique L-fuconate dehydratase has a poor phylogenetic relationship with other functional members of the D-altronate dehydratase/galactarate dehydratase protein family.

Structure-oriented engineering of nitrile hydratase: Reshaping of substrate access tunnel and binding pocket for efficient synthesis of cinnamamide.

Cinnamamide and its derivatives are the most common and important building blocks widely present in natural products. Currently, nitrile hydratase (NHase, EC 4.2.1.84) has been widely used in large-scale industrial production of nicotinamide and acrylamide, while its catalytic activity is extremely low or inactive for bulky nitrile substrates such as cinnamonitrile. Therefore, beneficial variant ßF37P/L48P/F51N were obtained from PtNHase of Pseudonocardia thermophila JCM3095 by reshaping of substrate access tunnel and binding pocket, which exhibited 14.88-fold improved catalytic efficiency compared to the wild-type PtNHase. Structure analysis, molecular dynamics simulations and dynamical cross-correlation matrix (DCCM) analysis revealed that the introduced mutations enlarged the substrate access tunnel and binding pocket, enhanced overall anti-correlated movements of enzymes, which would promote product release during the dynamic process of catalysis. In a hydration process, the complete conversion of 5 mM cinnamonitrile was achieved by ßF37P/L48P/F51N in a 50 mL reaction, with cinnamamide yield of almost 100 % and productivity of 0.736 g L-1 h-1. The study demonstrates the co-evolution of substrate access tunnel and binding pocket is an effective strategy, and provides a valuable reference for future research. Furthermore, NHases have huge potential for catalyzing bulky nitriles to form corresponding amides in large-scale industrial production.

Structural and Biochemical Analysis of 3-Dehydroquinate Dehydratase from Corynebacterium glutamicum.

Dehydroquinate dehydratase (DHQD) catalyzes the conversion of 3-dehydroquinic acid (DHQ) into 3-dehydroshikimic acid in the mid stage of the shikimate pathway, which is essential for the biosynthesis of aromatic amino acids and folates. Here, we report two the crystal structures of type II DHQD (CgDHQD) derived from Corynebacterium glutamicum, which is a widely used industrial platform organism. We determined the structures for CgDHQDWT with the citrate at a resolution of 1.80Å and CgDHQDR19A with DHQ complexed forms at a resolution of 2.00 Å, respectively. The enzyme forms a homododecamer consisting of four trimers with three interfacial active sites. We identified the DHQ-binding site of CgDHQD and observed an unusual binding mode of citrate inhibitor in the site with a half-opened lid loop. A structural comparison of CgDHQD with a homolog derived from Streptomyces coelicolor revealed differences in the terminal regions, lid loop, and active site. Particularly, CgDHQD, including some Corynebacterium species, possesses a distinctive residue P105, which is not conserved in other DHQDs at the position near the 5-hydroxyl group of DHQ. Replacements of P105 with isoleucine and valine, conserved in other DHQDs, caused an approximately 70% decrease in the activity, but replacement of S103 with threonine (CgDHQDS103T) caused a 10% increase in the activity. Our biochemical studies revealed the importance of key residues and enzyme kinetics for wild type and CgDHQDS103T, explaining the effect of the variation. This structural and biochemical study provides valuable information for understanding the reaction efficiency that varies due to structural differences caused by the unique sequences of CgDHQD.

Multi-omics analysis reveals PUS1 triggered malignancy and correlated with immune infiltrates in NSCLC.

Non-small cell lung cancer (NSCLC) is the main pathological type of lung cancer. In this study, multi-omics analysis revealed a significant increase of pseudouridine synthase 1 (PUS1) in NSCLC and the high expression of PUS1 was associated with shorter OS (Overall Survival), PFS (Progression Free Survival), and PPS (Post Progression Survival) of NSCLC patients. Clinical subgroup analysis showed that PUS1 may be involved in the occurrence and development of NSCLC. Besides, TIMER, ESTIMATE, and IPS analysis suggested that PUS1 expression was associated with immune cell infiltration, and the expression of PUS1 was significantly negatively correlated with DC cell infiltration. GESA analysis also indicated PUS1 may involve in DNA_REPAIR, E2F_TARGETS, MYC_TARGETS_V2, G2M_CHECKPOINT and MYC_TARGETS_V1 pathways and triggered NSCLC malignancy through MCM5 or XPO1. Furthermore, PUS1 may be a potential target for NSCLC therapy.

Exploring the Substrate-Assisted Dehydration of Chorismate Catalyzed by Dehydratase MqnA from QM/MM Calculations: The Role of Pocket Residues and the Hydrolysis Mechanism of N17D Mutant.

MqnA is the first enzyme on the futalosine pathway to menaquinone, which catalyzes the dehydration of chorismate to yield 3-enolpyruvyl-benzoate (3-EPB). MqnA is also the only chorismate dehydratase known so far. In this work, based on the recently determined crystal structures, we constructed the enzyme-substrate complex models and conducted quantum mechanics/molecular mechanics (QM/MM) calculations to elucidate the reaction details of MqnA and the critical roles of pocket residues. The calculation results confirm that the MqnA-catalyzed dehydration of chorismate follows the substrate-assisted E1cb mechanism, in which the enol carboxylate in the side chain of the substrate is responsible for deprotonating the C3 of chorismate. This proton transfer process is much slower than C4-OH departure. Calculations on different mutants reveal that S86 and N17 are important for anchoring the enol carboxylate of the substrate in a favorable conformation to extract the C3-proton. The strong H-bonds formed between the enol carboxylate of chorismate and S86/N17 play a key role in stabilizing the reaction intermediate. Consistent with the experimental observations, our calculations demonstrate that the MqnA N17D mutant also shows hydrolase activity and the typical enzyme-catalyzed hydrolysis mechanism is elucidated. The protonated D17 is responsible for saturating the methylene group of chorismate to start the hydrolysis reaction. The orientation of the carboxyl group of D17 is key in determining MqnA to be a dehydratase or hydrolase.

Metabolic Pathways for the Biosynthesis of Heptoses Used in the Construction of Capsular Polysaccharides in the Human Pathogen Campylobacter jejuni.

Campylobacter jejuni is the leading cause of food poisoning in North America. The exterior surface of this bacterium is coated with a capsular polysaccharide (CPS) that consists of a repeating sequence of 2-5 different carbohydrates that is anchored to the outer membrane. Heptoses of various configurations are among the most common monosaccharides that have been identified within the CPS. It is currently thought that all heptose variations derive from the modification of GDP-d-glycero-α-d-manno-heptose (GMH). From the associated gene clusters for CPS biosynthesis, we have identified 20 unique enzymes with different substrate profiles that are used by the various strains and serotypes of C. jejuni to make six different stereoisomers of GDP-6-deoxy-heptose, four stereoisomers of GDP-d-glycero-heptoses, and two stereoisomers of GDP-3,6-dideoxy-heptoses starting from d-sedoheptulose-7-phosphate. The modification enzymes include a C4-dehydrogenase, a C4,6-dehydratase, three C3- and/or C5-epimerases, a C3-dehydratase, eight C4-reductases, two pyranose/furanose mutases, and four enzymes for the formation of GMH from d-sedoheptulose-7-phosphate. We have mixed these enzymes in different combinations to make novel GDP-heptose modifications, including GDP-6-hydroxy-heptoses, GDP-3-deoxy-heptoses, and GDP-3,6-dideoxy-heptoses.