Alteration of the Route to Menaquinone towards Isochorismate-Derived Metabolites

Chorismate and isochorismate constitute branch-point intermediates in the biosynthesis of many aromatic metabolites in microorganisms and plants. To obtain unnatural compounds, we modified the route to menaquinone in Escherichia coli. We propose a model for the binding of isochorismate to the active site of MenD ((1R,2S, 5S,6S)-2-succinyl-5-enolpyruvyl-6-hydroxycyclohex-3-ene-1-carboxylate (SEPHCHC) synthase) that explains the outcome of the native reaction with α-ketoglutarate. We have rationally designed variants of MenD for the conversion of several isochorismate analogues. The double-variant Asn117Arg–Leu478Thr preferentially converts (5S,6S)-5,6-dihydroxycyclohexa-1,3-diene-1-carboxylate (2,3-trans-CHD), the hydrolysis product of isochorismate, with a >70-fold higher ratio than that for the wild type. The single-variant Arg107Ile uses (5S,6S)-6-amino-5-hydroxycyclohexa-1,3-diene-1-carboxylate (2,3-trans-CHA) as substrate with >6-fold conversion compared to wild-type MenD. The novel compounds have been made accessible in vivo (up to 5.3 g L⁻¹). Unexpectedly, as the identified residues such as Arg107 are highly conserved (>94 %), some of the designed variations can be found in wild-type SEPHCHC synthases from other bacteria (Arg107Lys, 0.3 %). This raises the question for the possible natural occurrence of as yet unexplored branches of the shikimate pathway.     

Cooking rice in excess water reduces both arsenic and enriched vitamins in the cooked grain

This paper reports the effects of rinsing rice and cooking it in variable amounts of water on total arsenic, inorganic arsenic, iron, cadmium, manganese, folate, thiamin and niacin in the cooked grain. We prepared multiple rice varietals both rinsed and unrinsed and with varying amounts of cooking water. Rinsing rice before cooking has a minimal effect on the arsenic (As) content of the cooked grain, but washes enriched iron, folate, thiamin and niacin from polished and parboiled rice. Cooking rice in excess water efficiently reduces the amount of As in the cooked grain. Excess water cooking reduces average inorganic As by 40% from long grain polished, 60% from parboiled and 50% from brown rice. Iron, folate, niacin and thiamin are reduced by 50–70% for enriched polished and parboiled rice, but significantly less so for brown rice, which is not enriched.     

Noncanonical Reactions of Flavoenzymes

Enzymes containing flavin cofactors are predominantly involved in redox reactions in numerous cellular processes where the protein environment modulates the chemical reactivity of the flavin to either transfer one or two electrons. Some flavoenzymes catalyze reactions with no net redox change. In these reactions, the protein environment modulates the reactivity of the flavin to perform novel chemistries. Recent mechanistic and structural data supporting novel flavin functionalities in reactions catalyzed by chorismate synthase, type II isopentenyl diphosphate isomerase, UDP-galactopyranose mutase, and alkyl-dihydroxyacetonephosphate synthase are presented in this review. In these enzymes, the flavin plays either a direct role in acid/base reactions or as a nucleophile or electrophile. In addition, the flavin cofactor is proposed to function as a “molecular scaffold” in the formation of UDP-galactofuranose and alkyl-dihydroxyacetonephosphate by forming a covalent adduct with reaction intermediates.     

Alteration of product specificity of Aeropyrum pernix farnesylgeranyl diphosphate synthase (Fgs) by directed evolution

Directed evolution of the C25 farnesylgeranyl diphosphate synthase of Aeropyrum pernix (Fgs) was carried out by error-prone PCR with an in vivo color complementation screen utilizing carotenoid biosynthetic pathway enzymes. Screening yielded 12 evolved clones with C20 geranylgeranyl diphosphate synthase activity which were isolated and characterized in order to understand better the chain elongation mechanism of this enzyme. Analysis of these mutants revealed three different mechanisms of product chain length specificity. Two mutants (A64T and A64V) have a single mutation at the 8th amino acid upstream of a conserved first aspartate-rich motif (FARM), which is involved in the mechanism for chain elongation reaction of all prenyl diphosphate synthases. One mutant (A135T) carries a single mutation at the 7th amino acid upstream of another conserved region (141GQ142), which was recently found to be another important region controlling chain elongation of a type III C20 geranylgeranyl diphosphate synthase and Escherichia coli C15 farnesyl diphosphate synthase. Finally, one mutant carrying four mutations (V84I, H88R, I177 M and M191V) is of interest. Molecular modeling, site-directed mutagenesis and in vitro assays of this mutant suggest that product chain-length distribution can be also controlled by a structural change provoked by a cooperative interaction of amino acids.     

Structure Conservation and Differential Expression of Farnesyl Diphosphate Synthase Genes in Euphorbiaceous Plants

Farnesyl diphosphate synthase (FPS) is a key enzyme of isoprenoids biosynthesis. However, knowledge of the FPSs of euphorbiaceous species is limited. In this study, ten FPSs were identified in four euphorbiaceous plants. These FPSs exhibited similar exon/intron structure. The deduced FPS proteins showed close identities and exhibited the typical structure of plant FPS. The members of the FPS family exhibit tissue expression patterns that vary among several euphorbiaceous plant species under normal growth conditions. The expression profiles reveal spatial and temporal variations in the expression of FPSs of different tissues from Euphorbiaceous plants. Our results revealed wide conservation of FPSs and diverse expression in euphorbiaceous plants during growth and development.     

Kinetics of Thiamin Degradation in Solutions under Ambient Storage Conditions

Thiamin is being incorporated into a variety of nutritionally enhanced beverages. Although thiamin degradation during thermal processing has been extensively studied, minimal data exist regarding its stability in solution during ambient storage. This study collected kinetic data for thiamin degradation in solutions as a function of pH, buffer type, and buffer concentration at 25 °C. Thiamin was incorporated into sodium phosphate and citrate buffer solutions, each prepared at 0.02 and 0.1 M and pH 4, 5, 6, and 7. Experimental solutions were stored in an incubator at 25 °C. Thiamin concentrations were analyzed at 9 time points over a 42‐wk period. Thiamin degradation data were modeled using pseudo‐1st‐order kinetics. Thiamin stability generally decreased as pH and buffer concentration increased in both phosphate and citrate buffers. In 0.1 M phosphate buffer, the time for a 10% loss of thiamin decreased from 79 wk at pH 4 to 3 wk at pH 7. At pH 6 and 7, thiamin stability was greater in citrate buffer than in phosphate buffer. However, at pH 4 and 5, thiamin stability was greater in phosphate buffer than citrate buffer. To optimize thiamin stability in low pH beverages, phosphate buffer would be more appropriate to use than citrate buffer, whereas citrate buffer should be used for better thiamin stability in high pH beverages.     

Exchanging the substrate specificities of pyruvate decarboxylase from Zymomonas mobilis and benzoylformate decarboxylase from Pseudomonas putida

Pyruvate decarboxylase from Zymomonas mobilis (PDC) and benzoylformate decarboxylase from Pseudomonas putida (BFD) are thiamine diphosphate-dependent enzymes that decarboxylate 2-keto acids. Although they share a common homotetrameric structure they have relatively low sequence similarity and different substrate spectra. PDC prefers short aliphatic substrates whereas BFD favours aromatic 2-keto acids. These preferences are also reflected in their carboligation reactions. PDC catalyses the conversion of benzaldehyde and acetaldehyde to (R)-phenylacetylcarbinol and predominantly (S)-acetoin, whereas (R)-benzoin and mainly (S)-2-hydroxypropiophenone are the products of BFD catalysis. Comparison of the X-ray structures of both enzymes identified two residues in each that were likely to be involved in determining substrate specificity. Site-directed mutagenesis was used to interchange these residues in both BFD and PDC. The substrate range and kinetic parameters for the decarboxylation reaction were studied for each variant. The most successful variants, PDCI472A and BFDA460I, catalysed the decarboxylation of benzoylformate and pyruvate, respectively, although both variants now preferred the long-chain aliphatic substrates, 2-ketopentanoic and 2-ketohexanoic acid. With respect to the carboligase activity, PDCI472A proved to be a real chimera between PDC and BFD whereas BFDA460I/F464I provided the most interesting result with an almost complete reversal of the stereochemistry of its 2-hydroxypropiophenone product.     

啤酒废酵母制备1,6-二磷酸果糖的研究

啤酒废酵母生产1,6-二磷酸果糖发酵工艺进行了试验性探讨。正交试验结果表明,啤酒废酵母发酵生产FDP的最佳工艺参数为:发酵体系中葡萄糖的加量为0.5mol/L、无机磷的浓度为0.4mol/L、pH6.5、转化时间6h。     

Global Concerns with B Vitamin Statuses: Biofortification,Fortification, Hidden Hunger,Interactions, and Toxicity

The prevalence of undernutrition due to insufficient energy intake has been reduced by nearly 50% since 1990. This reduction is largely attributed to improved yields of staple crops, such as wheat, rice, and maize; however, these improvements did little for micronutrient deficiencies that affect an estimated two billion people worldwide. Starchy staple crops are energy dense but are often lacking in one or more B vitamins, making resource‐constrained people who consume monotonous diets comprised predominantly of these staples at risk for developing deficiency. B vitamin deficiencies occur due to a poor overall nondiversified diet and rarely occur alone. Many B vitamins are essential cofactors involved in the metabolism of other nutrients, including other B vitamins, whereby the deficiency of one B vitamin affects the metabolism and status measurements of another. Food fortification efforts have nearly eradicated diseases of extreme B vitamin deficiency, such as beriberi from thiamin deficiency and pellagra from niacin deficiency. However, subclinical deficiency, sometimes referred to as hidden hunger, is still common especially in low‐income countries. Most dietary B vitamins, due to their water‐soluble nature, are not a concern for excessive intakes, but synthetic forms used for fortification and supplements sometimes can have adverse effects when consumed in high amounts. Biofortified crops offer a long‐term sustainable method to increase the amount of dietary B vitamins for people who rely on staple crops for most of their caloric intake. Efforts have been made to improve B vitamin content of crops, especially for thiamin, vitamin B₆, and folate, but none have undergone human feeding trials; therefore, more research is needed to provide sustainable and scalable solutions in many parts of the world.