Monooxygenation of aromatic compounds by flavin‐dependent monooxygenases

Many flavoenzymes catalyze hydroxylation of aromatic compounds especially phenolic compounds have been isolated and characterized. These enzymes can be classified as either single‐component or two‐component flavin‐dependent hydroxylases (monooxygenases). The hydroxylation reactions catalyzed by the enzymes in this group are useful for modifying the biological properties of phenolic compounds. This review aims to provide an in‐depth discussion of the current mechanistic understanding of representative flavin‐dependent monooxygenases including 3‐hydroxy‐benzoate 4‐hydroxylase (PHBH, a single‐component hydroxylase), 3‐hydroxyphenylacetate 4‐hydroxylase (HPAH, a two‐component hydroxylase), and other monooxygenases which catalyze reactions in addition to hydroxylation, including 2‐methyl‐3‐hydroxypyridine‐5‐carboxylate oxygenase (MHPCO, a single‐component enzyme that catalyzes aromatic‐ring cleavage), and HadA monooxygenase (a two‐component enzyme that catalyzes additional group elimination reaction). These enzymes have different unique structural features which dictate their reactivity toward various substrates and influence their ability to stabilize flavin intermediates such as C4a‐hydroperoxyflavin. Understanding the key catalytic residues and the active site environments important for governing enzyme reactivity will undoubtedly facilitate future work in enzyme engineering or enzyme redesign for the development of biocatalytic methods for the synthesis of valuable compounds.     

Serological response to the 2009 pandemic influenza A (H1N1) virus for disease diagnosis and estimating the infection rate in Thai population

Background

Individuals infected with the 2009 pandemic virus A(H1N1) developed serological response which can be measured by hemagglutination-inhibition (HI) and microneutralization (microNT) assays.

Methodology/Principal Findings

MicroNT and HI assays for specific antibody to the 2009 pandemic virus were conducted in serum samples collected at the end of the first epidemic wave from various groups of Thai people: laboratory confirmed cases, blood donors and health care workers (HCW) in Bangkok and neighboring province, general population in the North and the South, as well as archival sera collected at pre- and post-vaccination from vaccinees who received influenza vaccine of the 2006 season. This study demonstrated that goose erythrocytes yielded comparable HI antibody titer as compared to turkey erythrocytes. In contrast to the standard protocol, our investigation found out the necessity to eliminate nonspecific inhibitor present in the test sera by receptor destroying enzyme (RDE) prior to performing microNT assay. The investigation in pre-pandemic serum samples showed that HI antibody was more specific to the 2009 pandemic virus than NT antibody. Based on data from pre-pandemic sera together with those from the laboratory confirmed cases, HI antibody titers ≥40 for adults and ≥20 for children could be used as the cut-off level to differentiate between the individuals with or without past infection by the 2009 pandemic virus.

Conclusions/Significance

Based on the cut-off criteria, the infection rates of 7 and 12.8% were estimated in blood donors and HCW, respectively after the first wave of the 2009 influenza pandemic. Among general population, the infection rate of 58.6% was found in children versus 3.1% in adults.     

Optimised separation procedures for the simultaneous assay of three plant hormones in liquid biofertilisers

Introduction – The overuse of petrochemical‐based synthetic fertilisers has caused detrimental effects to soil, water supplies, foods and animal health. This, in addition to increased awareness of organic farming, has generated considerable interest in the evaluation of renewable biofertilisers. Objective – The three objectives of the current research were: (1) to evaluate and optimise a solid phase extraction procedure for extraction of three plant hormones, IAA, GA₃ and ABA from two model biofertilisers produced from coconut shells and pineapple peels; (2) to develop an HPLC analysis procedure for the simultaneous separation and quantification of three plant hormones (IAA, GA₃ and ABA); and (3) to evaluate the changes in three plant hormones levels at four different fermentation time periods and varying number of general bacteria, lactic acid bacteria and yeast. Result – An optimised procedure for sample preparation, separation and simultaneous analysis of three plant hormones [indole‐3‐acetic acid (IAA), gibberellic acid (GA₃) and abscisic acid (ABA)] produced in liquid biofertilisers was developed. This method involves sample cleanup using a Sep‐pack Oasis^®MAX cartridge containing mixed‐mode anion‐exchange and reverse‐phase sorbents that provided optimum recovery of 85.6, 91.9 and 94.3%, respectively, for the three hormones, IAA, GA₃, and ABA. Baseline separation of three hormones was achieved using mobile phase consisting of 1% acetic acid and acetonitrile (75:25, v/v) at pH 4.0. The amounts of hormones produced in liquid biofertilisers were influenced by fruit types, fermentation time and total number of general bacteria, lactic acid bacteria and yeasts. The quantities of three plant hormones produced during fermentation correlated well with the total number of microorganisms present in the liquid biofertilisers. Conclusion – A simple and rapid sample preparation procedure followed by RP‐HPLC with UV detection was optimised and developed for simultaneous quantification and identification of three plant hormones namely, IAA, GA₃ and ABA in the liquid biofertilisers. This procedure allows quantification of the three plant hormones in their natural states without any prior derivatisation step. The results presented illustrate that the contents of the three plant hormones depended on the type of fruit wastes, fermentation time and the number of microorganisms found in liquid biofertilisers. This method can be extended to determine the quantity of three hormones in other matrices. This assay procedure will aid in the development of liquid biofertilisers, a valuable alternative fertilisers to promote plant growth. This process will help farmers to reduce production cost and pollution problems. Copyright © 2009 John Wiley & Sons, Ltd.     

Stabilization of C4a-hydroperoxyflavin in a two-component flavin-dependent monooxygenase is achieved through interactions at flavin N5 and C4a atoms

p-Hydroxyphenylacetate (HPA) 3-hydroxylase is a two-component flavin-dependent monooxygenase. Based on the crystal structure of the oxygenase component (C₂), His-396 is 4.5 Å from the flavin C4a locus, whereas Ser-171 is 2.9 Å from the flavin N5 locus. We investigated the roles of these two residues in the stability of the C4a-hydroperoxy-FMN intermediate. The results indicated that the rate constant for C4a-hydroperoxy-FMN formation decreased ∼30-fold in H396N, 100-fold in H396A, and 300-fold in the H396V mutant, compared with the wild-type enzyme. Lesser effects of the mutations were found for the subsequent step of H₂O₂ elimination. Studies on pH dependence showed that the rate constant of H₂O₂ elimination in H396N and H396V increased when pH increased with pK_a >9.6 and >9.7, respectively, similar to the wild-type enzyme (pK_a >9.4). These data indicated that His-396 is important for the formation of the C4a-hydroperoxy-FMN intermediate but is not involved in H₂O₂ elimination. Transient kinetics of the Ser-171 mutants with oxygen showed that the rate constants for the H₂O₂ elimination in S171A and S171T were ∼1400-fold and 8-fold greater than the wild type, respectively. Studies on the pH dependence of S171A with oxygen showed that the rate constant of H₂O₂ elimination increased with pH rise and exhibited an approximate pK_a of 8.0. These results indicated that the interaction of the hydroxyl group side chain of Ser-171 and flavin N5 is required for the stabilization of C4a-hydroperoxy-FMN. The double mutant S171A/H396V reacted with oxygen to directly form the oxidized flavin without stabilizing the C4a-hydroperoxy-FMN intermediate, which confirmed the findings based on the single mutation that His-396 was important for formation and Ser-171 for stabilization of the C4a-hydroperoxy-FMN intermediate in C₂.     

Influenza Neuraminidase Subtype N1: Immunobiological Properties and Functional Assays for Specific Antibody Response

Influenza neuraminidase (NA) proteins expressed in TK⁻ cells infected with recombinant vaccinia virus carrying NA gene of highly pathogenic avian influenza H5N1 virus or 2009 pandemic H1N1 (H1N1pdm) virus were characterized for their biological properties, i.e., cell localization, molecular weight (MW), glycosylation and sialidase activity.Immune sera collected from BALB/c mice immunized with these recombinant viruses were assayed for binding and functional activities of anti-NA antibodies. Recombinant NA proteins were found localized in cytoplasm and cytoplasmic membrane of the infected cells. H1N1pdm NA protein had MW at about 75 kDa while it was 55 kDa for H5N1 NA protein. Hyperglycosylation was more pronounced in H1N1pdm NA compared to H5N1 NA according to N-glycosidase F treatment. Three dimensional structures also predicted that H1N1 NA globular head contained 4 and that of H5N1 contained 2 potential glycosylation sites. H5N1 NA protein had higher sialidase activity than H1N1pdm NA protein as measured by both MUNANA-based assay and fetuin-based enzyme-linked lectin assay (ELLA). Plaque reduction assay demonstrated that anti-NA antibody could reduce number of plaques and plaque size through inhibiting virus release, not virus entry. Assay for neuraminidase-inhibition (NI) antibody by ELLA showed specific and cross reactivity between H5N1 NA and H1N1pdm NA protein derived from reverse genetic viruses or wild type viruses. In contrast, replication-inhibition assay in MDCK cells showed that anti-H1N1 NA antibody moderately inhibited viruses with homologous NA gene only, while anti-H5N1 NA antibody modestly inhibited the replication of viruses containing homologous NA gene and NA gene derived from H1N1pdm virus. Anti-H1N1 NA antibody showed higher titers of inhibiting virus replication than anti-H5N1 NA antibody, which are consistent with the results on reduction in plaque numbers and sizes as well as in inhibiting NA enzymatic activity. No assay showed cross reactivity with reassorted PR8 (H1N1) virus and H3N2 wild type viruses.     

A novel pathogenic mechanism of highly pathogenic avian influenza H5N1 viruses involves hemagglutinin mediated resistance to serum innate inhibitors

In this study, the effect of innate serum inhibitors on influenza virus infection was addressed. Seasonal influenza A(H1N1) and A(H3N2), 2009 pandemic A(H1N1) (H1N1pdm) and highly pathogenic avian influenza (HPAI) A(H5N1) viruses were tested with guinea pig sera negative for antibodies against all of these viruses as evaluated by hemagglutination-inhibition and microneutralization assays. In the presence of serum inhibitors, the infection by each virus was inhibited differently as measured by the amount of viral nucleoprotein produced in Madin-Darby canine kidney cells. The serum inhibitors inhibited seasonal influenza A(H3N2) virus the most, while the effect was less in seasonal influenza A(H1N1) and H1N1pdm viruses. The suppression by serum inhibitors could be reduced by heat inactivation or treatment with receptor destroying enzyme. In contrast, all H5N1 strains tested were resistant to serum inhibitors. To determine which structure (hemagglutinin (HA) and/or neuraminidase (NA)) on the virus particles that provided the resistance, reverse genetics (rg) was applied to construct chimeric recombinant viruses from A/Puerto Rico/8/1934(H1N1) (PR8) plasmid vectors. rgPR8-H5 HA and rgPR8-H5 HANA were resistant to serum inhibitors while rgPR8-H5 NA and PR8 A(H1N1) parental viruses were sensitive, suggesting that HA of HPAI H5N1 viruses bestowed viral resistance to serum inhibition. These results suggested that the ability to resist serum inhibition might enable the viremic H5N1 viruses to disseminate to distal end organs. The present study also analyzed for correlation between susceptibility to serum inhibitors and number of glycosylation sites present on the globular heads of HA and NA. H3N2 viruses, the subtype with highest susceptibility to serum inhibitors, harbored the highest number of glycosylation sites on the HA globular head. However, this positive correlation cannot be drawn for the other influenza subtypes.