Structure and Function of the α-Hydroxylation Bimodule of the Mupirocin Polyketide Synthase

Mupirocin is a clinically important antibiotic produced by a trans-AT Type I polyketide synthase (PKS) in Pseudomonas fluorescens. The major bioactive metabolite, pseudomonic acid A (PA−A), is assembled on a tetrasubstituted tetrahydropyran (THP) core incorporating a 6-hydroxy group proposed to be introduced by α-hydroxylation of the thioester of the acyl carrier protein (ACP) bound polyketide chain. Herein, we describe an in vitro approach combining purified enzyme components, chemical synthesis, isotopic labelling, mass spectrometry and NMR in conjunction with in vivo studies leading to the first characterisation of the α-hydroxylation bimodule of the mupirocin biosynthetic pathway. These studies reveal the precise timing of hydroxylation by MupA, substrate specificity and the ACP dependency of the enzyme components that comprise this α-hydroxylation bimodule. Furthermore, using purified enzyme, it is shown that the MmpA KS⁰ shows relaxed substrate specificity, suggesting precise spatiotemporal control of in trans MupA recruitment in the context of the PKS. Finally, the detection of multiple intermodular MupA/ACP interactions suggests these bimodules may integrate MupA into their assembly.     

Flavinium Catalysed Photooxidation: Detection and Characterization of Elusive Peroxyflavinium Intermediates

Flavin‐based catalysts are photoactive in the visible range which makes them useful in biology and chemistry. Herein, we present electrospray‐ionization mass‐spectrometry detection of short‐lived intermediates in photooxidation of toluene catalysed by flavinium ions (Fl⁺). Previous studies have shown that photoexcited flavins react with aromates by proton‐coupled electron transfer (PCET) on the microsecond time scale. For Fl⁺, PCET leads to FlH^.+ with the H‐atom bound to the N5 position. We show that the reaction continues by coupling between FlH^.+ and hydroperoxy or benzylperoxy radicals at the C4a position of FlH^.+. These results demonstrate that the N5‐blocking effect reported for alkylated flavins is also active after PCET in these photocatalytic reactions. Structures of all intermediates were fully characterised by isotopic labelling and by photodissociation spectroscopy. These tools provide a new way to study reaction intermediates in the sub‐second time range.     

Inhibition mechanism of flavin by deprenyl as an acetylenic irreversible inhibitor

Possible inhibition mechanisms of flavin (isoalloxazine) with (−)-deprenyl as an acetylenic irreversible inhibitor have been investigated in detail by ab initio methods with the 6-31G* basis set through the simplified model compounds 3-formyl-2-imino-1-hydropyrazine and propargylamine. The resulting compounds have been verified by calculations with the 3-21G basis set using flavin itself and the model of (−)-deprenyl for confirmation of the reactions through the simplified models. Two cyclic O4,N5- and C4a,N5-covalent adducts have been found. The latter was the most stable and was considered to be the final irreversible product. The intermediates in the reaction, the acyclic C4a- or N5-allenic compounds and their hydrogen-transferred cyanine-type compounds, are in agreement with the results of experimental photochemical reactions. In most of the reaction processes, hydrogen migration played an important role. Received: 26 June 1998 / Accepted: 28 August 1998 / Published online: 11 November 1998     

Modification of glassy carbon electrode with a polymer/mediator composite and its application for the electrochemical detection of iodate

A modified glassy carbon electrode was prepared by depositing a composite of polymer and mediator on a glassy carbon electrode (GCE). The mediator, flavin adenine dinucleotide (FAD) and the polymer, poly(3,4-ethylenedioxythiophene) (PEDOT) were electrochemically deposited as a composite on the GCE by applying cyclic voltammetry (CV). This modified electrode is hereafter designated as GCE/PEDOT/FAD. FAD was found to significantly enhance the growth of PEDOT. Electrochemical quartz crystal microbalance (EQCM) analysis was performed to study the mass changes in the electrode during the electrodeposition of PEDOT, with and without the addition of FAD. The optimal cycle number for preparing the modified electrode was determined to be 9, and the corresponding surface coverage of FAD (Γ_FAD) was ca. 5.11 × 10⁻¹⁰ mol cm⁻². The amperometric detection of iodate was performed in a 100 mM buffer solution (pH 1.5). The GCE/PEDOT/FAD showed a sensitivity of 0.78 μA μM⁻¹ cm⁻², a linear range of 4–140 μM, and a limit of detection of 0.16 μM for iodate. The interference effects of 250-fold Na⁺, Mg²⁺, Ca²⁺, Zn²⁺, Fe²⁺, Cl⁻, NO₃⁻, I⁻, SO₄²⁻ and SO₃²⁻, with reference to the concentration of iodate were negligible. The long-term stability of GCE/PEDOT/FAD was also investigated. The GCE/PEDOT/FAD electrode retained 82% of its initial amperometric response to iodate after 7 days. The GCE/PEDOT/FAD was also applied to determine iodate in a commercial salt.     

First example of a molecularly imprinted polymer incorporating a difunctionalized alloxazine flavin isomer

High-quality alloxazine (a flavin isomer) imprinted polymers have been made for the first time. A molecularly imprinted polymer (MIP) for the N1,N3-di-functionalized alloxazine template 2 was made. The MIP prepared for 2 exhibited excellent, highly selective molecular recognition for template 2, as determined by HPLC analysis using columns prepared with the MIP. This has also demonstrated that the core flavin structure can survive the imprinting process.     

On the influence of dimerisation of lumiflavin in aqueous solution on its optical spectra – a quantum chemical study

The solvent effects on the photophysical and photochemical properties of lumiflavin are studied. By comparing different models for the aqueous environment including gas-phase ab-initio calculations, explicit solvation, continuum solvation model and QM/MM simulations the different aspects of solvation are singled out and analysed in detail. Dimerisation of flavins and its effects on the absorption and emission spectrum of flavins are studied using quantum chemical methods. The absorption and emission spectra of lumiflavin in water are compared to the properties of dimeric lumiflavin and to available experimental data.     

Determination of acid dissociation constants of flavin analogues by capillary zone electrophoresis

Acid dissociation constants (pK_a) of nine kinds of flavin analogues as molecular catalyst candidates were determined by CZE. Although some of the analogues are instable and degradable under the light exposure or in alkaline aqueous solutions, the effective electrophoretic mobility of the flavin analogue of interest has been measured with the residual substance. The pK_a values of the flavin analogues were analyzed through the changes in the effective electrophoretic mobility with varying pH of the separation buffer. One or two steps pK_a values were determined by the analysis. One of the degraded species from the flavin analogues, lumichrome, was also detected in the CZE analysis, and its pK_a values were also determined. While coexisting impurities generated over the storage conditions were found in some analogues, the pK_a values of the target analogues were successfully determined with the help of the CZE separations. A pressure-assisted CZE was utilized for the determination or the estimation of the pK_a values of such analogues as possessing carboxylic acid moiety.