The tandem of free radicals and methylglyoxal.

Mechanism of action of glutathione-dependent enzymes.

The individual steps of citrinin 1 biosynthesis in Monascus ruber M7 were determined by a combination of targeted gene knockout and heterologous gene expression in Aspergillus oryzae. The pathway involves the synthesis of an unreduced trimethylated pentaketide 10 by a non-reducing polyketide synthase (nrPKS) known as CitS. Reductive release yields the keto-aldehyde 2 as the first enzyme-free intermediate. The nrPKS appears to be assisted by an as-yet cryptic hydrolysis step catalysed by CitA which was previously wrongly annotated as an oxidase. CitB is a non-heme iron oxidase which oxidises the 12-methyl of 2 to an alcohol. Subsequent steps are catalysed by CitC which oxidises the 12-alcohol to an aldehyde and CitD which converts the 12-aldehyde to a carboxylic acid. Final reduction of C-3 by CitE yields citrinin. The pathway rules out alternatives involving intramolecular rearrangements, and fully defines the molecular steps for the first time and corrects previous errors in the literature. The activity of CitB links the pathway to fungal tropolone biosynthesis and the observation of aminated shunt products links the pathway to azaphilone biosynthesis. Production of citrinin by coordinated production of CitS + CitA–CitE in the heterologous host A. oryzae, in which each gene was driven by a constitutive promoter, was achieved in high yield.

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The molecular steps of citrinin biosynthesis in fungi

Publisher Summary This chapter introduces the concept of effective charge in its application to reactions in solution. A case is not being pressed for the application of charge derived from polar effects as the only quantity, whereby information is obtained about bonding; rather it is recognized that polar substituent effects on rates and equilibria in solution are easily and reliably obtained, and they constitute one of our most powerful mechanistic tools for studies of reactions in solution. The methods for charge measurement—namely, 13C nmr and esr spectroscopy—are only for ground or product states and the data refer to the solvated species; the field awaits comparative work. Knowledge of the development of charge on a substrate atom during catalysis is very important for understanding; its interpretation depends on the charge change in suitable calibration equilibria for the complexation process and also for the process occurring in the complex. These calibrating values are not generally available, but very early studies of the acylation of hydrolytic enzymes by substrates indicated that negative charge development on the leaving group atom is not as pronounced as in simple model reactions in aqueous solvent.

Effective Charge and Transition-state Structure in Solution

Glutathione was assessed individually, and in combination with ascorbic acid, for its ability to act as an antioxidant with respect to color development in an oxidizing model white wine system. Glutathione was utilized at concentrations normally found in wine (30 mg/L), as well as at concentrations 20-fold higher (860 mg/L), the latter to afford ascorbic acid (500 mg/L) to glutathione ratios of 1:1. The model wine systems were stored at 45 °C without sulfur dioxide and at saturated oxygen levels, thereby in conditions highly conducive to oxidation. Under these conditions the results demonstrated the higher concentration of glutathione could initially provide protection against oxidative coloration, but eventually induced color formation. In the period during which glutathione offered a protective effect, the production of xanthylium cation pigment precursors and o-quinone-derived phenolic compounds was limited. When glutathione induced coloration, polymeric pigments were formed, but these were different from those found in model wine solutions without glutathione. In the presence of ascorbic acid, high concentrations of glutathione were able to delay the decay in ascorbic acid and inhibit the reaction of ascorbic acid degradation products with the wine flavanol compound (+)-catechin. However, on depletion, the glutathione again induced the production of a range of different polymeric pigments. These results highlight new mechanisms through which glutathione can offer both protection and spoilage during the oxidative coloration of a model wine.

Marc S. Kanchuger

Papers

Acyl substituent effects on thiohemiacetal equilibriums

Interaction of the substrate phosphate substituent with glyceraldehyde-3-phosphate dehydrogenase.