The biogenesis of the topaquinone (TPQ) cofactor of copper amine oxidase (CAO) is self-catalyzed and requires copper and molecular oxygen. A dopaquinone intermediate has been proposed to undergo 1,4-addition of a copper-associated water molecule to form the reduced form of TPQ (TPQ(red)), followed by facile oxidation by O(2) to yield the mature TPQ (TPQ(ox)). In this study, we have incorporated a lysine residue in the active site of Arthrobacter globiformis CAO (AGAO) by site-directed mutagenesis to produce D298K-AGAO. The X-ray crystal structure of D298K-AGAO at 1.7-A resolution revealed that a covalent linkage formed between the epsilon-amino side chain of Lys298 and the C2 position of a dopaquinone derived from Tyr382, a precursor to TPQ(ox). We assigned the species as an iminoquinone tautomer (LTI) of lysine tyrosylquinone (LTQ), the organic cofactor of lysyl oxidase (LOX). The time course of the formation of LTI at pH 6.8 was followed by UV/vis and resonance Raman spectroscopies. In the early phase of the reaction, an LTQ-like intermediate was observed. This intermediate then slowly converted to LTI in an isosbestic manner. Not only is the presence of a dopaquinone intermediate in the TPQ biogenesis confirmed, but it also provides strong support for the proposed intermediacy of a dopaquinone in the biogenesis of LTQ in LOX. Further, this study indicates that the dopaquinone intermediate in AGAO is mobile and can swing from the copper site into the active-site wedge to react with Lys298.
On incubation with selected micro-organisms mycophenolic acid (1) has been found to undergo one or more of the following transformations: (a) oxygenation of the 4-methyl group giving an alcohol (5) and an aldehyde (6). ( 6 ) oxygenation of the 3'-methyl group and lactonisation giving a S-lactone (31). (c) oxidation at C-3 giving a lactol (3). ( d ) loss of the double bond and oxidation at C -4 , giving a P-hydroxy-acid (1 8). a methyl ketone (1 4). a carboxylic acid (1 0). and, by further hydroxylation, a lactol (1 l ) , a benzyl alcohol (1 2). and a dihydrobenzofuran (29), (e) oxygenation of the double bond giving a hydroxy-lactone (21). ( f ) oxygenation and cyclisation of the terpenoid substituent giving dihydrobenzofurans (26) and ( 28). (g) oxidative cyclisation of the terpenoid substituent giving a chromen (30). and ( h ) combination of the carboxy-group with the amino-groups of glycine and alanine giving amides (1 5) and (1 6). Lactones (20) and ( 22) and diphenylmethane (32) were also obtained from mycophenolic acid fermentations but these compounds may have been artefacts generated during work-up.Fermentations of mycophenolic acid methyl ether (21 and of comlsound (9) gave the benzyl alcohols (7) and (5) respectively, and fermenta'tions of dihydromycophenolic acid gave compounds (1 0). (1 l ) , and (29). The structures of the fermentation products have been established by spectroscopic methods and most of the compounds have been chemically related to mycophenolic acid. The biogenesis of the fermentation products is discussed and some preparations of cyclic ethers from mycophenolic acid derivatives are described.MYCOPHENOLIC ACID (l), a metabolite of several species of Peaicillium, was first isolated in 1896.1 Structure (1) was suggested in 1952 and confirmed in 1957. A total synthesis was reported in 1969. Recently mycophenolic acid has been shown to possess anti-tumour properties. As a way of making analogues of this compound for evaluation as anti-tumour drugs we have investigated its transformation by micro-organisms. More than five hundred micro-organisms, including fungi, yeasts, bacteria, streptomycetes, algae, and protozoa, were examined. Twenty-one micro-organisms modified mycophenolic acid, giving the products listed in Table 1. Compounds (21) and (30) had previously been obtained from a mycophenolic acid-producing strain of Pefiicillium brevicom$actum.Attention was concentrated initially on transformation products which retained the phenolic hydroxy-group, and material having the appropriate RP value was assumed to be unchanged substrate. Consequently it is possible that compound (10), whose cl~romatographic
An analysis of the structures of ®ve new cocrystals with saturated hydrogen bonding between amines and alcohols is presented. These ®ve are all cocrystals with p-phenylenediamine (PDA) and a mono-or diol. The cocrystals are. These crystals have two distinctly different supramolecular hydrogen-bond patterns, when considering only the hydroxyl and amine groups. There are, in addition, variations in the ways covalently bonded crosslinks connect these hydrogen-bond networks. The graph sets of the hydrogen-bond networks of these and other published saturated amine±alcohol crystals are compared and suggestions made on how to present the graph-set descriptions, especially those involving in®nite two-and three-dimensional networks of intersecting chains which are characteristic of most of these materials.
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