The origin recognition complex requires chromatin tethering by a hypervariable intrinsically disordered region that is functionally conserved from sponge to man.

The first step toward eukaryotic genome duplication is loading of the replicative helicase onto chromatin. This 'licensing' step initiates with the recruitment of the origin recognition complex (ORC) to chromatin, which is thought to occur via ORC's ATP-dependent DNA binding and encirclement activity. However, we have previously shown that ATP binding is dispensable for the chromatin recruitment of fly ORC, raising the question of how metazoan ORC binds chromosomes. We show here that the intrinsically disordered region (IDR) of fly Orc1 is both necessary and sufficient for recruitment of ORC to chromosomes in vivo and demonstrate that this is regulated by IDR phosphorylation. Consistently, we find that the IDR confers the ORC holocomplex with ATP-independent DNA binding activity in vitro. Using phylogenetic analysis, we make the surprising observation that metazoan Orc1 IDRs have diverged so markedly that they are unrecognizable as orthologs and yet we find that these compositionally homologous sequences are functionally conserved. Altogether, these data suggest that chromatin is recalcitrant to ORC's ATP-dependent DNA binding activity, necessitating IDR-dependent chromatin tethering, which we propose poises ORC to opportunistically encircle nucleosome-free regions as they become available.

UGT84F9 is the major flavonoid UDP-glucuronosyltransferase in Medicago truncatula.

Mammalian phase II metabolism of dietary plant flavonoid compounds generally involves substitution with glucuronic acid. In contrast, flavonoids mainly exist as glucose conjugates in plants, and few plant UDP-glucuronosyltransferase enzymes have been identified to date. In the model legume Medicago truncatula, the major flavonoid compounds in the aerial parts of the plant are glucuronides of the flavones apigenin and luteolin. Here we show that the M. truncatula glycosyltransferase UGT84F9 is a bi-functional glucosyl/glucuronosyl transferase in vitro, with activity against a wide range of flavonoid acceptor molecules including flavones. However, analysis of metabolite profiles in leaves and roots of M. truncatula ugt84f9 loss of function mutants revealed that the enzyme is essential for formation of flavonoid glucuronides, but not most flavonoid glucosides, in planta. We discuss the use of plant UGATs for the semi-synthesis of flavonoid phase II metabolites for clinical studies.

Glucuronidation of Methylated Quercetin Derivatives: Chemical and Biochemical Approaches.

Botanical supplements derived from grapes are functional in animal model systems for the amelioration of neurological conditions, including cognitive impairment. Rats fed with grape extracts accumulate 3'-O-methyl-quercetin-3-O-ß-d-glucuronide (3) in their brains, suggesting 3 as a potential therapeutic agent. To develop methods for the synthesis of 3 and the related 4'-O-methyl-quercetin-7-O-ß-d-glucuronide (4), 3-O-methyl-quercetin-3'-O-ß-d-glucuronide (5), and 4'-O-methyl-quercetin-3'-O-ß-d-glucuronide (6), which are not found in the brain, we have evaluated both enzymatic semisynthesis and full chemical synthetic approaches. Biocatalysis by mammalian UDP-glucuronosyltransferases generated multiple glucuronidated products from 4'-O-methylquercetin, and is not cost-effective. Chemical synthetic methods, on the other hand, provided good results; 3, 5, and 6 were obtained in six steps at 12, 18, and 30% overall yield, respectively, while 4 was synthesized in five steps at 34% overall yield. A mechanistic study on the unexpected regioselectivity observed in the quercetin glucuronide synthetic steps is also presented.