A pathogenesis-related 10 protein catalyzes the final step in thebaine biosynthesis.

The ultimate step in the formation of thebaine, a pentacyclic opiate alkaloid readily converted to the narcotic analgesics codeine and morphine in the opium poppy, has long been presumed to be a spontaneous reaction. We have detected and purified a novel enzyme from opium poppy latex that is capable of the efficient formation of thebaine from (7S)-salutaridinol 7-O-acetate at the expense of labile hydroxylated byproducts, which are preferentially produced by spontaneous allylic elimination. Remarkably, thebaine synthase (THS), a member of the pathogenesis-related 10 protein (PR10) superfamily, is encoded within a novel gene cluster in the opium poppy genome that also includes genes encoding the four biosynthetic enzymes immediately upstream. THS is a missing component that is crucial to the development of fermentation-based opiate production and dramatically improves thebaine yield in engineered yeast.

Efficient ethanol production from brown macroalgae sugars by a synthetic yeast platform.

The increasing demands placed on natural resources for fuel and food production require that we explore the use of efficient, sustainable feedstocks such as brown macroalgae. The full potential of brown macroalgae as feedstocks for commercial-scale fuel ethanol production, however, requires extensive re-engineering of the alginate and mannitol catabolic pathways in the standard industrial microbe Saccharomyces cerevisiae. Here we present the discovery of an alginate monomer (4-deoxy-L-erythro-5-hexoseulose uronate, or DEHU) transporter from the alginolytic eukaryote Asteromyces cruciatus. The genomic integration and overexpression of the gene encoding this transporter, together with the necessary bacterial alginate and deregulated native mannitol catabolism genes, conferred the ability of an S. cerevisiae strain to efficiently metabolize DEHU and mannitol. When this platform was further adapted to grow on mannitol and DEHU under anaerobic conditions, it was capable of ethanol fermentation from mannitol and DEHU, achieving titres of 4.6% (v/v) (36.2 g l(-1)) and yields up to 83% of the maximum theoretical yield from consumed sugars. These results show that all major sugars in brown macroalgae can be used as feedstocks for biofuels and value-added renewable chemicals in a manner that is comparable to traditional arable-land-based feedstocks.

Modulation of the mitotic regulatory network by APC-dependent destruction of the Cdh1 inhibitor Acm1.

The completion of mitosis depends on protein ubiquitination by the anaphase-promoting complex (APC). The APC is activated by association with Cdc20 in midmitosis and Cdh1 in late mitosis and G1. Here, we show that in budding yeast the activation of APC(Cdh1) is controlled in part by destruction of the Cdh1 inhibitor Acm1. We find that Acm1 uses pseudosubstrate and other sequence motifs to bind and inhibit Cdh1, but not Cdc20. Acm1 also contains a destruction sequence that promotes its ubiquitination by APC(Cdc20), resulting in the disappearance of Acm1 in early anaphase. Later in mitosis, Acm1 destruction is also promoted by APC(Cdh1). Finally, Cdk1-dependent phosphorylation of Acm1 modulates its localization and destruction. We conclude that ubiquitination of a Cdh1 inhibitor by APC(Cdc20) helps establish the order of activation of the two APC isoforms. We also speculate that the ability of APC(Cdh1) to target its own inhibitor enhances the bistability of the late mitotic regulatory system.

The APC subunit Doc1 promotes recognition of the substrate destruction box.

BACKGROUND: Accurate chromosome segregation during mitosis requires the coordinated destruction of the mitotic regulators securin and cyclins. The anaphase-promoting complex (APC) is a multisubunit ubiquitin-protein ligase that catalyzes the polyubiquitination of these and other proteins and thereby promotes their destruction. How the APC recognizes its substrates is not well understood. In mitosis, the APC activator Cdc20 binds to the APC and is thought to recruit substrates by interacting with a conserved target protein motif called the destruction box. A related protein, called Cdh1, performs a similar function during G1. Recent evidence, however, suggests that the core APC subunit Doc1 also contributes to substrate recognition. RESULTS: To better understand the mechanism by which Doc1 promotes substrate binding to the APC, we generated a series of point mutations in Doc1 and analyzed their effects on the processivity of substrate ubiquitination. Mutations that reduce Doc1 function fall into two classes that define spatially and functionally distinct regions of the protein. One region, which includes the carboxy terminus, anchors Doc1 to the APC but does not influence substrate recognition. The other region, located on the opposite face of Doc1, is required for Doc1 to enhance substrate binding to the APC. Importantly, stimulation of binding by Doc1 also requires that the substrate contain an intact destruction box. Cells carrying DOC1 mutations that eliminate substrate recognition delay in mitosis with high levels of APC substrates. CONCLUSIONS: Doc1 contributes to recognition of the substrate destruction box by the APC. This function of Doc1 is necessary for efficient substrate proteolysis in vivo.

Co-activator independent differences in how the metaphase and anaphase APC/C recognise the same substrate.

The Anaphase Promoting Complex or Cyclosome (APC/C) is critical to the control of mitosis. The APC/C is an ubiquitin ligase that targets specific mitotic regulators for proteolysis at distinct times in mitosis, but how this is achieved is not well understood. We have addressed this question by determining whether the same substrate, cyclin B1, is recognised in the same way by the APC/C at different times in mitosis. Unexpectedly, we find that distinct but overlapping motifs in cyclin B1 are recognised by the APC/C in metaphase compared with anaphase, and this does not depend on the exchange of Cdc20 for Cdh1. Thus, changes in APC/C substrate specificity in mitosis can potentially be conferred by altering interaction sites in addition to exchanging Cdc20 for Cdh1.