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The identification of a diterpene gene cluster is shown by using homology-based polymerase chain reaction (PCR) of geranylgeranyl diphosphate synthase (GGS) genes and the subsequent genome walking in the fungus Phomopsis amygdale N2. The structure determination of a novel diterpene hydrocarbon phomopsene provided by enzymatic synthesis with the recombinant terpene synthase PaPS and screening of fungal broth extracts with reference to NMR signals of phomopsene has helped in isolating a new diterpene, methyl phomopsenonate.

Highlights: [•] X-ray structure of P450 RauA in complex with aurachin RE intermediate is reported. [•] RauA exhibited high binding affinity to the aurachin RE intermediate. [•] The structure revealed aurachin binding mode enabling quinoline N-hydroxylation. [Copyright &y& Elsevier]

Proteasomes execute the degradation of most cellular proteins. Although the 205 core particle (CP) has been studied in great detail, the structure of the 195 regulatory particle (RP), which prepares ubiquitylated substrates for degradation, has remained elusive. Here, we report the crystal structure of one of the RP subunits, Rpn6, and we describe its integration into the cryo-EM density map of the 265 holocomplex at 9.1 A resolution. Rpn6 consists of an a-solenoid-like fold and a proteasome COP9/signalosome elF3 (PCI) module in a right-handed suprahelical configuration. Highly conserved surface areas of Rpn6 interact with the conserved surfaces of the Pre8 (alpha2) and Rpt6 subunits from the alpha and ATPase rings, respectively. The structure suggests that Rpn6 has a pivotal role in stabilizing the otherwise weak interaction between the CP and the RP. [ABSTRACT FROM AUTHOR]

Abstract: Vitamin D3 (VD3) is a fat-soluble prohormone in mammals. VD3 is inert and must be activated by hydroxylation at the C-25 and C-1α positions to exert its biological activity. We recently accomplished the bioconversion of VD3 to 25(OH)VD3 with a recombinant strain of Rhodococcus erythropolis and found that the permeability of VD3 into the cytoplasm may be the rate-limiting step of 25(OH)VD3 production (Sallam et al., 2010). When the cells were treated with the lipid II-targeting lantibiotic nisin, the permeability of green chemiluminescent cyclodextrin (GCCD), which is used as a model substrate instead of VD3-partially methylated-β-cyclodextrin (PMCD) complex, was drastically induced. Nisin also induced VD3 hydroxylation, and the rate was correlated with the expression levels of Vdh and its redox partner proteins. In the bioconversion reaction, the stability of the redox partner proteins and the additional NADH-regenerating system are crucial for VD3 hydroxylation. The degradation rate of the [2Fe–2S] cluster of ferredoxin ThcC from R. erythropolis NI86/21 is faster than that of AciB from Acinetobacter sp. OC4. Therefore, the nisin-treated R. erythropolis cells coexpressing Vdh and AciBC (1176.5μg) exhibited much greater 25(OH)VD3 production than the cells coexpressing Vdh and ThcCD (431.7μg) after four consecutive 16h reactions. These results suggest that nisin forms nisin-lipid II pore complexes in the Rhodococcus membrane that increase the accessibility of VD3–PMCD complexes to the inside of the cells. Furthermore, nisin-treated Rhodococcus cells can be utilized for the bioconversion of other fat-soluble chemicals. [Copyright &y& Elsevier]

The D-aldohexose dehydrogenase from the thermoacidophilic archaeon Thermoplasma acidophilum (AldT) is a homotetrameric enzyme that catalyzes the oxidation of several D-aldohexoses, especially D-mannose. AldT comprises a unique C-terminal tail motif (residues 247-255) that shuts the active-site pocket of the neighboring subunit. The functional role of the C-terminal tail of AldT has been investigated using mutational and crystallographic analyses. A total of four C-terminal deletion mutants (Δ254, Δ253, Δ252, and Δ249) and two site-specific mutants (Y86G and P254G) were expressed by Escherichia coli and purified. Enzymatic characterization of these mutants revealed that the C-terminal tail is a requisite and that the interaction between Tyr86 and Pro254 is critical for enzyme activity. The crystal structure of the Δ249 mutant was also determined. The structure showed that the active-site loops undergo a significant conformational change, which leads to the structural deformation of the substrate-binding pocket. Proteins 2009. © 2008 Wiley-Liss, Inc. [ABSTRACT FROM AUTHOR]