A cDNA encoding a cytochrome P450-dependent oxidase, berbamunine synthase (EC 1.1.3.34; CYP80), from cell suspension cultures of the higher plant Berberis stolonifera Koehne and Wolf (barberry) has been isolated and heterologously expressed in functional form in insect cell culture using a baculovirus-based expression system. This cytochrome P450-dependent enzyme is unusual in that it catalyzes the regio-and stereoselective formation of a C-0 phenol couple in bisbenzylisoquinoline alkaloid biosynthesis without concomitant incorporation of activated oxygen into the product. Consistent with the function of an oxidase rather than a monooxygenase, an essential glycine residue in the distal helix, which forms the oxygen-binding pocket in the well-studied bacterial enzyme P-450cam, is replaced by proline at the equivalent position in berbamunine synthase. This oxidase was accumulated in an active form in insect cell microsomes and accepted electrons from the endogenous NADPH-cytochrome P450 reductase. The heterologously expressed enzyme oxidatively couples either two molecules of (R)-N-methylcoclaurine to form the (R,R) dimer guattegaumerine or one molecule each of (R)-and (S)-Nmethylcoclaurine to form the (R,S) dimer berbamunine. The ratio of the two bisbenzylisoquinolines formed could be altered by reductase source or by varying the enantiomer composition of the substrates.

Inactivation of the Bacillus subtilis sspF gene had no effect on sporulation, spore resistance, or germination in a wild-type strain or one lacking DNA protective ␣/␤-type small, acid-soluble proteins (SASP). Overexpression of SspF in wild-type spores or in spores lacking major ␣/␤-type SASP (␣ ؊ ␤ ؊ spores) had no effect on sporulation but slowed spore outgrowth and restored a small amount of UV and heat resistance to ␣ ؊ ␤ ؊ spores. In vitro analyses showed that SspF is a DNA binding protein and is cleaved by the SASP-specific protease (GPR) at a site similar to that cleaved in ␣/␤-type SASP. SspF was also degraded during spore germination and outgrowth, and this degradation was initiated by GPR.The Bacillus subtilis sspF gene (originally termed 0.3 kb) is highly transcribed only in the forespore compartment of sporulating cells, but SspF has not been identified in spores (9, 18). The sspF gene codes for a 61-residue protein which shows some sequence homology to small, acid-soluble spore proteins (SASP) of the ␣/␤ type (16). Spores of all Bacillus species examined to date contain multiple members of the ␣/␤-type SASP family (so named for the two major members of this family in B. subtilis, ␣ and ␤) which exhibit an average of 67% sequence identity in pairwise comparisons either within species or among Bacillus cereus, Bacillus megaterium, and B. subtilis (17). The ␣/␤-type SASP are bound to spore DNA in vivo and play important roles in spore resistance to heat, radiation, and chemicals such as hydrogen peroxide, although the third major SASP in B. subtilis spores, ␥, plays no role in spore resistance (17); SASP-␥ is also quite different from ␣/␤-type SASP and SspF in amino acid sequence (16). The divergence of B. subtilis SspF's amino acid sequence from those of ␣/␤-type SASP (14% identity and 48% similarity) indicates that sspF is a rather distant relative of ␣/␤-type SASP. However, the SspFs of B. cereus, B. megaterium, and B. subtilis exhibit an average of 74% sequence identity in pairwise comparisons (7); this high degree of sequence conservation is consistent with some role for SspF in sporulation or spore properties. In order to assess this possibility, we have analyzed the sporulation, spore properties, and spore germination and outgrowth of B. subtilis strains with an inactivated sspF gene or expressing high levels of SspF in developing forespores and have also analyzed some properties of SspF.In order to inactivate the sspF gene, the 2-kb EcoRI fragment containing sspF from plasmid p213-1 (9) was isolated, and the ends were filled in with the large fragment of Escherichia coli DNA polymerase and cloned into the SmaI site of plasmid pUC19 in E. coli. This plasmid (pPS2367) was cut with ApaI and SmaI to remove a 0.1-kb fragment encompassing the carboxyl-terminal half of the sspF coding region (9). The remaining 4.6-kb fragment was ligated with a 1.2-kb ApaIBamHI fragment from pJL73 (6) containing the spectinomycin gene in which the BamHI end had been filled in, and the ligation mix was used to transform...