1994). Transcription of the SpaC protein started in the late logarithmic growth phase, reaching a maximum in the early stationary growth phase. No SpaC was detectable in the early logarithmic growth phase. Deletions within the spaR and spaK genes, which act as a two-component regulatory system, resulted in failure to express SpaB and SpaC, indicating that these two genes are the regulatory targets. Western blot analysis of vesicle preparations of B. subtilis revealed that the SpaB, SpaT, and SpaC proteins are membrane bound, although some of the protein was also detectable in cell extracts. By using the yeast two-hybrid analysis system for protein interactions, we showed that a complex of at least two each of SpaT, SpaB, and SpaC is most probably associated with the substrate SpaS. These results were also confirmed by coimmunoprecipitation experiments. In these cosedimentation experiments, SpaB and SpaC were coprecipitated by antisera against SpaC, SpaB, and SpaT, as well as by a monoclonal antibody against epitope-tagged SpaS, indicating that these four proteins are associated.Lantibiotics such as subtilin, nisin, and epidermin are ribosomally synthesized peptide antibiotics. They contain the unusual amino acids meso-lanthionine, dehydrobutyrine, and dehydroalanine (36). The major characteristic of lantibiotics is the occurrence of the thioether amino acids meso-lanthionine and 3-methyllanthionine, which are generated during maturation of the lantibiotic prepeptides. Lantibiotics can be divided into two subgroups (19): (i) linear lantibiotics, including subtilin (16), nisin (28, 32), epidermin (1, 2), gallidermin (20), and Pep5 (33), and (ii) globular lantibiotics, including cinnamycin (RoO9-0198 or lanthiopeptin) (6, 21, 30), duramycin (15), and ancovenin (40).Epidermin and gallidermin are potentially applicable in the treatment of acne disease because of their high activity against Propionibacterium acnes. Subtilin is very similar to nisin, which is the most important member of the group of linear lantibiotics. Since the realization of the mutagenic effect of nitrite, which is used against clostridia in canned food, there has been an increasing interest in the use of nisin as a food preservative. The main difficulty in exploiting lantibiotics commercially is the low production rates. To overcome this problem, the biosynthesis of lantibiotics has to be elucidated.The structural genes of linear lantibiotics encode prepeptides which consist of an N-terminal leader sequence followed by the C-terminal propeptide from which the lantibiotics mature (36). Several genes essential for the synthesis of the lantibiotics subtilin (5, 23, 24, 25), epidermin (4, 35), and nisin (10, 38) have been identified. Sequence analysis of the DNA regions adjacent to the subtilin structural gene spaS revealed eight open reading frames (13). The genes spaB, spaT, and spaC are located upstream of spaS. Downstream to the spaS gene, spaI, spaF, spaG, spaR, and spaK were identified. All of these genes have been shown to be similar to respe...

A green enzyme from Clostridium aminovalericum with valeryl-CoA dehydrogenase activity was purified to homogeneity (169 5 3 kDa) and crystallized. By SDS/PAGE, one type of subunit (42 kDa) was detected indicating a homotetrameric structure. The unusual ultraviolet/visible spectrum of the green enzyme (maxima at 394 nm, 438 nm and 715 nm) was converted to a normal flavoprotein spectrum either by reduction with dithionite and reoxidation under air, or by removal of the prosthetic group at pH 2 and reconstitution with FAD (not FMN). Besides FAD (4 mo1/169 kDa), the enzyme contained 4 mol of a CoA ester which was similar but not identical to 5-hydroxy-2-pentenoyl-CoA. The reconstituted holoenzyme as well as the native green enzyme, but not the apoenzyme, catalysed the reversible dehydration of 5-hydroxyvaleryl-CoA to 4-pentenoyl-CoA in the absence of an external electron acceptor. In its presence (preferentially ferricenium ion), the green or yellow enzyme catalysed the formation of (@-5-hydroxy-2-pentenoyl-CoA and 2,4-pentadienoyl-CoA either from 4-pentenoyl-CoA or from 5-hydroxyvaleryl-CoA. The reversible hydration of 2,4-pentadienoyl-CoA to (@-5-hydroxy-2-pentenoylCoA was mediated by both enzymes as well as by the apoenzyme in the absence of FAD. Hydration of 4-pentenoate in 2 H z 0 yielded optically active 5-hydro~y[2,4-~H~]valerate by the combined action of 5-hydroxyvalerate CoAtransferase, the green dehydratase and catalytical amounts of acetyl-CoA. The data show that the reversible hydration of the isolated double bond of 4-pentenoyl-CoA to 5-hydroxyvaleryl-CoA, which apparently violates the Markovnikov rule, is preceded by oxidation to 2,4-pentadienoyl-CoA. The latter compound, a vinyl analogue of 2-enoyl-CoA, is then easily hydrated to (E)-S-hydroxy-2-pentenoyl-CoA and finally reduced to 5-hydroxyvaleryl-Co A.

2,CPentadienoyl-CoA reductase from Clostridium aminovalericum was purified to homogeneity (170 -182 kDa). PAGE in the presence of SDS revealed a single band (44 kDa) indicating a homotetrameric structure. The native enzyme had a green colour and contained 0.4 mol FAD/subunit. Its unusual ultraviolet/visiblespectrum showed absorption maxima at 270,402 and 715 nm as well as shoulders at 278, 360,450 and 500 nm. Removal of the prosthetic group at pH 2 in the presence of salt and charcoal yielded a colourless and completely inactive apoenzyme, which could be reconstituted with FAD (not with FMN) to an active holoenzyme showing a normal flavoprotein spectrum (peaks at 369 nm and 436 nm). Thereby the FAD content increased to 0.9 mol/ subunit with a concomitant rise in activity to 200% of the original value. Anaerobic reduction of the green enzyme by dithionite and reoxidation by air afforded a green preparation with a spectrum similar to that of the native enzyme. Addition of excess FAD to the green reductase also increased the activity by a factor of two.The green enzyme catalysed the oxidation of (E)-3-pentenoyl-CoA or (E)-3-hexenoyl-CoA to 2,4-pentadienoylCoA or 2,4-hexenoyl-CoA, respectively. 2-Pentenoyl-CoA or 4-pentenoyl-CoA were not oxidised. Meldola blue (8-dimethylamino-2,3-benzophenoxazine) and 2-(4-iodophenyl)-3-(4-nitrophenyl)-5-phenyltetrazolium chloride ( V = 26 nkat/mg protein) or ferricenium hexafluorophosphate ( V = 1900 nkat/mg), but not NAD(P), served as electron acceptors. Reduction of 2,4-pentadienoyl-CoA ( V = 370 nkat/mg) was observed with reduced benzyl viologen, but not with NAD(P)H as an electron donor. Although the enzyme had some pentenoyl-CoA A-isomerase activity (1.2 nkat/mg), the only product of the reduction was 3-pentenoyl-CoA rather than 2-pentenoylCoA.

5-Hydroxyvalerate CoA-transferase fromClostridium aminovalericum, strainT2-7, was purified approximately 100-fold to homogeneity. The molecular mass of the native enzyme was determined by three different methods to be 178 ± 11 kDa; that of the subunit was 47 kDa, indicating a homotetrameric structure. The following Co A esters acted as substrates (decreasing specificity, V/K m ): 5-hydroxyvaleryl-CoA > propionyl-CoA > acetyl-CoA > (Z)-5-hydroxy-2-pentenoyl-CoA > butyryl-CoA > valeryl-CoA. 4-Pentenoate and 3-pentenoate were also activated by acetyl-CoA to the corresponding CoAesters, whereas crotonate, (£)-5-hydroxy-2-pentenoate, (E)-2-pentenoate and 2,4-pentadienoate were not attacked. 5-Hydroxyvalerate CoA-transferase showed ping-pong kinetics and was inactivated by sodium boranate only in the presence of a CoA substrate. This indicated the formation of a thiolester between a specific carboxyl group of the enzyme and Co ASH during the course of the reaction. The CoAtransferase was inhibited by ATP and CTP, slightly by ADP, GTP and UTP, but not by AMP. The inhibition by ATP was competitive towards Co A esters and noncompetitive towards acetate. Eigenschaften der 5-Hydroxyvalerate CoA-Transferase aus Clostridium aminovalericumZusammenfassung: 5-Hydroxyvalerat-CoA-Transferase aus Clostridium aminovalericum, Stamm T2-7, wurde etwa lOOfach zur Homogenität gereinigt. Die molekulare Masse des nativen Enzyms wurde zu 178 ± 11 kDa, die der Untereinheit zu 47 kDa bestimmt, woraus sich eine tetramere Struktur errechnete (3.8 Untereinheiten/178 kDa). Folgende CoA-Ester wurden als Substrate gefunden (abnehmende Spezifität, V/K m ): 5-HydroxyvalerylCoA > Propionyl-CoA > Acetyl-CoA > (Z)-5-Hydroxy-2-pentenoyl-CoA > Butyryl-CoA > ValerylCoA. Die entsprechenden Säuren sowie 4-Pentenoat und 3-Pentenoat wurden mit Acetyl-CoA zu den CoA-Estern aktiviert, während Crotonat, (£)-5-Hydroxy-2-pentenoat, (£)-2-Pentenoat and 2,4-Pentadienoat nicht reagierten. 5-Hydroxyvalerat-CoATransferase zeigte Ping-Pong-Kinetiken und wurde durch Natriumboranat in Gegenwart von PropionylCoA inaktiviert. Diese beiden Befunde lassen sich mit der Bildung eines Enzym-CoA-Esters als Zwischenstufe erklären. Das Enzym wurde durch ATP und CTP in seiner Aktivität gehemmt. ADP, GTP und UTP waren weniger wirksam, während AMP keinen Einfluß hatte. Die Hemmung durch ATP war kompetitiv gegenüber CoA-Estern und nicht-kompetitiv gegenüber Acetat.

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