An enzyme which we call carboxylic acid reductase (aldehyde dehydrogenase) seems to be the first which is able to reduce non‐activated carboxylic acids to aldehydes at the expense of reduced viologens. There is no further reduction of the aldehydes to the corresponding alcohols. In the presence of oxidized viologens aldehydes can be dehydrogenated to carboxylic acids roughly 20 times faster than the latter are reduced. The specific enzyme activity in crude extracts is about 100 times increased if 10 μM tungstate and a sulphur source in addition to sulphate is given to the growth medium of Clostridium thermoaceticum. Carboxylic acid reductase seems to be present in two forms. One has an apparent molecular mass of about 240 kDa and is bound to red‐Sepharose, whereas, the other, a form of an apparent molecular mass of about 60 kDa, is not bound. SDS gel electrophoresis shows a higher complexity. The very labile enzyme has been enriched by a factor of about 145 by binding to octyl‐Sepharose and further chromatographic separation by red‐Sepharose and FPLC using Mono‐Q and phenyl‐Superose columns. After cell growth in the presence of [185W]tungstate, radioactivity coincides with the two forms of enzyme activity during all purification steps. This is also the case when the enzyme is electrophoretically separated on polyacrylamide slab gels.

The genes moaABC of Escherichia coli were ligated into the expression vector pNCO113. The resulting plasmid was transformed into a moeA mutant of E. coli. From cultures of the recombinant strain, a pteridine designated compound Z could be isolated at 5 mg/liter. Compound Z is a product of precursor Z, a biosynthetic precursor of molybdopterin. Cultures of the recombinant E. coli strain were supplied with [U-13 C 6 ]glucose, [U-13 C 5 ]ribulose 5-phosphate, or [7-15 N,8-13 C]guanine. The culture medium also contained a large excess of unlabeled glucose. Compound Z as well as nucleosides obtained by hydrolysis of RNA were isolated from the bacterial cultures, and their heavy isotope distribution was investigated by one-dimensional and two-dimensional NMR spectroscopy. The labelling patterns of compound Z show that the carbon atoms of a pentose or pentulose are diverted to the ring atoms C6 and C7 and to the side chain atoms C2′, C3′ and C4′ of compound Z. Carbon atom C1′ of compound Z is derived from carbon atom C8 of a guanine derivative. The remodeling of the carbon skeleton of the pentose and purine moieties proceed via intramolecular rearrangement reactions.Keywords : biosynthesis of molybdopterin; NMR; rearrangement reaction ; Escherichia coli. Fig. 1) is a nonconjugated tetrahy-acts as cofactor of xanthine dehydrogenase and sulphite oxidase, among others [7]. Genetic defects of the molybdopterin pathway dropterin derivative complexed to a molybdenum ion and serves as cofactor for a variety of enzyme-catalysed redox reactions are conducive to severe neurological deficiencies in man (for review see [14]

From the thermophilicBacillusacidocaldarius^ a membrane bound cyclase catalysing the formation of 22(29)-hopene (diploptene) and 22-hopanol (diplopterol) from squalene was enriched 670-fold to a purity of 95% as judged by gel chromatography. The specific activity of the purified enzyme in the presence of Triton X-100 is 0.22 product formation per min and mg protein at 54 °C. The molecular mass is 150 kDa and that of the subunits 80 kDa as determined by FPLC gel chromatography and sodium dodecyl sulfate gel electrophoresis, respectively.Not only squalene but also £,£-homofarnesol, homogeraniol and homofarnesyl (1,5,9-trimethyl-4,8-decadienyl) ether are substrates. The ether bond of the latter substrate is split by the enzyme. The products of the aforementioned three additional substrates are ambroxan (dodecahydro-3a,6,6,9a-tetramethylnaphtho[2,1 -b ]-furan), octahydro-4,4,7a-trimethylbenzofuran and ambroxan together with 1,5,9-trimethyl-4,8-decadienol. The cyclization rates of these substrates compared to squalene are 3%, 0.05% and 0.3%, respectively. The half-life of the enzyme activity is 80 h at 45 °C and 18 h at 54 °C. Reinigung, partielle Charakterisierung und Substratspezifität einer Squalene Cyclase aus Bacillus acidocaldarius Zusammenfassung: Aus dem thermophilen Bacillus acidocaldarius wurde eine Membran-gebundene Cyclase 670-fach angereichert. Nach Gelchromatographie war das Material zu mehr als 95% rein. Die Cyclase katalysiert die Bildung von 22(29)-Hopen (Diploptene) und 22-Hopanol (Diplopterol) aus Squalen. Die spezifische Aktivität des gereinigten Enzyms beträgt in Gegenwart von Triton X-100 0.22 Produkte pro min und mg Protein bei 54 °C. Die beiden Hopanoide entstehen im Verhältnis von ca. 6:1. Die apparente Molmasse der Cyclase, bestimmt durch FPLC-Gelchromatographie, ist 150 kDa und die durch Natriumdodecylsulfat-Polyacrylamidgel-Elektrophorese bestimmte Molmasse der Untereinheiten beträgt 80 kDa. Nicht nur Squalen ist Substrat, sondern aucĥ^-Homofarnesol, Homogeraniol und Homo farnesyl-( l ,5,9-trimethyl-4,8-decadienyl)-ether werden cyclisiert. Dabei wird die Etherbindung des letztgenannten Substrats durch das Enzym gespalten. Die Produkte dieser Substrate sind Ambroxan (Dodecahydro-3a,6,6,9a-tetramethylnaphto[2, l-fc]furan), Octahydro-4,4,7a-trimethylbenzofuran sowie Ambroxan zusammen mit l,5,9-Trimethyl-4,8-decadienol. Die Cyclisierungsgeschwindigkeiten dieser Substrate sind 3%, 0.05% und 0.3% der des Squalens. Die Halbwertszeit der Enzymaktivität beträgt 80 h bei 45 °C und 18 h bei 54 °C.

No abstract

Judged by properties observed during the purification and based on the sequence of the first 25 amino acids, the enzyme from Clostridium formicoaceticum catalysing the reversible reduction of nonactivated carboxylic acids to aldehydes at the expense of reduced viologens, is astonishingly different from that found by us in C. thermoaceticum.According to native and SDS gel electrophoresis the reductase is nearly homogeneous after only 26-fold purification. The specificity for various substrates and artificial electron carriers is also broad, but Vot the purified aldehyde dehydrogenase activity (54 U/mg enzyme for butanal) is about 1 order of magnitude lower than that of the enzyme from C. thermoaceticum.