Isolation from Clostridium

The simplest of these proteins are rubredoxins, which are bacterial proteins having a characteristic red colour (from which their name is derived) containing an FeS4 assembly, consisting of an Fe(III) ion coordinated to four cysteine groups. The typical tetrahedral structure of this group is illustrated in Figure 17 for the rubredoxin isolated from Clostridium pasteurianum (FW 6100).35... 

Another selenium-containing molybdenum hydroxylase that has been isolated from Clostridium barkeri (identical to Eubacterium barkeri) is nicotinic acid hydroxylase (NAH). Clostridium barkeri was isolated initially as a fermentor of nicotinic acid and thus NAH is a key enzyme in the efficient fermentation of nicotinic acid as a source of carbon and energy. NAH contained selenium when purified from cells labeled with Se-selenite. However, this label was lost during denaturing gel electrophoresis and also on heating of the enzyme (Dilworth 1982). Exhaustive analysis of selenium-labeled alkylation products of NAH under various conditions revealed selenium was bound as a labile cofactor (Dilworth 1982), and not as seleno-cysteine. This report was the first to describe a selenium-dependent enzyme that did not contain selenium in the form of selenocysteine. 

Sliwkowski MX, StadtmanTC. 1988. Selenium-dependent glycine reductase differences in physicochemical properties and biological activities of selenoprotein A components isolated from Clostridium sticklandii and Clostridium purinolyticum. Biofactors 1 293-6. 

This enzyme [EC 1.2.99.2], also known as acetyl-CoA synthase, catalyzes the reaction of carbon monoxide with water and an acceptor to produce carbon dioxide and the reduced acceptor. The cofactors of this enzyme include nickel and zinc ions as well as non-heme iron. Methyl viologen can act as the acceptor substrate. The enzyme is isolated from Clostridium sp. Interestingly, it also catalyzes an exchange reaction of carbon between Cl of acetyl-CoA and carbon monoxide. The protein participates in the synthesis of acetyl-CoA from carbon dioxide and hydrogen in the organisms. 

Lysine mutase is the first of a group of AdoCbl-dependent enzymes that catalyses the 1,2-migration of an amino group (Fig. 26). It has been isolated from Clostridium sticklandii [39] and consists of a cobalamin-binding orange protein and a smaller yellow protein. Apart from AdoCbl, several other essential cofactors have been identified, such as pyridoxal phosphate, ATP, FAD, thiols, Mg2+ and K+ [38]. The function of the yellow protein and some of these cofactors is to renew continuously... 

A variety of 3-substituted aspartic acids 7 can be prepared by stereospecific addition of ammonia to substituted fumaric acids 6 catalyzed by /(-methylaspartase (EC 4.3.1.2). The enzyme, which was first isolated from Clostridium tetanomorphum (ATCC 15920)42, aminates... 

The two nitrogenase proteins, Fe-protein and MoFe-protein, are composed of a total of three different types of subunits and contain three different types of metal centers. The properties of the nitrogenase proteins have been extensively studied and are summarized below. To distinguish the two nitrogenase proteins isolated from different bacterial sources, the MoFe-protein and Fe-protein are designated as components 1 and 2, respectively, preceded by a two-letter abbreviation of the source species and genus i.e., Avl is MoFe-protein isolated from Azotobacter vinelandii and Cp2 is Fe-protein isolated from Clostridium pasteurianum, etc. 

The name ferredoxin was first proposed for a non haem iron-redox-protein (hence its name) isolated from Clostridium pasteurianum and presumably involved in the hydrogen gas evolution from pyruvate by this bacterium (254). The smallest of the known iron-sulfur proteins, 6,000 dalton, the clostridial ferredoxins are, however, the most complex in terms of the iron and inorganic sulfur content 8Fe 8S. They are single chain polypeptides of about 55 residues of which eight are cysteines (Fig. 18). 

Stabilities of GDH s from microorganisms vary greatly. Whereas the NADP-GDH from Neurospora retains activity for several days at 50° and pH 7.2 (152), the NAD-GDH inactivates rapidly at raised temperatures and low ionic strengths, but is more stable in the presence of NAD (1 mAf) and the competitive inhibitor isophthalate (64). NAD-GDH s from lower fungi are also reported to be unstable (71), but those isolated from Clostridium SBj (35) and Peptococcus aerogenes (31) are stable at 50°, and so are the NADP-dependent enzymes of Salmonella... 

Du Toit, P.J. Potgieter, D.J.J. De Villiers, V. Properties of 1-phosphofructo-kinase isolated from Clostridium pasteurianum. Enzymologia, 43, 285-300 (1972)... 

A 3-D-glucosidase has been isolated from Clostridium thermocellum the enzyme was localized in the periplasmic space. ° It was purified in a five-step procedure including ion-exchange chromatography, gel filtration, and isoelectric... 

Martin el al. (1961) also demonstrated that their enzyme system catalyzed the fixation of HCOj in malonyl CoA in a reaction dependent on malonyl CoA and caproyl CoA. The formulation suggested for a similar reaction catalyzed by an enzyme isolated from Clostridium kluyveri was ... 

Clostripain [9028-00-6] Mr -55,000 [EC 3.4.22.8]. Clostripain is isolated from Clostridium histolyticum callogenase by extraction in pH 6.7 buffer, followed by hydroxylapatite chromatography with a 0.1-0.2 M phosphate gradient, then Sephadex G-75 gel filtration with 0.05M phosphate pH 6.7, dialysis and a second... 

Clostiipain [9028-00-6] 55,000 [EC 3.4.22.8]. Clostripain is isolated from Clostridium histolyticum... 

Recently, a fusion protein between a cellulose binding domain (CBOeios)> isolated from Clostridium cellulovorans, and OPH was shown to be capable of immobilization onto various cellulose materials (24). The use of cellulose as an immobilization matrix is advantageous due to its low cost, wide spread availability and non-toxic nature. The kinetic parameters of OPH fused to die CBD domain were essentially identical to die soluble protein with a 3.8 fold increase in from 0.058 to 0.220 and a 10.4% decrease in Kd from 3170 to 2840. Additionally, the immobilized fusion protein offered superior stability over that of soluble OPH, retaining over 85% activity over a period of 45 days (24). 

Enoate reductase. Enoate reductase isolated from Clostridium tyrobutyricum catalyses the NADH or methylviologen-dependent reduction of the a,p carbon-carbon double bond of non-activated 2-enoates and in a reversible way that of 2-enals. The enzyme appears to be a multimer of identical subunits. The total molecular mass is 940 kDa ( 73 kDa per subunit). Sedimentation equilibrium experiments, molecular mass data, and electron microscopy indicate that the native enzyme is composed of a tetramer of trimers. Each enzyme subunit contains one FAD, 0.6 FMN and one [4Fe-4S] cluster. 

Nicotinic acid hydroxylase. Nicotinic acid hydroxylase consists of four dissimilar subunits and occurs in forms with different molecular masses. There are 5-7 Fe, one FAD, and one molybdenum molybdopterin per 160 kDa protein. Unusually, nicotinic acid hydroxylase as isolated from Clostridium barkeri contains Mo(V) rather than Mo(VI). By EPR spectroscopy of the enzyme containing either natural-abundance Se or Se (/ = Gladyshev and co-workers showed that Se is directly coordinated, although in an unidentified form to Mo. Furthermore, a flavin radical and two [2Fe-2S] clusters could be observed with EPR spectroscopy. ... 

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