Cheese flavour formation by amino acid catabolism

The crystal structure of the nitrogenase molybdenum–iron protein from Azotobacter vinelandii has been determined at 2.7 A resolution. The α- and β-subunits in this α_2β_2 tetramer have similar polypeptide folds. The FeMo-cofactor is completely encompassed by the α-subunit, whereas the P-cluster pair occurs at the interface between α- and β-subunits. Structural similarities are apparent between nitrogenase and other electron transfer systems, including hydrogenases and the photosynthetic reaction centre.

Crystallographic structure and functional implications of the nitrogenase molybdenum-iron protein from azotobacter vinelandii.

Domains, motifs, and linkers in 2-oxo acid dehydrogenase multienzyme complexes: a paradigm in the design of a multifunctional protein.

There was a long-held belief that the gram-positive soil bacterium Bacillus subtilis is a strict aerobe. But recent studies have shown that B. subtilis will grow anaerobically, either by using nitrate or nitrite as a terminal electron acceptor, or by fermentation. How B. subtilis alters its metabolic activity according to the availability of oxygen and alternative electron acceptors is but one focus of study. A two-component signal transduction system composed of a sensor kinase, ResE, and a response regulator, ResD, occupies an early stage in the regulatory pathway governing anaerobic respiration. One of the essential roles of ResD and ResE in anaerobic gene regulation is induction of fnr transcription upon oxygen limitation. FNR is a transcriptional activator for anaerobically induced genes, including those for respiratory nitrate reductase, narGHJI.B. subtilis has two distinct nitrate reductases, one for the assimilation of nitrate nitrogen and the other for nitrate respiration. In contrast, one nitrite reductase functions both in nitrite nitrogen assimilation and nitrite respiration. Unlike many anaerobes, which use pyruvate formate lyase, B. subtilis can carry out fermentation in the absence of external electron acceptors wherein pyruvate dehydrogenase is utilized to metabolize pyruvate.

Anaerobic growth of a “strict aerobe” (bacillus subtilis)

Acetoin is an important physiological metabolite excreted by many microorganisms. The excretion of acetoin, which can be diagnosed by the Voges Proskauer test and serves as a microbial classification marker, has its vital physiological meanings to these microbes mainly including avoiding acification, participating in the regulation of NAD/NADH ratio, and storaging carbon. The well-known anabolism of acetoin involves α-acetolactat synthase and α-acetolactate decarboxylase; yet its catabolism still contains some differing views, although much attention has been focused on it and great advances have been achieved. Current findings in catabolite control protein A (CcpA) mediated carbon catabolite repression may provide a fuller understanding of the control mechanism in bacteria. In this review, we first examine the acetoin synthesis pathways and its physiological meanings and relevancies; then we discuss the relationship between the two conflicting acetoin cleavage pathways, the enzymes of the acetoin dehydro...

Acetoin Metabolism in Bacteria

The pyruvate dehydrogenase and branched-chain 2-oxo acid dehydrogenase activities of Bacillus subtilis were found to co-purify as a single multienzyme complex. Mutants of B. subtilis with defects in the pyruvate decarboxylase (E1) and dihydrolipoamide dehydrogenase (E3) components of the pyruvate dehydrogenase complex were correspondingly affected in branched-chain 2-oxo acid dehydrogenase complex activity. Selective inhibition of the E1 or lipoate acetyltransferase (E2) components in vitro led to parallel losses in pyruvate dehydrogenase and branched-chain 2-oxo acid dehydrogenase complex activity. The pyruvate dehydrogenase and branched-chain 2-oxo acid dehydrogenase complexes of B. subtilis at the very least share many structural components, and are probably one and the same. The E3 component appeared to be identical for the pyruvate dehydrogenase, 2-oxoglutarate dehydrogenase and branched-chain 2-oxo acid dehydrogenase complexes in this organism and to be the product of a single structural gene. Long-chain branched fatty acids are thought to be essential for maintaining membrane fluidity in B. subtilis, and it was observed that the ace (pyruvate dehydrogenase complex) mutant 61142 was unable rapidly to take up acetoacetate, unlike the wild-type, indicative of a defect in membrane permeability. A single pyruvate dehydrogenase and branched-chain 2-oxo acid dehydrogenase complex can be seen as an economical means of supplying two different sets of essential metabolites.

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Dual role of a single multienzyme complex in the oxidative decarboxylation of pyruvate and branched-chain 2-oxo acids in Bacillus subtilis

A simple procedure is described for the purification of the pyruvate dehydrogenase complex and dihydrolipoamide dehydrogenase from Bacillus subtilis. The method is rapid and applicable to small quantities of bacterial cells. The purified pyruvate dehydrogenase complex (s0(20),w = 73S) comprises multiple copies of four different types of polypeptide chain, with apparent Mr values of 59 500, 55 000, 42 500 and 36 000: these were identified as the polypeptide chains of the lipoate acetyltransferase (E2), dihydrolipoamide dehydrogenase (E3) and the two types of subunit of the pyruvate decarboxylase (E1) components respectively. Pyruvate dehydrogenase complexes were also purified from two ace (acetate-requiring) mutants of B. subtilis. That from mutant 61142 was found to be inactive, owing to an inactive E1 component, which was bound less tightly than wild-type E1 and was gradually lost from the E2E3 subcomplex during purification. Subunit-exchange experiments demonstrated that the E2E3 subcomplex retained full enzymic activity, suggesting that the lesion was limited to the E1 component. Mutant 61141R elaborated a functional pyruvate dehydrogenase complex, but this also contained a defective E1 component, the Km for pyruvate being raised from 0.4 mM to 4.3 mM. The E1 component rapidly dissociated from the E2E3 subcomplex at low temperature (0-4 degrees C), leaving an E2E3 subcomplex which by subunit-exchange experiments was judged to retain full enzymic activity. These ace mutants provide interesting opportunities to analyse defects in the self-assembly and catalytic activity of the pyruvate dehydrogenase complex.

Wild-type and mutant forms of the pyruvate dehydrogenase multienzyme complex from Bacillus subtilis.

Limited digestion of the pyruvate dehydrogenase complex of Bacillus subtilis with either trypsin or chymotrypsin at 0 degrees C inhibited its ability to decarboxylate pyruvate and 2-oxoisovalerate oxidatively, without causing disassembly of the complex. The proteinases selectively cleaved the E1 alpha subunits to form two fragments of Mr 31500 and approx. 9500, as judged by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, both fragments remaining bound to the complex. Trypsin also caused a much slower cleavage of the E2 subunits, to form a fragment of apparent Mr 34000. The inhibition of overall dehydrogenase-complex activity was accompanied by the apparent loss of the pyruvate-driven and 2-oxoisovalerate-driven E1 activities, which was found to be due to a large increase in the Km for the 2-oxo acids: this change was correlated with the cleavage of the E1 alpha subunit.

R N Perham

Papers

Dual role of a single multienzyme complex in the oxidative decarboxylation of pyruvate and branched-chain 2-oxo acids in Bacillus subtilis

Wild-type and mutant forms of the pyruvate dehydrogenase multienzyme complex from Bacillus subtilis.

Limited proteolysis of the pyruvate dehydrogenase multienzyme complex of Bacillus subtilis.