[8] Purification and properties of interference factor i: a cistron-specific translation control factor and a subunit of phage RNA replicase

The purification and characterization of AAT1, one of two aromatic amino acid aminotransferase (EC 2.6.1.57) in Azospirillum brasilense, is described. Purified AAT1 had a subunit mass of 33 kDa and a nondenatured molecular mass of 66 kDa, suggesting a dimeric structure. Other properties include a pI of 5.04, an optimum temperature of 45 °C, and optimum pH of 8.5. AAT1 utilized all aromatic amino acids, the L-tryptophan derivatives such as L-5-methyl tryptophan and L-flourtryptophan, and L-histidine. The apparent Km values for L-tyrosine, L-phenylalanine, and L-tryptophan were 0.19, 0.43, and 1.05 mM, respectively. The enzyme was competive inhibited by indole-3-pyruvic acid with a Ki of 0.17 mM.Key words: aromatic aminotransferase, Azospirillum brasilense, indole acetic acid production.

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4-Sulphobenzoate 3,4-dioxygenase. Purification and properties of a desulphonative two-component enzyme system from Comamonas testosteroni T-2

Biochemical Journal ◽

10.1042/bj2740833 ◽

1991 ◽

Vol 274 (3) ◽

pp. 833-842 ◽

Cited By ~ 50

Author(s):

H H Locher ◽

T Leisinger ◽

A M Cook

Keyword(s):

Gel Filtration ◽

Hydrophobic Interaction Chromatography ◽

Anion Exchange Chromatography ◽

Exchange Chromatography ◽

Comamonas Testosteroni ◽

Visible Absorption ◽

Mononuclear Iron ◽

Purification And Properties ◽

A Subunit ◽

Protein Components

Cell-free extracts of Comamonas testosteroni T-2 grown in toluene-p-sulphonate/salts medium catalyse the conversion of p-sulphobenzoate (PSB) into protocatechuate and sulphite by an NADH-requiring and Fe2(+)-activated dioxygenase. Anion-exchange chromatography of extracts yielded red (A) and yellow (B) protein fractions, both of which were necessary for dioxygenative activity. Further purification of each fraction by hydrophobic interaction chromatography and gel filtration led to two homogeneous protein components (A and B), which together converted 1 mol each of PSB, O2 and NADH into 1 mol each of protocatechuate, sulphite and, presumably, NAD+. The system was named 4-sulphobenzoate 3,4-dioxygenase (PSB dioxygenase system). Monomeric component B (Mr 36,000) was determined to be a reductase that contained 1 mol of FMN and about 2 mol each of iron and inorganic sulphur per mol. This component transferred electrons from NADH to the oxygenase component (A) or to, e.g., cytochrome c. Homodimeric component A (subunit Mr 50,000) of the PSB dioxygenase system contained one [2Fe-2S] centre per subunit and its u.v.-visible-absorption spectrum corresponded to a Rieske-type iron-sulphur centre. The requirement for activation by iron was interpreted as partial loss of mononuclear iron during purification of component A. Component A could be reduced by dithionite or by NADH plus catalytic amounts of component B. The PSB dioxygenase system displayed a narrow substrate range: none of 18 sulphonated or non-sulphonated analogues of PSB showed significant substrate-dependent O2 uptake. The physical properties of the PSB dioxygenase system resemble those of other bacterial multi-component dioxygenase, especially phthalate dioxygenase. However, it differs from most characterized systems in its overall reaction; the product is a vicinal diphenol, and not a dihydrodiol.

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