Purification and characterization of a novel lactonohydrolase, catalyzing the hydrolysis of aldonate lactones and aromatic lactones, from Fusarium oxysporum

Abstract

A novel lactonohydrolase, an enzyme that catalyzes the hydrolysis of aldonate lactones to the corresponding aldonic acids, was purified 10-fold to apparent homogeneity, with a 61 % overall recovery, from Fusarium oxysporum AKU 3702, through a purification procedure comprising DEAESephacel, octyl-Sepharose CL-4B and hydroxyapatite chromatographies and crystallization. The molecular mass of the native enzyme, as estimated by high-performance gel-permeation chromatography, is 125 kDa, and the subunit molecular mass is 60 kDa. The enzyme contains 15.4% (by mass) glucose equivalent of carbohydrate, and about 1 mol calcium/subunit. The enzyme hydrolyzes aldonate lactones, such as D-galactono-y-lactone and L-mannono-y-lactone, stereospecifically. Furthermore, it can catalyze the asymmetric hydrolysis of D-pantoyl lactone, which is a promising chiral building block for the chemical synthesis of D-pantothenate. These reactions are reversible, and the reaction equilibrium at pH 6.0 has a molar ratio of nearly 1 : 1 with D-pantoyl lactone and D-pantoic acid. The K, and V,,, for D-galactono-y-lactone are 3.6 mM and 1440 U/mg, respectively, and those for D-galactonate are 52.6 mM and 216 U/mg, respectively. The enzyme also irreversibly hydrolyzes several aromatic lactones, such as dihydrocoumarin and homogentisic-acid lactone.Lactonohydrolases (lactonases) reversibly catalyze the hydrolysis of lactone compounds into hydroxy acids, i.e. they mediate the interconversion between the lactone and acid forms of hydroxy carboxylic acids. Among them, gluconolactonase (aldonolactonase), L-arabinonolactonase and D-arabinonolactonase specifically hydrolyze aldonate lactones to their respective aldonic acids. Although the activities of these enzymes have been detected in various organisms [l -81, there has been no report of the purification of one of these enzymes to homogeneity, except for bovine liver aldonolactonase [5], and so ceI1-free extracts or partially purified preparations have been used for characterization of the enzymes in most cases. Therefore, there has not been sufficient study of the enzyme chemistry. On the other hand, as to the roles of these enzymes in vivo, Bulitz and Lehninger suggested that aldonolactonase participates in the formation of L-gulono-y-lactone from L-gulonate in L-ascorbate biosynthesis [3]. Recently, L-rhamnonolactonase and D-xylOnOlactonase were found in Pulluluriu pulluluns and Pseudomonas frugi, respectively, being involved in the oxidative degradation of L-rhamnose and D-xylose, respectively [9, 101.

Crystallization and preliminary X-ray diffraction study of lactonohydrolase from Fusarium oxysporum

“…Therefore, elucidation of the three-dimensional structure of the lactonohydrolase from F. oxysporum is important for the elucidation of the mechanisms underlying the stereoselectivity and substrate speci®city of the enzyme. The lactonohydrolase was easily crystallized by desalting (Shimizu et al, 1992), but the crystals obtained showed poor stability with respect to X-ray irradiation. In this study, we report the crystallization of the lactonohydrolase of F. oxysporum to give crystals suitable for atomic resolution X-ray structure analysis, and the preliminary crystallographic characterization of the lactonohydrolase.…”

“…Recently, we found that Fusarium oxysporum produced a novel lactonohydrolase catalyzing the hydrolysis of aldonate lactones and aromatic lactones (Shimizu et al, 1992). The relative molecular mass of the native enzyme is 125 000, and the subunit molecular mass is 60 000.…”

“…The enzyme was puri®ed as described by Shimizu et al (1992). The crystallization conditions were preliminarily screened with a Crystal Screen reagent kit (Hampton Research, USA).…”

Solubilization, Purification, and Properties of Membrane-BoundD-Glucono-δ-lactone Hydrolase fromGluconobacter oxydans

“…2). specificity D-xylono--lactone (90%) L-pantoyl lactone and aromatic lactones but not for aldonolactones 10) synthetic lactones but not for aldonolactones 11) L--hydroxyglutaric acid--lactone 12) (R)-5-oxo-2-tetrahydrofran carboxylic acid and -butyrolactone 2,800 dihydrocumarin 9) 13.7 L-pantolactone 10) 230 (R)-5-oxo-2-tetrahydrofran carboxylic acid 11) 95 (S)-5-oxo-2-tetahydrofran carboxylic acid 11) 39.3 (S)-5-oxo-2-tetahydrofran carboxylic acid 3.6 D-galactonolactone 9) 6.3 dihydrocumarin 9) 3.59 L-pantoyl lactone 10) 1.9 (R)-5-oxo-2-tetrahydrofran carboxylic acid 11) 39.8 (S)-5-oxo-2-tetahydrofran carboxylic acid 11) 53 (S)-5-oxo-2-tetahydrofran carboxylic acid 12) EDTA/EGTA inhibited à inhibited 7,9,10) not inhibited 11,12) Restoration of lost Ca = Mg>Zn>Fe = Co, Ca, Mn, Zn 7) enzyme activity with Mn, Ni, Cu>Mo …”

“…[1][2][3][4][5][6][7][8][9][10][11][12][13] When D-glucose or D-glucose-6-phosphate is oxidized in cytoplasm by NAD(P)-dependent D-glucose dehydrogenase or NADP-dependent D-glucose-6-phosphate dehydrogenase, the corresponding lactone is formed and subsequently hydrolyzed to D-gluconic acid or 6-phospho-D-gluconic acid by the action of SGL.…”