[$2]

MANNOSIDOSTREPTOMYCIN HYDROLASE

637

Activity has not been detected in a number of strains which do not secrete streptomycin. Specificity. ATP cannot be replaced by UTP, GTP, or CTP. [6-32p]3'-Deoxydihydrostreptomycin-6-P is also a good phosphate accepfor. 2 As noted earlier, it is not known whether acceptors must be phosphorylated at position 6. The following compounds cannot serve as phosphate acceptors: streptomycin, streptomycin-6-P, dihydrostreptomycin-3"-P, and dihydrostreptobiosamine. Phosphate is presumed to be esterified at position 3'a since 3'-deoxydihydrostreptomycin-6-P, but not streptomycin-6-P, can serve as phosphate acceptor. 2

[52]

Mannosidostreptomycin Hydrolase

By

EDWARDINAMINEand ARNOLDL. DEMAIN NH II HN-- C --NH 2

\ ! O

L/OH

Streptidine 1

l Streptose OH

Streptomycin Mannosido-

l

streptomycin

O

l

N-Methyl-Lglucosamine

/ ~H

CII2OH

l D-Mannose Mannosidostreptomycin+ H20

* streptomycin+ mannose

638

ANTIBIOTIC INACTIVATION AND MODIFICATION

[52]

Mannosidostreptomycin hydrolase is an enzyme that transforms mannosidostreptomycin to streptomycin by the hydrolytic removal of the mannose moiety. 1 The two antibiotics are made concurrently during the course of a fermentation by strains of S t r e p t o m y c e s griseus. ~ The hydrolase is an inducible enzyme whose formation is subject to catabolite repression so t h a t it is usually synthesized late in a fermentation when the repressible carbon source is near depletion. 3-~

Assay Method Principle. The a-D-mannosidase enzyme is most conveniently assayed by use of the chromogenic substrate p-nitrophenyl-a-D-mannopyranoside. The p-nitrophenol liberated is determined spectrophotometrically at 400 nm2, 4 Reagents

Potassium phosphate buffer, 0.1 M, pH 7.2 Manganese acetate, 80 mM, prepared in 50 m M potassium phosphate buffer and the p H is adjusted to 7.2 with 20% K O H p-Nitrophenyl-a-D-mannopyranoside, 16.6 mM, in water Potassium carbonate, 0.2 M and 1.0 M Procedure. A reaction mixture containing 1.0 ml enzyme source, 4.0 ml buffer, 1.0 ml of manganese acetate, and 1.0 ml of water (which can be replaced by other additives) is equilibrated at 28 ° for 5 min in a r o t a r y water bath shaker at 275 rpm. The reaction is started by the addition of 1.0 ml of the substrate. Two controls, one without enzyme and the other without substrate, are run simultaneously. After 30 min, the incubation is terminated and color is developed by the addition of 2.0 ml of 1.0 M potassium carbonate. The mixture is centrifuged at 10,000 rpm for 3-5 rain. One milliliter of the clear supernatant fluid is diluted with 9.0 ml of 0.2 M potassium carbonate and the p-nitrophenol concentration determined on a Spectronic 20 colorimeter at 400 nm against the enzyme blank. Since the substrate itself undergoes a very slow hydrolysis during the incubation period, a substrate blank correction is applied. The standard curve is constructed with reagent grade p-nitrophenol. The com-

D. Perlman and A. F. Langlykke, J. Amer. Chem. Soc. 70, 3968 (1948). ~J. Fried and E. Titus, J. Biol. Chem. 168, 391 (1947). 8D. J. D. Hockenhull, G. C. Ashton, K. H. Fantes, and B. Y. Whitehead, Biochem. J. 57, 93 (1954). E. Inamine, B. Lago, and A. L. Demain, in "Fermentation Advances" (D. Perlman, ed.), p. 199. Academic Press, New York, 1969. 5G. Kollar, Acta Microbiol. Acad. Sci. Hung. 5, 19 (1958).

[52]

MANNOSIDOSTREPTOMYCI N HYDROLASE

639

pound is dissolved in 0.2 M potassium carbonate and its optical density at 400 nm is determined over a 0-20 n m o l e / m l range. Definition o] Unit Activity. One unit of activity is defined as t h a t amount of enzyme which catalyzes the release of 1 nmole of p-nitrophenol in 30 min. Substrate Source. p-Nitrophenyl-a-D-mannopyranoside can either t)e prepared by the method of Conehie and L e v v y ~ or purchased commercially from any number of sources. The natural substrate, mannosidostreptomycin, is not commercially available.

Properties Specificity. Streptomyces mannosidase hydrolyzes phenyl-~-D-mannopyranoside, mannosidostreptomycin, mannosidodihydrostreptomyein in addition to p-nitrophenyl-a-D-mannopyranoside. ',~,4 All the available d a t a on the properties of the enzyme are derived from studies done with crude preparations. Location. The Streptomyces enzyme is bound to the cell until lysis occurs. The cell-bound enzyme can be extracted into water, but its release is inhibited by sodium chloride, phosphate, or Tris. The enzyme t h a t is released by water is still attached to particulate matter which remains suspended at 10,000 r p m but is sedimented when centrifuged at 50,000 rpm. The activity of the cell-bound enzyme is not increased by lysozyme lysis, toluene treatment, or grinding with sand, so t h a t the enzyme appears to be bound at or near the cell surface and is readily accessible to the substrate. 7 Temperature and pH Optima. Optimmn temperature for the Streptomyces enzyme is 40 ° and it is rapidly inactivated at 50 ° while the p H for o p t i m u m activity is in the range of 7.0-8.0 depending on the strain of organism used. 1,:~,s Metal Requirement. The a-D-mannosidase of Streptomyces requires a divalent cation for activity. 7 The activity t h a t is lost by E D T A treatment is completely restored by Mn '-'+while Zn '-'+ and Ca ~+ are not as effective. Metals such as Cu -°+, H g 2+, and Fe ~+ inhibit the enzyme while K +, Mg ~+, and Ba '-'+ are without effect. ~,:~ Stimulators and Inhibitors. Arsenate is reported to stimulate the hydrolysis of phenyl-a-D-mannopyranoside while phosphate is without effect. :~,s Cyanide, fluoride, azide, bisulfite, and eysteine are reported to inhibit the enzyme to various degrees. 3,s Several sugars inhibit the activ"J. Conehie and G. A. Levvy, Methods Carbohyd. Chem. 2, 345 (1963). ' E. Inamine and A. L. Demain, Biotechnol. Bioeng. 12, 159 (1970). 8 G. Kollar, Acta Microbiol. Acad. Sci. Hung. 5, 11 (1958).

640

ANTIBIOTIC INACTIVATION AND MODIFICATION

[53a]

ity of the enzyme; the inhibition elicited by the sugars varies depending on whether the synthetic or natural substrate is used. 3,7,9 Adequate aeration is required for maximum enzyme activity2 ,s Pretreatment of enzyme-containing cells with N-ethylmaleimide is reported to eliminate the aeration requirement. 7 , D. J. D. Hockenhull, Progr. Ind. Microbiol. 2, 133 (1960).

[53a] f l - L a c t a m a s e ( B a c i l l u s c e r e u s ) By DAvm R. THATCHER

The reactions catalyzed by the fl-lactamases and the methods for assaying these enzymes are described elsewhere in this volume. 1 Bacillus cereus produces two distinct fl-lactamases in high yield. These two enzymes have been called fl-lactamase I (EC 3.5.2.6) and fl-lactamase II (EC 3.5.2.8) 2'3 and are coded by two separate genes. 4 No immunological affinity, as tested by antibody absorption or by the effect of antisera on activity, has been demonstrated between them. 5 Both enzymes are found as cellular and extracellular types and are coordinately induced in the presence of low levels of penicillin. 6 Cell bound fl-lactamase is also found in an immunologically distinguishable, iodine sensitive state (y-penicillinase)7,s This distinct fl-lactamase species must not be confused with a number of "y-state" derivatives of extracellular fl-lactamase I which have been reported in the literature. 9,1o Although displaying many of the properties of y-penicillinase, these induced conformational states of fl-lactamase I are not y-penicillinase sensu stricto. The presence of a separate cephalosporinase activity in culture supernatants and cells of B. cereus has been knbwn since 19626 and the enzyme responsible for this activity, f~-lactamase II, was isolated from fl-lac1 This volume [5]. S. Kuwabara and E. P. Abraham, Biochem. J. 103, 27 (1967). s M. R. Pollock, Ann. N.Y. Acad. Sci. 151, 502 (1968). ' M. R. Pollock and J. Fleming, J. Gen. Microbiol. 59, 303 (1969). M. R. Pollock and J. Fleming, unpublished results, 1968. 6 B. Crompton, M. Jago, K. Crawford, G. G. F. Newton, and E. P. Abraham, Biochem. J. 83, 52 (1962). M. R. Pollock, J. Gen. Microbiol. 15, 154 (1956). " N. Citri and A. Kalkstein, Arch. Biochem. Biophys. 121, 720 (1967). "M. B. Rudzik, and J. Imsande, J. Biol. Chem. 245, 3556 (1970). 10N. Citri, Biochim. Biophys. Acla 27, 277 (1958).

Mannosidostreptomycin hydrolase.

[$2] MANNOSIDOSTREPTOMYCIN HYDROLASE 637 Activity has not been detected in a number of strains which do not secrete streptomycin. Specificity. ATP...
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