ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, Apr. 1989, p. 452-4590066-4804/89/040452-08$02.00/0Copyright © 1989, American Society for Microbiology

Reactions Catalyzed by Purified L-Glutamine:Keto-Scyllo-InositolAminotransferase, an Enzyme Required for Biosynthesis

of Aminocyclitol AntibioticsLYNNE A. LUCHER,t* YU-MING CHEN, AND JAMES B. WALKER

Department of Biochemistry, Rice University, Houston, Texas 77251

Received 10 June 1988/Accepted 23 December 1988

Dialyzed extracts of the gentamicin producer Micromonospora purpurea catalyze reactions which representtransaminations proposed for 2-deoxystreptamine biosynthesis. To determine whether these transaminationswere catalyzed by a single aminotransferase or by multiple enzymes, we purified and characterized an

L-glutamine:keto-scyllo-inositol aminotransferase from M. purpurea. This enzyme was purified 130- to 150-foldfrom late-log-phase mycelia of both wild-type M. purpurea and a 2-deoxystreptamine-less idiotroph. Thecofactor pyridoxal phosphate was found to be tightly bound to the enzyme, and spectral analysis demonstratedits participation in the transamination reactions of this enzyme. The major physiological amino donor for theenzyme appears to be L-glutamine; the keto acid product derived from glutamine was characterized as2-ketoglutaramate, indicating that the alpha amino group of glutamine participates in the transamination. Wefound that crude extracts contained omega-amidase activity, which may render transaminations withglutamine irreversible in vivo. The substrate specificity of the aminotransferase was shown to be restricted todeoxycyclitols, monoaminocyclitols, and diaminocyclitols, glutamine, and 2-ketoglutaramate, which contrastswith the broader substrate specificity of mammalian glutamine aminotransferase. The appearance of theenzyme in late-log phase, coupled with its narrow substrate specificity, indicates that it participatespredominantly in 2-deoxystreptamine biosynthesis rather than in general metabolism. The enzyme catalyzesreactions which represent both transamination steps of 2-deoxystreptamine biosynthesis. Although copurifi-cation of two aminotransferases cannot be ruled out, our data are consistent with the participation of a singleaminotransferase in the formation of both amino groups of 2-deoxystreptamine during biosynthesis by M.purpurea. We propose that this aminotransferase participates in a key initial step in the biosynthesis of mostaminocyclitol antibiotics.

2-Deoxystreptamine is a component of numerous clini-cally important antibiotics such as gentamicin, neomycin,tobramycin, amikacin, and hygromycin, while streptamine isknown to occur naturally in place of 2-deoxystreptamine inat least one member (SS-56C) of the destomycin group (forreviews, see references 19 and 21). A pathway for 2-deoxystreptamine biosynthesis has been proposed (Fig. 1)on the basis of mutasynthesis and isotopic competitionstudies (9, 13) and in vitro enzymatic studies (5). The twoaminotransferase steps of this pathway may be analogous tothe two transaminations which occur in the biosynthesis ofthe streptidine moiety of streptomycin (27). These reactionsin streptidine biosynthesis are catalyzed by separate en-zymes; L-glutamine:keto-scyllo-inositol aminotransferase(glutamine-scyllo-inosose aminotransferase; EC 2.6.1.50)catalyzes the first transamination reaction (26). This enzy-matic activity has also been detected in extracts of actino-mycetes that produce bluensomycin (26) and the 2-deoxys-treptamine-containing antibiotics neomycin and gentamicin(5).Our laboratory has been concerned with the enzy-

matic reactions responsible for formation of the N' and N3groups of 2-deoxystreptamine and streptamine. Dialyzedextracts of Micromonospora purpurea, a gentamicin pro-ducer, and Streptomyces fradiae, a neomycin producer,were previously found to catalyze transaminations involving

* Corresponding author.t Present address: Microbiology Group, Department of Biological

Sciences, Illinois State University, Normal, IL 61761.

L-glutamine, keto-scyllo-inositol, aminodeoxy-scyllo-inosi-tol, streptamine, and 2-deoxystreptamine (5), reactions rep-resentative of both aminotransferase steps in the proposedpathway. It was important to determine whether the multipletransaminations catalyzed by crude extracts of these organ-isms were catalyzed by a single enzyme or whether two ormore different aminotransferases were involved. In thispaper, we report on the purification and substrate specificityof the enzyme responsible for catalyzing the L-glutamine:keto-scyllo-inositol activity in M. purpurea.

MATERIALS AND METHODS

Materials. Microorganisms were obtained from the Amer-ican Type Culture Collection. All microbiological mediawere from Difco Laboratories. L-[U-'4C]glutamine (248 to251 mCi/mmol), 2-[U-_4C]ketoglutarate (250 mCi/mmol), and2-[U-_4C]pyrrolidone-5-carboxylic acid (pyroglutamate, 289mCi/mmol) were obtained from New England Nuclear Corp.Aminodeoxy-scyllo-[1-_4C]inositol (3.5 mCi/mmol) was fromCalifornia Bionuclear Corp. D,L-epi-Inosose was from Ma-kor Chemicals, Ltd. Streptamine, 2-deoxystreptamine, andN3-methyl-2-deoxystreptamine were gifts from K. Koch ofEli Lilly. Keto-scyllo-inositol and all other amino acids andketo acids were from Sigma Chemical Co. Dowex 50 (H+)and Dowex 1 (Cl-) (both analytical grade, 200/400 mesh),DEAE-agarose gel, hydroxyapatite powder, and Bio-GelP-300 were obtained from Bio-Rad Laboratories. The follow-ing were also from Sigma: glutamate dehydrogenase (EC1.4.1.3, type 2, bovine liver); glutaminase (EC 3.5.1.2, grade5, Escherichia coli); catalase (EC 1.11.1.6, bovine liver);

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AMINOCYCLITOL AMINOTRANSFERASE FROM M. PURPUREA

op

I

o=0

IB

NHH NNH2 NNH22 ~ ~

2. D ZcV'1 20/n2 lE

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FIG. 1. Proposed pathway of 2-deoxystreptamine biosynthesis.I, viboquercitol; II, 2-deoxyinosose; III, 2-deoxyinosamine; IV,aminodeoxyinosose; V, 2-deoxystreptamine.

L-amino acid oxidase (EC 1.4.3.2, type 6, Crotalus atrox);and all other proteins, cofactors, and chemicals.Growth of microorganisms. M. purpurea ATCC 15835 and

M. purpurea ATCC 31119 were maintained at room temper-ature on slants of 1% glucose, 2% soluble starch, 0.5% yeastextract, 0.5% peptone, 0.5% casein hydrolyzate, and 1.5%agar. Suspension cultures were grown in 0.5% yeast extract,0.3% beef extract, 0.5% tryptone, 2.4% soluble starch, 0.5%glucose, and 0.4% calcium carbonate (suspension medium;modified from reference 20). To cultivate M. purpurea insuspension, tubes of suspension medium were inoculatedwith a loopful of vegetative mycelia from slant cultures andwere grown at 35°C for 2 to 4 days with shaking at 230 rpm

(New Brunswick rotary shaker-incubator). Samples (2 to 5ml) of these starter cultures were then transferred to 2-literflasks containing 500 ml of suspension medium and were

grown with shaking. Mycelia were harvested by suctionfiltration with Whatman no. 4 filter paper or by centrifuga-tion at 19,000 x g for 15 min at 4°C. Harvested mycelia were

stored as a paste at -200C.Preparation of cell extracts. All steps were performed at

4°C, unless otherwise stated. For routine analysis of ami-notransferase activity, 5 to 6 g of frozen mycelial paste was

suspended in 5 to 10 ml of 100 mM potassium phosphate (pH7.4)-0.1 mM phenylmethylsulfonyl fluoride (buffer A) andsonicated with a Branson sonifier for 2 to 3 min total time.The broken-cell suspension was centrifuged at 27,000 x g,

and the supernatant was dialyzed against two changes of 10mM potassium phosphate (pH 7.4)-0.1 mM phenylmethyl-sulfonyl fluoride (buffer B) for 24 h. The dialyzed extractswere stored at -200C.For large-scale preparation of cell extracts, 200 g of frozen

mycelial paste was first stirred into 1,000 ml of 1 M KCI andcentrifuged at 19,600 x g for 20 min. The KCl wash was

performed three times to reduce the extracellular proteaseactivity associated with actinomycetes (15). The washedcells were suspended in 1,000 ml of buffer A and passedtwice through a prechilled Maulton-Gaulin mill at 6,000lb/in2. The resulting broken-cell suspension was centrifugedat 19,600 x g for 30 min, and the supernatant was used forthe purification of aminocyclitol aminotransferase. This ex-

tract typically showed an 80% reduction in general proteaseactivity (as measured by the method of Millet; 17) as a

consequence of the KCI washes described above.Purification of L-glutamine:keto-scyllo-inositol aminotrans-

ferase. All steps were performed at 4°C, and all buffers

contained 0.7 mM mercaptoethanol. Crude extract preparedfrom 200 g of M. purpurea 15835 or 31119 was fractionatedwith solid ammonium sulfate; the fraction precipitating be-tween 40 and 70% saturation was dissolved in buffer A anddialyzed for 20 to 24 h against two changes of buffer B. Theammonium sulfate extract was applied to a DEAE-agarosecolumn (2.6 x 30 cm) equilibrated in buffer B. The ami-notransferase was eluted with a linear gradient of 0 to 0.3 MNaCI in buffer B (900 ml, total volume) at a flow rate of 41ml/h. Fractions of 8 ml were collected and analyzed foraminotransferase activity; the active fractions were pooledand dialyzed as described above. The aminotransferase wastypically located between fractions 80 and 90, eluting justprior to a section of pink effluent.The DEAE extract was applied to a hydroxyapatite col-

umn (2.5 by 35 cm) equilibrated in buffer B. The aminotrans-ferase was eluted with a linear gradient of 10 to 100 mMpotassium phosphate (pH 7.4) (450 ml, total volume) at 41ml/h. Fractions of 4 ml were collected and analyzed foraminotransferase activity. The pooled active fractions wereconcentrated by dialysis against 20% polyethylene glycol(molecular weight, 20,000) in buffer A, followed by dialysisagainst buffer B. The aminotransferase was typically locatedbetween fractions 65 and 85, on the trailing edge of thesecond of two protein peaks.The hydroxyapatite extract was then applied to an omega-

aminobutyl-agarose column equilibrated in buffer B; approx-imately 1 ml of agarose was used for every 10 mg of protein.The column was washed with buffer B, and the aminotrans-ferase was eluted with 6 ml of buffer B containing 0.5 MNaCl. The yellow effluent containing the aminotransferasewas dialyzed against buffer B.

Molecular weight estimation. The molecular weight of theM. purpurea aminocyclitol aminotransferase was estimatedby gel filtration chromatography with a Bio-Gel P-300 col-umn (1.6 by 72 cm). The following protein standards (mo-lecular weight) were used: ferritin (440,000), aldolase(158,000), albumin (67,000), ovalbumin (43,000), chymotryp-sinogen A (25,000), and lysozyme (14,300).

SDS-polyacrylamide gel electrophoresis. Sodium dodecylsulfate (SDS) gel electrophoresis was performed as previ-ously described (29) with separating gels of 10% acrylamidein 1 M Tris (pH 9.0)-0.1% SDS, stacking gels of 3.3%acrylamide in 0.2 M Tris (pH 6.8)-0.1% SDS, and a runningbuffer of 25 mM Tris (pH 8.1)-0.2 M glycine-0.1% SDS.

Preparation of aminotransferase substrates. L-Glutaminewas recrystallized prior to use to remove contaminating2-pyrrolidone-5-carboxylic acid. An aqueous solution ofL-glutamine was warmed to 60°C, and 5 volumes of 95%ethanol at 60°C was added. After cooling, L-glutamine crys-tals were collected by filtration. L-[U-14C]glutamine wassimilarly purified before use by cocrystallization of 50 ,uCiwith 22 mg of recrystallized L-glutamine. The resultingspecific activity typically ranged from 0.25 to 0.33 mCi/mmol.

2-Ketoglutaramate was synthesized enzymatically by themethod of Meister (10, 16) with L-amino acid oxidase andcatalase. 2-Ketoglutaramate cyclizes in solution to 2-hy-droxy-5-oxo-proline with an equilibrium constant (at pH 7.5)of 3 x 10-3, favoring cyclization (11). Concentrations of2-ketoglutaramate are given as the concentration of uncyc-lized molecule, on the basis of the equilibrium constant.Following deproteinization with perchloric acid and neutral-ization with KOH, the 2-ketoglutaramate product was iso-lated from the reaction mixture by elution from a Dowex 50(H') column with water. The acidic effluent was concen-

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ANTIMICROB. AGENTS CHEMOTHER.

trated by rotary evaporation at 40°C and decolorized whennecessary with activated charcoal. To isolate 2-ketoglutara-mate as its barium salt, the pH was adjusted to 4.5 with solidBaOH, and barium 2-ketoglutaramate was then precipitatedwith 5 volumes of cold 95% ethanol. Alternatively, the pHwas adjusted to 7.0 with NaOH to isolate 2-ketoglutaramateas its sodium salt.

2-[U-_4C]ketoglutaramate was synthesized enzymaticallyby the same procedure, beginning with 30 to 40 RCi ofpurified L-[U-_4C]glutamine. Once eluted from a Dowex 50(H+) column, the 2-[U-14C]ketoglutaramate was neutralizedwith NaOH and concentrated in vacuo.

Aminodeoxy-scyllo-[1-_4C]inositol was treated to removedecomposition material by application to a Dowex 50 (H+)column; after extensive washing with water, aminodeoxy-scyllo-[1-_4C]inositol was eluted with 0.5 N HCl (26). Theacidic effluent was dried in vacuo in the presence of solidNaOH and redissolved in a minimal volume of water.

Keto-scyllo-[1-14C]inositol was prepared enzymaticallywith the M. purpurea aminocyclitol aminotransferase (5).The reaction mixture contained 0.6 Rmol of aminodeoxy-scyllo-[1-14C]inositol, 1.8 ,umol of keto-scyllo-inositol, and100 ,ug of purified M. purpurea aminotransferase, in 100 mMpotassium phosphate (pH 7.4) (700 RI, total volume). Afterincubation (6 h) at 37°C, the reaction mixture was applied toa Dowex 50 (H+) column, and keto-scyllo-[1-_4C]inositolwas eluted with water. The product was further purified byelution from a Dowex 1 (CI-) column with water. The finaleffluent was dried in vacuo in the presence of solid NaOHand dissolved in a minimal volume of water.

All other amino and keto acids were used as obtained fromthe supplier.

Assays for aminotransferase activity. Assays were per-formed to measure the following aminocyclitol aminotrans-ferase reactions:

keto-scyllo-inositol + R-NH2 aminodeoxy- (1)scyllo-inositol + R=O

L-glutamine + R=O = 2-ketoglutaramate (2)+ R-NH2

Keto-scyllo-inositol and aminodeoxy-scyllo-inositol wereused as 2-hydroxyl analogs of the presumed natural sub-strates, since the 2-deoxycyclitols were unavailable for thesestudies. Reaction mixtures contained amino donor, aminoacceptor, aminotransferase, and either 50 mM potassiumphosphate (pH 7.4) or 25 mM Tris (pH 8.5) in a final volumeof 30 ,ul; reaction blanks lacked enzyme. The mixtures wereincubated for up to 2 h at 37°C. When the substrate donor oracceptor was radiolabeled, the labeled products were mea-sured by liquid scintillation counting after separation byhigh-voltage paper electrophoresis (26). Alternatively, 2-[U-14C]ketoglutaramate from reaction 2 (forward reactionwith L-[U-_4C]glutamine as donor) was eluted from a Dowex50 (H+) column with water and counted. Liquid scintillationcounting was performed with a Beckman LS 100-C scintil-lation counter.When nonradioactive 2-ketoglutaramate was the acceptor,

the L-glutamine product from reaction 2, reverse direction,was converted to glutamate by glutaminase, and the gluta-mate was then detected spectrophotometrically with gluta-mate dehydrogenase (method adapted from reference 2).The transamination reaction was stopped by the addition of30 IlI of 0.5 M acetate (pH 5.0), and 0.05 U (10 pul) of E. coliglutaminase was added. After further incubation at 37°C for1 h, the entire reaction mixture was added to 0.9 ml of 0.5 M

glycine-0.4 M hydrazine (pH 9.0)-i mM ADP with 10 U (10pI) of glutamate dehydrogenase. The reaction was initiatedby the addition of NAD+ to 1 mM; after incubation for 1 h atroom temperature, the increase in A340 due to NAD+ reduc-tion was determined. Aminotransferase blanks lacked one ofthe substrates. Up to 60 nmol of L-glutamine were convertedto 2-ketoglutaramate under these conditions. The opticaldata were used to calculate the amount of glutamine pro-duced during transamination, as follows: (AA340/6.22) xcuvette volume in ml x 103 = nmol of glutamine.Crude aminotransferase extracts exhibited precipitation in

acetate buffer, but this did not interfere with the glutaminasereaction. To avoid interference with the absorbance mea-surements, these reaction mixtures were clarified prior to theglutamate dehydrogenase step by adding 25 pul of cold 7%perchloric acid and centrifuging. The acidic supernatantswere then neutralized with KOH and analyzed with gluta-mate dehydrogenase.

Preparation of rat liver omega-amidase. Rat liver omega-amidase was partially purified by a modification of theprocedure of Hersh (11). Fresh rat livers were homogenizedin 40 mM potassium phosphate (pH 7.5)-50 mM KCI-100mM sucrose-1 mM EDTA-25 mM NaF. After centrifugationat 34,000 x g for 1 h, the supernatant solution was treatedwith solid ammonium sulfate, and the protein precipitatingbetween 50 and 70% saturation was dissolved in and dia-lyzed against 10 mM potassium phosphate (pH 7.2)-0.1 mMEDTA-20 mM mercaptoethanol (buffer C). The ammoniumsulfate extract was applied to a DEAE-cellulose columnequilibrated in buffer C, and the amidase was eluted in theflowthrough with buffer C. The amidase was then precip-itated with 0.5 g of ammonium sulfate per ml and stored as asuspension at 4°C. Under these conditions, the amidasepreparation is stable for at least 2 years. When needed, 1 mlof the suspension was centrifuged and the pellet was dis-solved in 100 mM potassium phosphate (pH 7.4) and dia-lyzed against 10 mM potassium phosphate (pH 7.4)-20 mMmercaptoethanol. In solution, the amidase preparation isstable at 4°C for only a few weeks.Amidase assay. 2-Ketoglutaramate-hydrolyzing amidase

activity was assayed with glutamate dehydrogenase. Theamidase reaction mixture typically contained 50 mM potas-sium phosphate (pH 7.4) or 25 mM Tris (pH 8.5), up to 0.33mM 2-ketoglutaramate, and rat liver amidase or crude ex-tract of M. purpurea in a final volume of 60 pl. The mixturewas incubated at 37°C for 15 to 60 min, and the reaction wasstopped by the addition of 25 pAl of cold 7% perchloric acid.The mixture was neutralized with 20 pI of 2 N KOH andcentrifuged. The 2-ketoglutarate produced by hydrolysis wasmeasured with glutamate dehydrogenase (1). The superna-tant (50 pul) was added to 0.9 ml of 50 mM Tris (pH 8.5)-10mM ammonium chloride-0.14 mM NADH-1.6 mM ADP,and the reaction was begun with the addition of 10 U (10 ,Il)of glutamate dehydrogenase. The decrease in A340 due toNADH oxidation was followed.

Protein assay. Protein concentrations were determined bythe method of Lowry et al. (14).

RESULTS

Aminotransferase activity during M. purpurea growth cycle.For aminotransferase purification, it was desirable to obtainM. purpurea mycelia containing the highest amount ofaminotransferase activity. The L-glutamine:keto-scyllo-ino-sitol aminotransferase specific activity was initially very lowbut peaked during late-log phase (Fig. 2), which is charac-

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AMINOCYCLITOL AMINOTRANSFERASE FROM M. PURPUREA

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FIG. 2. Appearance of aminocyclitol aminotransferase activitiesduring the growth cycle of M. purpurea 15835. All assays wereperformed in Tris (pH 8.5) as described in Materials and Methodswith crude extract. The pH of the growth medium rose from 6.5 to7.0 between 0.5 and 1 day and gradually rose from 7.0 to 7.2throughout the rest of the experiment (not shown). Curve 1: 0,transamination between 10 mM L-[U-'4C]glutamine and 0.33 mMketo-scyllo-inositol (nanomoles of product per minute per milligramof protein); curve 2: 0, transamination between 5 mM streptamineand 0.33 mM 2-ketoglutaramate (nanomoles of product per minuteper milligram of protein); curve 3: A, amidase hydrolysis of 0.33 mM2-ketoglutaramate (nanomoles of product per minute per milligramof protein); curve 4: *, mycelial dry weight (milligrams per millili-ter).

teristic of enzymes involved in antibiotic biosynthesis (28).Purification of the aminotransferase. The L-glutamine: keto-

scyllo-inositol aminotransferase activity was purified about130-fold from late-log-phase mycelia of M. purpurea 15835(Table 1). The 2-deoxystreptamine-less idiotroph M. pur-purea 31119 was a good alternative organism for the isolationof the aminotransferase; a 150-fold purification of the activ-ity from the idiotroph was achieved. The aminotransferasedid not copurify with some commonly occurring aminotrans-ferases, as shown in Table 2. Samples of the purifiedaminotransferase generally exhibited five major bands inSDS gels.

General properties of the aminotransferase. The molecularweight of the aminotransferase was estimated by gel filtra-tion chromatography to be between 64,000 and 76,000.

TABLE 1. Purification of L-glutamine:keto-scyllo-inositolaminotransferase from M. purpurea 15835

Total Fold ToaExtract protein Sp acta purifi- actityb Yield

(mg) cation

Crude (dialyzed) 4,030 1.85 7,455 100Ammonium sulfate 1,870 3.41 1.8 6,377 85DEAE-agarose 120 53 29 6,360 85Hydroxyapatite 19 150 79 2,850 37Aminobutyl-agarose 7.8 240 130 1,872 26

a Sp act, Nanomoles of product per minute per milligram of protein.Aminotransferase assay was performed at pH 8.5 with 10 mM L-[U-_4C]$lutamine and 0.33 mM keto-scyllo-inositol.

b Total activity, Nanomoles of product per minute (specific activity timestotal milligrams of protein).

TABLE 2. Purification of L-glutamine:keto-scyllo-inositolaminotransferase away from other aminotransferase

activities in M. purpurea 15835

L-Gln:IOa L-Glu:Oxa' L-Glu:Pyr'Extract Totald % Total % Total %

activity Yield activity Yield activity Yield

Crude (dialyzed) 82.52 100 4,010 100 6.9 100Ammonium sulfate 78.06 95 96 2.4 4.6 67DEAE-agarose 43.86 53 0.6 0.01 0.84 121lydroxyapatite 29.88 36 0.2 <0.01 0.16 2.3Aminobutyl-agarose 27.75 34 0.2 <0.01 0.14 2.0

10mM L-[U-14C]glutamine plus 0.33 mM keto-scyllo-inositol (10), pH 8.5.b 17 mM L-[U-14C]glutamate plus 5 mM oxaloacetate (Oxa), pH 7.4.

2-[U-'4Cjketoglutarate was detected following elution from a Dowex 50 (H+)column.

' 17 mM L-[U-14C]glutamate plus 5 mM pyruvate (Pyr), pH 7.4. Productwas detected as for L-Glu:Oxa.

d Total activity, Micromoles of product per minute.

The purified aminotransferase was stable at -20°C (atconcentrations greater than 1 mg/ml) for several months withlittle loss of activity but was inactivated within a monthwhen stored at 4°C. Cruder preparations were less stableunder either condition. Activity could not be restored by theaddition of mercaptoethanol or the cofactor pyridoxal phos-phate.The aminotransferase at the ammonium sulfate stage was

heat labile, losing all activity upon incubation at 50°C for 20min. Neither keto-scyllo-inositol nor pyridoxal phosphateserved to stabilize the aminotransferase when included withthe extract during treatment.The L-glutamine: keto-scyllo-inositol transamination rate

in purified preparations exhibited a marked dependence onpH. Activity increased 40% from pH 7.5 to 8.5 (Table 3). Thereaction exhibited a 60% increase between pH 7.0 and 7.5but no additional increase between pH 8.5 and 9.0 (notshown). There was no difference in activity between pH 7.5and 8.5 when the 2-deoxystreptamine:2-ketoglutaramatetransamination was tested (Table 3).

Preliminary evidence of essential sulfhydryl groups in theaminotransferase was obtained by reacting the enzyme withp-hydroxymercuribenzoate and 5,5'-dithio-bis(2-nitroben-zoic acid). These compounds reduced aminotransferase spe-cific activity by 50%; activity was restored upon the additionof mercaptoethanol or dithiothreitol.

Presence of pyridoxal phosphate at the active site. Exoge-nous pyridoxal phosphate in the assay mixtures had little orno stimulatory effect on aminocyclitol aminotransferase ac-tivity. In addition, the enzyme did not require the presence

TABLE 3. Effect of pH on activities of purified L-glutamine:keto-scyllo-inositol aminotransferase from M. purpurea 15835

ATase sp act (nmol/min per mg)aATase P04 HEPES Tris TAPS

(pH 7.5) (pH 7.5) (pH 8.5) (pH 8.5)

GLN + job 258 239 415 488M-DS + KGMC 58 44 44 55

" ATase, Aminotransferase; HEPES, N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid; TAPS, 3-{[tris-(hydroxymethyl)-methyl]-amino}-propane-sulfonic acid.

b 10 mM L-[U-14C]glutamine (GLN)-0.33 mM keto-scyllo-inositol (IO)-25mM buffer-1.1 ,ug of ATase; 10 min; 37'C.

5S mM N3-methyl-2-deoxystreptamine (M-DS)-0.11 mM 2-ketoglutaramate(KGM)-25 mM buffer-7.4 p.g of ATase; 40 min; 37°C.

2

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ANTIMICROB. AGENTS CHEMOTHER.

w

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350 450 350 450WAVELENGTH, NM

FIG. 3. Changes in aminotransferase absorbance upon additionof amino donors or amino acceptors. Purified L-glutamine:keto-scyllo-inositol aminotransferase from M. purpurea 15835 (2.4 mg/ml)was used. Substrate additions were made directly in the cuvette, andafter the addition of one substrate, the enzyme solution was desaltedby using a 4-ml Sephadex G-25 column before the addition of thenext substrate. (A) Native curve, Spectrum of purified aminotrans-ferase prior to substrate addition (A280 = 2.75). +GLN curve,Spectrum of aminotransferase after addition of L-glutamine to 1.5mM. (B) Curve A, Spectrum of glutamine-treated aminotransferase(from panel A) after desalting (A280 = 1.60). +10 curve, Spectrumafter subsequent addition of keto-scyllo-inositol to 3 mM. (C) CurveB, Spectrum of keto-scyllo-inositol-treated aminotransferase (frompanel B) after desalting (A280 = 0.85). +DS curve, Spectrum aftersubsequent addition of 2-deoxystreptamine to 0.3 mM.

of exogenous cofactor for retention of activity during puri-fication. However, the yellow of the purified aminotrans-ferase preparations was consistent with the presence ofpyridoxal phosphate. To confirm the involvement of thecofactor in reactions catalyzed by this enzyme, as would beexpected for an aminotransferase, the presence of pyridoxalphosphate at the enzyme active site was investigated.The purified aminotransferase exhibited an absorbance

spectrum which is typical of aminotransferases containingpyridoxal phosphate at the active site (4), with a maximumaround A415 (Fig. 3A). The participation of the boundcofactor in reactions catalyzed by the aminotransferase wasindicated most strongly by changes in the absorbance spec-trum following substrate addition (Fig. 3). The absorbancepeak was abolished upon addition of either of the aminodonors L-glutamine or 2-deoxystreptamine. The absorbancepeak was restored upon the subsequent addition of theamino acceptor keto-scyllo-inositol.The aminotransferase was also sensitive to the action of

carbonyl reagents, and pyridoxal phosphate was more effec-tive than pyridoxal for reactivation (Table 4).

Evidence that 2-ketoglutaramate is the keto acid productwhen L-glutamine is the amino donor. Participation of pyri-doxal phosphate in the reactions catalyzed by the ami-notransferase indicated that the alpha amino group of L-glutamine was transferred to the acceptors, rather than theamide group. It was therefore anticipated that the keto acidproduct of transamination with L-glutamine would be 2-ketoglutaramate, as is the case with mammalian L-glutamine:keto acid aminotransferase (10). Following incubation ofL-glutamine, keto-scyllo-inositol, and aminotransferase, the

TABLE 4. Effect of carbonyl reagents on L-glutamine:keto-scyllo-inositol aminotransferase purified from M. purpurea

Sp acta following treatmentb with:Assay condition

Water HydroxaSemiylamine carbazide Hydrazine

Standardc 100 44 63 54+ 0.33 mM pyridoxal-Pd 112 66 71 83+ 0.33 mM pyridoxal 103 49

24-h preincubation+ 0.5 mM pyridoxal-P 117 80+ 0.5 mM pyridoxal 105 54

+ S mM IOe 100 42 40 37a Specific activity was normalized to the value obtained for the "standard"

assay condition without addition of reagents.b Aminotransferase sample was incubated at 4°C with 0.1 M neutralized

reagent (or water) for 1 h (semicarbazide and hydrazine) or 2 h (hydroxyl-amine). L-[U-_4C]glutamine:keto-scyllo-inositol activity was determined fol-lowing desalting of the treated enzyme.

c 5 mM L-[U-14C]glutamine plus 2.5 mM keto-scyllo-inositol (10) at pH 8.5.d pyridoxal-P, Pyridoxal phosphate.I Any reagent left in a treated enzyme sample would be evidenced by an

increase in aminotransferase activity upon increasing keto-scyllo-inositol (10)concentration.

keto acid product was isolated from the reaction mixtures byelution from Dowex 50 (H+) columns with water. Theproduct was compared to authentic 2-ketoglutaramate, syn-thesized as described in Materials and Methods.The reaction product and authentic 2-ketoglutaramate

both reacted with Ehrlich reagent to yield an orange-redcolor, which is characteristic for 2-ketoglutaramate (6).During high-voltage paper electrophoresis, the productcomigrated with 2-ketoglutaramate but not with 2-ketoglut-arate (Fig. 4). The product was converted to 2-ketoglutarateby the action of rat liver omega-amidase, as was authentic2-ketoglutaramate (Fig. 5) (16). Even though the productcomigrated with 2-pyrrolidone-5-carboxylic acid, the car-boxylic acid was not acted upon by the amidase (Fig. 5).

Aminotransferase substrate specificity. The purified ami-notransferase exhibited a very narrow substrate specificity,which was dominated by cyclitol compounds (Table 5). Themajor amino donors to keto-scyllo-inositol were L-gluta-mine, 2-deoxystreptamine, N3-methyl-2-deoxystreptamine,streptamine, and aminodeoxy-scyllo-inositol; these samecompounds were amino donors to 2-ketoglutaramate. Themajor amino acceptors from glutamine were 2-ketoglutara-mate, keto-scyllo-inositol, and D,L-epi-inosose. A variety ofamino and keto acids did not serve as substrates for theaminotransferase, even at elevated concentrations. Aminoacids inactive as donors at physiological concentrations butnot included in Table 5 were as follows: L-alanine, L-glutamic acid, L-asparagine, L-aspartic acid, D,L-homo-serine, D,L-2-aminobutyric acid, L-arginine, glycine, glycyl-glycine, glycinamide, L-methionine, L-glutamic--y-methyl es-ter, D,L-homocysteine, L-cysteine, O-carbamoyl-L-serine,L-citrulline, L-norvaline, L-norleucine, L-2-aminoadipic acid,and L-glutamic-y-ethyl ester.The purified aminotransferase catalyzed reactions repre-

sentative of both transaminations of the 2-deoxystreptaminebiosynthetic pathway. For example, reaction 3 representsthe forward direction of the first transamination, whilereaction 4 represents the reverse direction of the secondtransamination. (Streptamine or N3-methyl-2-deoxystrepta-mine may be substituted for 2-deoxystreptamine in reaction4.)

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AMINOCYCLITOL AMINOTRANSFERASE FROM M. PURPUREA

C.

0

R 10 20 30CENTIMETER

0

40oRFIG. 4. Synthesis and electrophoresis of the keto acid product of

L-glutamine:keto-scyllo-inositol transamination catalyzed by pun-fied aminotransferase from M. purpurea 15835. High-voltage paperelectrophoresis was performed at pH 7.4. Reaction mixtures werespotted at the 10-cm mark (indicated by an arrow) and migratedtoward the positive electrode (indicated by +). GLN, L-[U-14C]Glutamine; PCA, 2-[U-14C]pyrrolidone-5-carboxylic acid; KGM,2-[U-14C]ketoglutaramate standard; KG, 2-[U-14C]ketoglutarate; P,[14C]keto acid product. (A) Migration of standard compounds.Symbols: 0, glutamine standard; 0, 2-pyrrolidone-5-carboxylicacid standard; A, 2-ketoglutarate standard. (B) Synthesis and comi-gration of aminotransferase product with 2-ketoglutaramate stan-dard. Reaction mixtures contained 10 mM L-[U-14C]glutamine and0.33 mM keto-scyllo-inositol in Tris (pH 8.5) incubated at 37°C withor without purified aminotransferase. Symbols: 0, reaction mixtureplus aminotransferase; A, reaction mixture plus aminotransferaseplus overspotted 2-ketoglutaramate standard; 0, reaction mixtureminus aminotransferase.

L-glutamine + keto-scyllo-inositol2-ketoglutaramate + aminodeoxy-scyllo-inositol (3)

2-deoxystreptamine + 2-ketoglutaramate -

2-deoxy-keto-inosamine + L-glutamine (4)Both activities followed the same time course of appearanceduring M. purpurea growth (Fig. 2).

2-Ketoglutaramate-hydrolyzing amidase activity in M. pur-purea. The presence of a 2-ketoglutaramate-hydrolyzingamidase activity was investigated, since a similar enzyme ispresent in mammalian tissues containing an L-glutamineaminotransferase (10). An amidase activity was indeedpresent in extracts of M. purpurea, and its specific activityduring growth paralleled that of the aminotransferase (Fig.2), thus implicating it also as an idiophase enzyme. Theamidase was extremely labile in crude extracts; dialysisalone abolished amidase activity, but this could be preventedby including 2-ketoglutarate in the dialysis buffer. Oncedialyzed, however, even in the presence of 2-ketoglutarate,the amidase activity was rapidly lost. Unlike the mammalianomega-amidase (11), the M. purpurea amidase activity wasnot stimulated by the addition of mercaptoethanol just priorto assay.

DISCUSSIONWe have purified 130- to 150-fold the enzyme which is

responsible for the L-glutamine:keto-scyllo-inositol trans-

FIG. 5. Deamidation by rat liver omega-amidase of the keto acidproduct of L-glutamine:keto-scyllo-inositol transamination. High-voltage paper electrophoresis was performed as for Fig. 4. (A)Conversion of aminotransferase product (P) to 2-ketoglutarate (KG)by rat liver amidase. The [14C]keto acid product was isolated afterL-[U-14C]glutamine transamination by elution from a Dowex 50 (H+)column. Reaction mixtures contained isolated 14C-labeled productin 25 mM Tris (pH 8.5) incubated at 37°C for up to 1 h, with orwithout amidase. Symbols: 0, reaction mixture plus amidase; A,reaction mixture plus amidase plus overspotted 2-ketoglutaratestandard; 0, reaction mixture minus amidase. (B) Conversion of2-[U-14C]ketoglutaramate (KGM) standard to 2-ketoglutarate (KG)by rat liver amidase. Reaction mixtures were incubated as in panelA but with authentic 2-ketoglutaramate replacing the aminotrans-ferase product. Curve symbols are the same as in panel A. (C)Inability of amidase to catalyze reaction with 2-pyrrolidone-5-carboxylic acid (PCA). Reaction mixtures contained 2-[U-_4C]pyrrolidone-5-carboxylic acid, with or without amidase.Symbols: 0, reaction mixture plus amidase; 0, reaction mixtureminus amidase.

amination activity previously observed in crude extracts ofM. purpurea (5). By virtue of its ability to catalyze reactionsrepresentative of both transaminations of 2-deoxystrep-tamine biosynthesis (reactions 3 and 4), the aminotrans-ferase may function at both of these steps in the pathway.However, since the aminotransferase was not purified tohomogeneity, it is possible that our preparation contains twoaminotransferases, each catalyzing one of the representativereactions. Less extensive attempts at separating activitiesrepresenting both 2-deoxystreptamine transaminations froma neomycin-producing S. fradiae were also unsuccessful(24). This is in contrast to the situation with streptomycinproducers, in which two aminotransferases were readilyseparated by gel filtration chromatography of relativelycrude preparations (27).The narrow substrate specificity of the M. purpurea L-

glutamine:keto-scyllo-inositol aminotransferase, coupledwith its time course of synthesis during growth, indicatesthat it is an idiophase enzyme specific for the biosynthesis of2-deoxystreptamine. It is not known whether any of the

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ANTIMICROB. AGENTS CHEMOTHER.

TABLE 5. Substrate specificity of purified L-glutamine:keto-scyllo-inositol aminotransferase from M. purpurea

Compound Sp act'

Amino acceptor from L-glutamine'Keto-scyllo-inositol .......................................... 100D,L-epi-Inosose ......................... ................. 80 (109)2-Ketoglutaramate .......................................... 273Pyruvate ............. ............................. 5 (23)Oxaloacetate .......................................... 3Glyoxylate.......................................... 0 (29)2-Ketoglutarate ......................... ................. 0 (2)2-Ketovalerate ....................... ................... 0 (4)2-Ketocaproate.......................................... 0 (2)2-Ketobutyrate .......................................... 02-Ketoadipate .......................................... 02-Keto--y-methiolbutyrate .......................................ND (4)

Amino donor to keto-scyllo-inositol1Aminodeoxy-scyllo-inositol .................................... 59Streptamine .......................................... 1042-Deoxystreptamine .......................................... 164N3-Methyl-2-deoxy-streptamine ............................... 168L-Glutamine ................................................... 100

Amino donor to 2-ketoglutaramatedAminodeoxy-scyllo-inositol .................................... 19Streptamine .......................................... 772-Deoxystreptamine .......................................... 59N3-Methyl-2-deoxy-streptamine ............................... 100L-Glutamine ............................................... 199

a Specific activity within each group was normalized to the value shown as100. Value in parentheses from using 5 mM amino acceptor. ND, Notdetermined.

b 10 mM L-[U-_4C]glutamine plus 0.33 mM amino acceptor at pH 8.5.c 0.42 mM Keto-scyllo-[14C]inositol plus 0.33 mM amino donor at pH 8.5.d0.13 mM 2-Ketoglutaramate plus 0.86 mM aminodeoxy-scyllo-[U-'4C]

inositol or 0.13 mM 2-[U-14C]ketoglutaramate plus 5 mM amino donor atpH 8.5.

cyclitol substrates of the enzyme are used by M. purpurea(or other producers) in metabolic reactions besides antibioticproduction.

It was necessary to use keto-scyllo-inositol for thesestudies because L-2-deoxyketo-scyllo-inositol, the probablenatural substrate for the aminotransferase, was unavailable.The aminotransferase will accept inosose and inosaminesubstrates with a hydroxyl group present at position 2 of thecyclitol ring. This is consistent with in vivo studies (9, 22) inwhich a 2-deoxystreptamine-less idiotroph of a gentamicinproducer incorporated keto-scyllo-inositol or aminodeoxy-scyllo-inositol into 2-hydroxygentamicin. A similar tolerancefor either the presence or absence of a 2-hydroxyl on thecyclitol substrate has been observed with the aminotrans-ferase of neomycin-producing S. fradiae (5), with the ami-notransferase of the bluensidine pathway (26), and with bothaminotransferases of the streptidine pathway (27). Thistolerance for an equatorial 2-hydroxyl group on the cyclitolring may be a general characteristic of aminocyclitol-synthe-sizing aminotransferases.The M. purpurea aminotransferase was also able to utilize

epi-inosose as an amino acceptor. This indicates that atposition 3 of the cyclitol ring, the aminotransferase cantolerate a hydroxyl group in either the axial or equatorialposition. It would be of interest to determine whetherepi-inosose can be utilized by the rest of the enzymes of 2-deoxystreptamine biosynthesis, since idiotrophs of S. fra-diae incorporate 2-epi-streptamine and an idiotroph ofMicro-monospora inyoensis incorporates 5-epi-2-deoxystreptamineinto antibiotic analogs (8, 23).

The ability of the aminotransferase to catalyze the trans-amination between N3-methyl-2-deoxystreptamine and 2-ketoglutaramate is consistent with the addition of the firstamino group at position 3, as had been previously proposed(5). Addition in this order is also supported by the isolationof L-2-deoxy-scyllo-inosamine from a 2-deoxystreptamine-less idiotroph of the butirosin producer Bacillus circulans(12).

It had been proposed (5) that during L-glutamine:keto-scyllo-inositol transamination the amino nitrogen of glu-tamine is transferred to the cyclitol ring; however, the ketoacid product of this reaction had not been characterized. Wehave shown that the keto acid product of glutamine trans-amination by this aminotransferase is 2-ketoglutaramate.The isolated keto acid product mimicked authentic 2-keto-glutaramate in its reactivity with Ehrlich reagent, in itsmobility during high-voltage electrophoresis, and in its con-version to 2-ketoglutarate by rat amidase.

L-Glutamine is the major physiological amino donor forthe M. purpurea L-glutamine:keto-scyllo-inositol aminotrans-ferase. Using L-glutamine as the donor would favor theformation of the inosamine during transamination, becauseof cyclization of the 2-ketoglutaramate product (reducing theconcentration of the uncyclized molecule for the reversetransamination) and because of hydrolysis of 2-ketoglutara-mate to 2-ketoglutarate by the amidase present in idiophasecells of M. purpurea.The apparent narrow metabolic function of the L-glu-

tamine:keto-scyllo-inositol aminotransferase of M. purpureaand of streptidine producers (27) is in contrast to the functionof the mammalian L-glutamine:keto acid aminotransferase.The mammalian enzyme appears to function in generalmetabolism to salvage the carbon chains of keto acids bytransamination to amino acids for protein synthesis, sincethe enzyme exhibits a broad substrate specificity (7). M.purpurea may also contain a glutamine aminotransferasewhich is used for salvage in general metabolism.We were able to demonstrate that pyridoxal phosphate

participated in the reactions catalyzed by the M. purpureaaminotransferase, as was expected. The cofactor must betightly bound to the active site, since the enzyme was stableto dialysis and did not require exogenous cofactor foractivity (3). In contrast, the cofactor must be more looselyattached to the aminotransferases of streptidine biosynthe-sis, which require exogenous pyridoxal phosphate duringdialysis (27).

Since its initial discovery in streptomycin producers,L-glutamine:keto-scyllo-inositol aminotransferase has beenfound in other aminocyclitol producers as well. We proposethat this is an enzyme which is central to the synthesis ofmany of the aminocyclitol antibiotics. For example, wewould predict the presence of this enzyme in extracts of theastromicin producer Micromonospora olivasterospora,since astromicin biosynthesis begins with the synthesis of apseudodisaccharide containing aminodeoxy-scyllo-inositol(18). We would also expect to detect the L-glutamine:keto-scyllo-inositol aminotransferase in extracts of Strepto-myces hygroscopicus, which synthesizes the inosamycincomplex of antibiotics containing 2-deoxy-scyllo-inosaminein place of 2-deoxystreptamine (25). Because the ami-notransferase can catalyze reactions using cyclitols with orwithout a 2-hydroxyl group, the specificities of other, per-haps earlier, enzymes must determine whether the endproduct will be a 2-hydroxyaminocyclitol or a 2-deoxyami-nocyclitol. Further characterization of the enzymes of ami-nocyclitol biosynthesis from many producers, coupled with

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genetic studies, is necessary to understand completely thebiosynthesis of the aminocyclitol antibiotics and how thesepathways evolved.

ACKNOWLEDGMENTS

This work was supported by grant C-153 from the Robert A.Welch Foundation, Public Health Service grant AI-12278 from theNational Institutes of Health, and grant PCM 7809755 from theNational Science Foundation. L.A.L. was a predoctoral fellow ofthe Robert A. Welch Foundation.

LITERATURE CITED1. Bergmeyer, H. U., and E. Bernt. 1974. 2-Oxoglutarate. UV

spectrophotometric determination, p. 1577-1580. In H. U.Bergmeyer (ed.), Methods of enzymatic analysis, vol. 3. Aca-demic Press, Inc., New York.

2. Bernt, E., and H. U. Bergmeyer. 1974. L-Glutamate. UV-assaywith glutamate dehydrogenase and NAD+, p. 1704-1708. InH. U. Bergmeyer (ed.), Methods of enzymatic analysis, vol. 4.Academic Press, Inc., New York.

3. Braunstein, A. E. 1960. Pyridoxal phosphate, p. 113-184. In P.Boyer, H. Lardy, and K. Myrback (ed.), The enzymes, 2nd ed.,vol. 2. Academic Press, Inc., New York.

4. Braunstein, A. E. 1973. Amino group transfer, p. 379-481. In P.Boyer (ed.), The enzymes, 3rd ed., vol. 9. Academic Press,Inc., New York.

5. Chen, Y., and J. B. Walker. 1977. Transaminations involvingketo- and amino-inositols and glutamine in actinomycetes whichproduce gentamicin and neomycin. Biochem. Biophys. Res.Commun. 77:688-692.

6. Cooper, A. J. L. 1978. Spot test for the detection of 2-ketoglu-taramic acid in human cerebrospinal fluid. Anal. Biochem.90:444-446.

7. Cooper, A. J. L., and A. Meister. 1974. Isolation and propertiesof a new glutamine transaminase from rat kidney. J. Biol. Chem.249:2554-2561.

8. Daum, S. J., and J. R. Lemke. 1979. Mutational biosynthesis ofnew antibiotics. Annu. Rev. Microbiol. 33:241-265.

9. Daum, S. J., D. Rosi, and W. A. Goss. 1977. Mutationalbiosynthesis by idiotrophs of Micromonospora purpurea. II.

Conversion of non-amino containing cyclitols to aminoglycosideantibiotics. J. Antibiot. 30:98-105.

10. Duffy, T. E., A. J. L. Cooper, and A. Meister. 1974. Identifica-tion of 2-ketoglutaramate in rat liver, kidney, and brain. Rela-tionship to glutamine transaminase and omega-amidase activi-ties. J. Biol. Chem. 249:7603-7606.

11. Hersh, L. B. 1971. Rat liver omega-amidase. Purification andproperties. Biochemistry 10:2884-2891.

12. Igarishi, K., T. Honma, T. Fujiwara, and E. Kondo. 1980.Structure elucidation of an intermediate of 2-deoxystreptaminebiosynthesis. J. Antibiot. 33:830-835.

13. Kakinuma, K., Y. Ogawa, T. Sasaki, H. Seto, and N. Otake.1981. Stereochemistry of ribostamycin biosynthesis. An appli-cation of 2H NMR spectroscopy. J. Am. Chem. Soc. 103:5614-5616.

14. Lowry, 0. H., N. J. Rosebrough, A. L. Farr, and R. J. Randall.1951. Protein measurement with the Folin phenol reagent. J.Biol. Chem. 193:265-275.

15. Maier, S., and H. Grisebach. 1979. Biosynthesis of streptomy-cin. Enzymic oxidation of dihydrostreptomycin(6-phosphate) tostreptomycin(6-phosphate) with a particulate fraction of Strep-tomyces griseus. Biochim. Biophys. Acta 586:231-241.

16. Meister, A. 1953. Preparation and enzymatic reactions of theketo analogues of asparagine and glutamine. J. Biol. Chem.200:571-589.

17. Millet, J. 1970. Characterization of proteinases excreted byBacillus subtilis Marburg strain during sporulation. J. Appl.Bacteriol. 33:207-219.

18. Odakura, Y., H. Kase, S. Itoh, S. Satoh, S. Takasawa, K.Takahashi, K. Shirahata, and K. Nakayama. 1984. Biosynthesisof astromicin and related antibiotics. II. Biosynthetic studieswith blocked mutants of Micromonospora olivasterospora. J.Antibiot. 37:1670-1680.

19. Pearce, C. J., and K. L. Rinehart, Jr. 1981. Biosynthesis ofaminocyclitol antibiotics, p. 73-100. In J. W. Corcoran (ed.),Antibiotics IV. Biosynthesis. Springer-Verlag KG, Berlin.

20. Porter, J. N. 1975. Cultural conditions for antibiotic-producingmicroorganisms. Methods Enzymol. 43:3-23.

21. Rinehart, K. L., Jr., J.-R. Fang, W.-Z. Jin, C. J. Pearce, K.-I.Tadano, and T. Toyokuni. 1985. Biotransformations and biosyn-thesis of aminocyclitol antibiotics. Dev. Ind. Microbiol. 26:117-128.

22. Rosi, D., W. A. Goss, and S. J. Daum. 1977. Mutationalbiosynthesis by idiotrophs of Micromonospora purpurea. I.Conversion of aminocyclitols to new aminoglycoside antibiot-ics. J. Antibiot. 30:88-97.

23. Shier, W. T., K. L. Rinehart, Jr., and D. Gottlieb. 1969.Preparation of four new antibiotics from a mutant of Strepto-myces fradiae. Proc. Natl. Acad. Sci. USA 63:198-204.

24. Suzukake, K., K. Tokunaga, H. Hayashi, M. Hori, Y. Uehara, D.Ikeda, and H. Umezawa. 1985. Biosynthesis of 2-deoxystrep-tamine. J. Antibiot. 38:1211-1218.

25. Tsunakawa, M., M. Hanada, H. Tsukjura, M. Konishi, and H.Kawaguchi. 1985. Inosamycin, a complex of new aminoglyco-side antibiotics. II. Structure determination. J. Antibiot. 38:1313-1321.

26. Walker, J. B. 1975. Pathways of biosynthesis of the guanidi-nated inositol moieties of streptomycin and bluensomycin.Methods Enzymol. 43:429-470.

27. Walker, J. B., and M. S. Walker. 1969. Streptomycin biosyn-thesis. Transamination reactions involving inosamines andinosadiamines. Biochemistry 8:763-770.

28. Walker, J. B., and M. S. Walker. 1982. Enzymatic synthesis ofstreptomycin as a model system for study of the regulation andevolution of antibiotic biosynthetic pathways, p. 271-281. In V.Krumphanzl, B. Sikyta, and Z. Vanek (ed.), Overproduction ofmicrobial metabolites. Academic Press, Inc., New York.

29. Weber, K., J. R. Pringle, and M. Osborn. 1972. Measurement ofmolecular weights by electrophoresis on SDS-acrylamide gels.Methods Enzymol. 26:3-27.

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