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C1 Assimilation in Obligate Methylotrophs and Restricted Facultative Methylotrophs by JOHN COLBY* and LEONARD J

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C1 Assimilation in Obligate Methylotrophs and Restricted Facultative Methylotrophs by JOHN COLBY* and LEONARD J Biochem. J. (1975) 148, 513-520 513 Printed in Great Britain Enzymological Aspects ofthe Pathways for Trimethylamine Oxidation and C1 Assimilation in Obligate Methylotrophs and Restricted Facultative Methylotrophs By JOHN COLBY* and LEONARD J. ZATMAN Department ofMicrobiology, University ofReading, Reading RG1 5AQ, U.K. (Received8 January 1975) 1. Extracts of trimethylamine-grown W6A and W3A1 (type M restricted facultative methylotrophs) contain trimethylamine dehydrogenase whereas similar extracts of Bacillus PM6 and Bacillus S2A1 (type L restricted facultative methylotrophs) contain trimethylamine mono-oxygenase and trimethylamine N-oxide demethylase but no trimethylamine dehydrogenase. 2. Extracts oftherestricted facultatives and ofthe obligate methylotroph C2A1 contain hexulose phosphate synthase-hexulose phosphate isomerase activity; hydroxypyruvate reductase was not detected. 3. Neither the restricted facultatives nor the obligates 4B6 and C2A1 contain all the enzymes ofthe hexulose phos- phatecycleofformaldehyde assimilation as originallyproposed byKemp & Quayle (1967). 4. Organisms PM6 and S2A1 lack transaldolase and use a modified cycle involving sedoheptulose 1,7-diphosphate and sedoheptulose diphosphatase. 5. The obligates 4B6 and C2A1, and the type M organisms W6A and W3A1, use a different modification of the assimilatory hexulose phosphate cycle involving the Entner-Doudoroff-pathway enzymes phosphogluconate dehydratase and phospho-2-keto-3-deoxygluconate aldolase. Thelackoffructosediphosphatealdolaseandhexosediphosphataseintheseorganismsmay be a partial explanation of their restricted growth-substrate range. 6. Enzymological evidence suggests that all the obligates and the restricted facultatives use a dissimilatory hexulose phosphate cycle to accomplish the complete oxidation of formaldehyde to CO2 and water. Colby & Zatman (1975) described four pure bac- chloride, paraformaldehyde, methanol, sodium terial cultures each ofwhich grows on trimethylamine formate and 2,6-dichlorophenol-indophenol were and on a restricted range of other compounds; obtained from BDH Chemicals Ltd., Poole, Dorset, such organisms were designated restricted facul- U.K.; trimethylamine hydrochloride was obtained tative methylotrophs. Thus the type M isolates W6A from Ralph N. Emmanuel Ltd., Wembley, Middx., and W3A1 grow only on glucose out of 50 non-C1 U.K. Phenazine methosulphate, GSH, D-ribose compounds tested, whereas the type L isolates 5-phosphate (disodium salt), sedoheptulose 1,7- Bacillus PM6 and Bacillus S2A1 grow on betaine, diphosphate (sodium salt), lithium hydroxypyruvate, glucose, gluconate, citrate, glutamate, alanine and thiamine pyrophosphate chloride, cysteine hydro- nutrient agar, but not on any of the other 56 non-C1 chloride, ribose phosphate isomerase (spinach) compounds tested. The present paper describes (EC 5.3.1.6), glucose phosphate isomerase (rabbit investigations of the mechanisms used by these muscle) (EC 5.3.1.9), transketolase (yeast, type IV) organisms for the oxidation of trimethylamine and (EC 2.2.1.1) and ribulose phosphate 3-epimerase for the assimilation of trimethylamine carbon. The (yeast) (EC 5.1.3.1) were purchased from Sigma results are compared with those obtained from two (London) Chemical Co. Ltd., Kingston-upon- previously described obligate methylotrophs, 4B6 Thames, Surrey, U.K. The Boehringer Corporation and C2A1 (Colby & Zatman, 1972, 1973). (London) Ltd., London W.5, U.K. supplied NAD+, NADP+, NADPH, NADH, ATP, fructose 6-phos- Materials and Methods phate (disodium salt), fructose 1,6-diphosphate (trisodium salt), glucose 6-phosphate (disodium salt), Materials gluconate 6-phosphate (trisodium salt), glucose Dimethylamine hydrochloride, methylamine 6-phosphate dehydrogenase (yeast, grade I) (EC hydrochloride, trimethylamine N-oxide hydro- 1.1.1.49), aldolase (rabbit muscle) (EC 4.1.2.13), * Present address: Department of Biological Sciences, lactate dehydrogenase (pig heart) (EC 1.1.1.27) and University of Warwick, Coventry CV4 7AL, U.K. glycerol 3-phosphate dehydrogenase-triose phos- Vol. 148 17 514 J. COLBY AND L. J. ZATMAN phate isomerase mixture (rabbit muscle) (EC 1.1.1.8 Spectrophotometry. All spectrophotometric meas- and EC 5.3.1.1. respectively). Dithiothreitol was urements were made in a Hitachi Perkin-Elmer 124 obtained from Koch-Light Laboratories Ltd., double-beam grating instrument (Perkin-Elmer Colnbrook, Bucks., U.K. Cultures of Hypho- Ltd., Beaconsfield, Bucks., U.K.) fitted with a microbium strains X and G (Attwood & Harder, constant-temperature cuvette housing and coupled to 1972) were kindly supplied by Dr. Margaret Attwood. a Servoscribe chart recorder (Smiths Industries Ltd., Wembley, Middx., U.K.). Methods Measurement of enzyme activities in crude sonic extracts. All assays were done at 30°C. The assay Preparation ofcrude sonic extracts. Organisms were methods for trimethylamine dehydrogenase, tri- grown, harvested in mid-exponential phase and crude methylamine mono-oxygenase (spectrophotometric sonic homogenates prepared from the washed method), dimethylamine mono-oxygenase, primary suspensions as described by Colby & Zatman (1972, amine dehydrogenase, methanol dehydrogenase 1975). Enzyme assays were done on crude sonic (EC 1.1.99.8), formaldehyde dehydrogenase (NAD+) extracts prepared by centrifuging the homogenates (EC 1.2.1.1), formaldehyde dehydrogenase (2,6- at 10000g for 20min. Extracts forhexulose phosphate dichlorophenol-indophenol), formate dehydrogenase synthase-hexulose phosphate isomerase, hexose (EC 1.2.1.2) and hydroxypyruvate reductase (EC diphosphatase (EC 3.1.3.11) and sedoheptulose 1.1.1.29) were those described by Colby & Zatman diphosphatase assays were prepared from cells (1972, 1973). Hexulose phosphate synthase and washed twice with 50mM-Tris-HCI buffer, pH7.0, hexulose phosphate isomerase (see Cox & Zatman, and resuspended in 50mM-triethanolamine-HCI 1974) were assayed together spectrophotometrically buffer, pH7.5, containing 5mM-MgCI2. as described by Dahl et aL (1972), except that assays Protein estimations. The concentrations of protein were done in 50mM-triethanolamine-HCl-NaOH in crude sonic extracts were determined with the buffer, pH7.5, and contained 1.5units of ribose- Folin-phenol reagent (Kennedy & Fewson, 1968) phosphate isomerase. These modifications ensure with crystalline bovine plasma albumin (Armour respectively that interference by glyceraldehyde Pharmaceutical Co. Ltd., Eastbourne, Sussex, U.K.) phosphate dehydrogenase which might be present in as the standard. the crude extracts would be minimized by excluding Buffer solutions. These were prepared as described phosphate, and that the production of ribulose by Dawson et al. (1969). 5-phosphate (see Kemp, 1972) from ribose 5-phos- Spectrophotometric estimation of pyruvate, triose phate would not be rate-limiting. The activities of the phosphate and glycerol 3-phosphate in reaction following enzymes were determined by the methods mixtures. Samples (0.5ml) were first deproteinized by quoted: glucose phosphate isomerase (Wu & Racker, the addition of 0.5ml of 1 M-HC104, and the 1959); 6-phosphofructokinase (EC 2.7.1.11; Ling denatured protein was removed by centrifugation. et al., 1966); ribose phosphate isomerase (Axelrod & Tripotassium phosphate (0.7M, 0.25nml) was then Jang, 1954); glucokinase (EC 2.7.1.2; Anderson & added to 0.5ml samples of the supematants at 0°C Kamel, 1966); glucose 6-phosphate dehydrogenase and, after allowing the KC104 to sediment, the (Kornberg & Horecker, 1955); phosphogluconate resulting solutions were used for the estimations. dehydrogenase (EC 1.1.1.44; Horecker & Smyrniotis, Pyruvate was estimated in assay mixtures containing 1955). in 1 ml total volume: 50,umol of sodium phosphate Trimethylamine N-oxide demethylase. This enzyme buffer, pH7.0; 0.34umol of NADH; lactate dehydro- was assayed by the colorimetric method B of Myers genase (5units); test solution (0.1 ml, 0-0.2,umol of (1971). Reaction mixtures (1.5ml total volume) con- pyruvate). Triose phosphate was estimated in assay tained: 100,amol of triethanolamine-HCI-NaOH mixtures (1 ml) containing: 751umol of glycylglycine- buffer, pfI8.0; 10,umol of GSH; 100umol of FeSO4; NaOH buffer, pH7.6; 0.3umol of NADH; glycerol 25 umol of sodium L-ascorbate; crude sonic extract; 3-phosphate dehydrogenase-triose phosphate iso- SOumol of trimethylamine N-oxide hydrochloride merase mixture (5 and 30 units respectively); test (adjusted to pH8). The reaction was started by the solution (0.1 ml, 0-0.2#mol of triose phosphate). addition of substrate and samples were then removed In both cases the decrease in E340 on addition of the at intervals for the determination of formaldehyde test solution was measured at 30°C. Glycerol 3- as described by Colby & Zatman (1973). phosphate was estimated by measuring the increase Transketolase. Reaction mixtures contained in in E340 at 30°C on addition of a test solution 1 ml: 50,umol of glycylglycine-NaOH buffer, pH7.6; (0.1ml, 0-0.2,umol of glycerol 3-phosphate) to an 1, mol of MgC92; 0.1,umol of thiamine pyrophos- assay mixture (0.9ml) containing: 200,umol of phate; 0.17pmol of NADH; glycerol 3-phosphate glycine-NaOH buffer, pH9.8; 500,umol ofhydrazine dehydrogenase-triose phosphate isomerase mixture hydrate; 20,umol of NAD+; glycerol 3-phosphate (0.5 and 3 units respectively); ribose phosphate -dehydrogenase (5 units). isomerase (1.5 units); ribulose phosphate 3-epimerase 1975 TRIMETHYLAMINE OXIDATION AND ASSIMILATION IN METHYLOTROPHS 515 (1 unit); crude sonic
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