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Биология Биология

The production of 2H-labeled amino acids by a new mutant of RuMP fucultative methylotroph Вrevibacterium methylicum

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he produc io of 2H-labeled ami o acids by a ew mu a of RuMP fucul a ive me hylo roph Вrevibac erium me hylicum Oleg V. Mosi 1 1 Depar me of Bio ech ology, M. V. Lomo osov S a e Academy of Fi e Chemical ech ology, Ver adskogo Prospek 86, 117571, Moscow, Russia Summary he biosy hesis of 2H-labeled phe ylala i e was do e by co verse of low molecular weigh subs ra es (me ha ol a d 2H2O) i a ew RuMP facul a ive me hylo rophic mu a Brevibac erium me hylicum. o make he process work, adap ed cells wi h improved grow h charac eris ics were used o mi imal medium M9 wi h he maximum co e of 2H-labeled subs ra es. Ala i e, vali e, a d leuci e/isoleuci e were produced a d accumula ed exoge eously i addi io o he mai produc of biosy hesis. Elec ro impac mass spec rome ry of me hyl es ers of he -D s-ami o acid mix ure ob ai ed af er he chemical deriva iza io of grow h medium wi h da syl chloride a d diazome ha e, was do e o calcula e he deu erium e richme of he ami o acids sy hesized. he experime al da a es ified o he charac er of labeli g of ami o acid molecules as he eroge eous; however, high levels of deu erium e richme were de ec ed i all prese ed molecules - for phe ylala i e he e richme was six, leuci e/isoleuci e - 5.1, vali e - 4.7, a d ala i e - 3.1 deu erium a oms. Keywords: Brevibac erium me hylicum - Heavy wa er - Biosy hesis - 2H-Labeled ami o acids - Phe ylala i e - EIMS Abbrevia io s: EI MS: elec ro impac mass spec rome ry; LC: hi layer chroma ography; D SCl: da sylchloride; DZM: diazome ha e; - MU: - i roso-me hylurea; RuMP: rybolose mo ophospha e; Pe P: pe ose phospha e; PEP: phosphoe olpyruva e; ERP: ery hrose-4-phospha e. I roduc io Labeli g of ami o acid molecules wi h deu erium is becomi g a esse ial par for various biochemical s udies wi h 2H-labeled molecules a d i ves iga io of cer ai aspec s of heir biosy hesis(LeMas er, 1990). For i roduc io of deu erium i o ami o acid molecules ei her chemical or biosy he ical me hods may be used. Chemical sy hesis of hese compou ds has o e sig ifica limi a io ; i is a very laborious a d cos ly mul is ep process resul i g i a mix ure of dl-racema es. his major disadva age, however esse ially delayi g i s developme is a difficul y i prepari g he appropria e 2H-labeled ami o acids. Chemical sy hesis usually resul s i ob ai i g a mix ure of d,l-racema es (Daub, 1979). Al hough chemomicrobiological sy hesis overcomes his problem (Walker, 1986), he amou of purified e zymes required is prohibi ive (Faleev, 1989). By growi g algae o media wi h 96% (v/v) 2H2O, he desired 2H-labeled biochemicals ca be produced bo h a high yields a d e richme s (Cox, 1988), bu he process i volves algae is limi ed by he expe se of a mix ure of 2H-labeled ami o acids isola ed from hydrolysa es of biomass (Daboll, 1962). he usi g for his purpose a cer ai me hylo rophs which assimila es Me OH as a source of carbo a d e ergy via RuMP cycle has a grea prac ical adva age because heir abili y o produce a d acumula e a gram qua i ies of 2H-labeled ami o acids duri g he grow h o media wi h 2H2O a d Me OH a d he compara ively low price of Me OH (Kar aukhova, 1994).

he biosy hesis of 2H-labeled ami o acids usually i volves grow h of a orga ism o selec ive media co ai i g he labeled subs ra es: e.g., grow h of algae au o rophically o media wi h co e of 2H2O 90% a d more, is a well es ablished me hod for biosy hesis of umerous highly deu era ed molecules. Bu his me hod, while bei g ge erally applicable, is limi ed by he low resis a ce of pla cells o 2H2O a d expe se of 2H-labeled ami o acids isola ed from algae hydrolysa es. Al er a ive a d rela ively i expe sive objec s for biosy hesis of 2H-labeled ami o acids seem cer ai auxo rophic mu a s of me hylo rophic bac eria usi g me ha ol as a mai source of carbo a d e ergy via he ribulose-5-mo ophospha e (RuMP) a d he seri e cycle of carbo assimila io . hese bac eria have a big adva age because of heir abili y o produce a d accumula e gram qua i ies of highly e riched, 2H-labeled ami o acids duri g grow h o mi imal sal media wi h me ha ol a d 2H2O a d he compara ively low price of me ha ol. I is o ly i rece years ha some progress was made i he isola io of a umber of versa ile he RuMP cycle me hylo rophic bac eria, sui able for such s udies, hough he research ha has bee do e wi h me hylo rophs was limi ed a d suffered from low grow h charac eris ics o 2H2O-co ai i g media. Al hough he produc io of 2H-labeled ami o acids by obliga e me hylo roph Me hylobacillus flagella um described by Kar aukhova i volves he grow h o media wi h approxima ely 75% (v/v) 2H2O. We have rece ly selec ed a ew mu a of facul a ive me hylo roph Brevibac erium me hylicum, realizi g he AD depe de me ha ol gehydroge ase (EC 1.6.99.3) varia of RuMP cycle of carbo assimila io , which seems more co vi ie for he prepara io of 2H-labeled ami o acids ha M. flagella um because i s abili y o grow o liquid M9 wi h 98% (v/v) 2H2O (Mosi , 1995). hus, we have previously s udied he applicabili y of he RuMP cycle obliga e me hylo rophic bac erium Me hylobacillus flagella um for biosy hesis of 2H-labeled leuci e 8). his approach is o ye prac ical for he biosy hesis of 2H-labeled phe ylala i e, mai ly because of he abse ce of sui able me hylo rophic producer of his ami o acid. Af er selec i g a ew he RuMP cycle me hylo rophic producer of phe ylala i e, leuci e auxo roph Brevibac erium me hylicum, we have used his s rai for his research. Ma erial a d me hods 2H2O (99.9 a .% 2H) was purchased from Russia Scie ific E erprises, Sa k Pe ersburg. Me OH (97.5 a .% 2H) was from Biophysic Ce er, Pushi o. D SCl of seque ial grade was from Sigma Chemicals Corp., USA. DZM was prepared from - MU, Pierce Chemicals, Corp., USA. A gram-posi ive pare al s rai of RuMP facul a ive me hylo roph Brevibac erium me hylicum # 5662 was ob ai ed from Russia S a e Scie ific Ce er for Ge e ics a d Selec io of I dus rial Microorga isms G IIGE E IKA ( esvera, 1991). Basal sal medium M9 (Miller, 1976) wi h Me OH as a carbo a d e ergy source (2%, v/v) a d suppleme ed wi h Leu (100 mg/l) was used for bac erial grow h. For iso opic experime s M9 was e riched wi h Me OH a d 2H2O of various co e (see able below). he bac erial grow h was carried ou u der ba ch co di io s (Kar aukhova, 1994).

he expo e ially growi g cells (cell de si y 2.0 a absorba ce 540 m) were pelle ed by ce rifuga io (1200 g for 15 mi ), he super a a was lyophilized a d used for chemical deriva iza io . he amou of Phe was de ermi ed for 10 ml aliquo es of liquid M9 by LC wi h solve of iso-PrOH-ammo ia (7:3, v/v) usi g pure commercial available Phe as a s a dard. he spo s were de ec ed by 0.1% i hydri e solu io i ace o e, elu ed by 0.5% CdCl2 solu io i 50% E OH (2 ml). he absorba ce of he elua es was measured a 540 m, he co ce ra io was calcula ed usi g a s a dard curve. he samples of lyophilized M9 were da syla ed i 1 M sodium hydrohycarbo a e-ace o e (1:2, v/v) solu io (pH 10-11) wi h e fold excess of D SCl, a d rea ed accordi g o Deve yi (1976). he deriva iza io o me hyl es ers of -D S-ami o acids was performed i a s a dard procedure wi h DZM (Gree s ei , 1976). EI MS was performed o Hi achi MB 80 spec rome er a io izi g e ergy 70 eV a d a io source empera ure of 180oC. Resul s a d discussio Phe is sy hesized i mos bac eria via shikimic acid pa hway (Co , 1986). he precursors for he biosy hesis of Phe are PEP a d ERP. he la er compou d is a i ermedia e i he Pe P pa hway a d, i some me hylo rophs, he RuMP cycle of carbo assimila io (A o y, 1982; Kle sova, 1988). I is widely accep ed, ha he a ive bac erial s rai s ca o o be a s ro g producers of Phe owi g o he effec ive mecha isms of i s me abolic regula io , al hough cer ai bac erial mu a s wi h mu a io s of prephe a e dehydroge ase (EC 1.3.1.12), prephe a e hydra ase (EC 4.2.1.51), chorisma e mu ase (EC 5.4.99.5) a d a umber of o her several e zymes are proved o be a ac ive producers of his ami o acid (Umbarger, 1978). ha is why he bes Phe produci g s rai s o ce selec ed were he mu a s par ially or comple ely depe de o yr or rp for grow h. he repor s abou he o her regula ive mecha isms of Phe biosy hesis i bac erial cell are qui e u commo , hough oday i is k ow a large umber of RuMP cycle auxo rophic mu a s of me hylo rophs, coveri g umerious s eps i aroma ic ami o acid biosy hesis (Dijkhuize , 1996). he selec io of ew producers of Phe has a big impor a ce for s udies of heir regula i g pa hways a d possible produc io of 2H-labeled Phe. For our s udies we have used a ew o - radi io al producer of phe ylala i e: a leuci e auxo roph of he facul a ive me hylo rophic bac erium Brevibac erium me hylicum ob ai i g he AD depe de me ha ol dehydroge ase (EC 1.6.99.3) varia of he RuMP cycle of carbo assimila io , wi h maximum produc ivi y of phe ylala i e o pro o a ed medium M9 - 0.95 gram per li er of grow h medium. Accordi g o experime s, various composi io s of me ha ol a d 2H2O were added o he grow h media as hydroge (deu erium) a oms could be assimila ed bo h from me ha ol a d H2O. he grow h charac eris ics of he o -adap ed bac eria a d produc io of phe ylala i e i he prese ce of i creasi g co e of 2H2O are give i able (Exp s. 3-10) rela ive o he co rol (1) o pro o a ed medium a d o he adap ed bac eria (Exp . 10’). he odd umbers of experime were chose o i ves iga e whe her he replaceme of me ha ol of i s pro o a ed a alogue has a posi ive effec o grow h charac eris ics i he prese ce of 2H2O.

Rutherford: Simple Genius. Cambridge, Mass.: MIT Press, 1983. Wilson, Edward O. The Diversity of Life. Cambridge, Mass.: Belknap Press/Harvard University Press, 1992. Winchester, Simon. The Map That Changed the World: The Tale of William Smith and the Birth of a Science. London: Viking, 2001. Woolfson, Adrian. Life Without Genes: The History and Future of Genomes. London: Flamingo, 2000. INDEX *The following items may be used as a guide to search for information in this eBook. A ABC of Relativity, The (Russell) adenosine triphosphate (ATP) Agassiz, Louis AgassizБЂ™s theory Ager, Derek V. Aharanov, Yakir AIDS; HIV virus albedo (reflectiveness) alchemy All Species Foundation Alpha Centauri aluminum Alvarez, Walter and Luis America (New World): Ashfall Fossil Beds, Nebraska; asteroid craters; BuffonБЂ™s assertion of inferiority of; dinosaur bones, tracks, locations; dinosaurs, discovery of; human-caused extinctions; as land of opportunity; Lewis and Clark expedition; mammoth bones in; Sierra Nevada mountains; volcanoes American Museum of Natural History American Ornithology (Peale) amino acids Anderson, Ray anesthesia animal world: categories; unknown or new species

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98. Population of the UK

99. The art of conversation


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