CA2214458A1 - Enzymatic process for producing gdp-alpha-d-mannose, a gdp mannose pyrophosphorylase and phosphomannomutase suitable for that process, the extraction of the said enzymes, and an enzyme test - Google Patents

Abstract

The invention concerns a GDP-mannose-pyrophosphorylase. The aim of the invention is to produce a GDP-mannose-pyrophosphorylase which can be obtained for an acceptable outlay and does not cause problems, in particular because of its monofunctionality, in continuous multiple stage processes. To that end, a mannose- or mannose-derivative-specific GDP-mannose-pyrophosphorylase, which can be isolated from microorganisms and has a specific activity of 2 U/mg, is prepared.

Description

, u<~ ~v~ v l r~~ u ~ vJ oc ~~ vvv Enzymat~c synthesis o~ GDP-a-D-manno~a _ _ ~ n 0il ~~P
3-MallROS~ O ll~l~ose 6- P

s ~3 ~-. ~ rr, G

~ ? . r k . t~C ~ S r~ ,' ' 1~31LLl~
3 ~ . O ~ a;u7 The ob~ect of the inven~ion i-~ a new G~P-mannos~-pyrophosphorylase ~GDPMan-PP) that is mono*~nctional wlth re~p~ct to the hexose re~iduer of mi~robial origin, whi~h hao a specific activity >2 U/mg~ a~d it comprises a method for ~he preparation of said enzyme as well a~ u~e in the preparation o~ GDP-~annose.
GDP-mannose is one of th~ activated sugars that ~t thi~ timo have be~n ~xtensivelv Qxami~ed, an~ which can ~e r~ac~d with glyco~yl tran-~erases to form oligosaccharide3. Moreover, it forms the starting mater~al i'or the preparation of GDP-fuco~e.
GDP-mannose pyropho~phorylase has been known ~or a long time. It has been isolated from various sources: in 1964 by Preiss et al. ~ Biol. Chem., Vol. Z39, pp, 3119-26, 1964) via "6, rr~ u r~ ~ alp~ Y~ rOY ~ bl;O~ ~ UU ~
, .

the isolation of the enzyme ~rom Arthro~act~r ~p. D. Shlnabarger et al. (J. Biol. Chem., VolO 2~6, pp. 2080-88, l9gl) describe the isolation o~ a multif~nctional GDP-Man-PP ~rom ~soudomonas aeruginosa with pho~sph~-m~nn~se isomera:3e and py~ophosphorylase activity .
A GDP-~an-~?P isolated iErom mammalian glands catalyze~ both the synthesis of GDP-mannos~ and GDP-~lucose. A cr)p-M~-pp with 70, 000-~old purification wa~3 prepared from porcine thyroid ~lands, which pre~ented no ~;t;P-g~ucose synthesis acti~lty.
T. Szumilo et al. (J. Biol Chen~., Vol. 268, pp. 17943-50, 1993) reports on the isolatlon and 5000-~old purification of ~DP-Man-PP from porcine liver, whe~ce 4 mg o~ enzyme with a SpQCif~iC
ac~ivity of 9 . 25 U~mg was isolated fro~ 1 kg o~ l~ ver in a multistap purlfication. This enzyme catalyzes bo~h the for~ation of CDP-manno~e and of GDP-glucosQ.
More recen~ly, GDP-~an-PP-ouelle [sic~ possibly quelle =
~ource~ has been obtained prlmarily from yea~t ~S. cereYi~iae~
as a rulQ, it is not subiected to a specific purification ~.
Wang ct al. in J. O~g. Chem., Vol. 58, pp. 3~85-9~, 1993). In W093/0820 Al, a report 1s made o~ the purification of GDP-Man-PP
from yeast, where an enzyme solution with an activity of 0.1 U~ml w~s obtained from a yeast cell extract by fr~ction~ted (NH~2So~
precipitation ~nd dialysis.
In ~ummary it can now be obser~ed that the commercially unavailable GDP-Man-PP is either isolatQd with very great exp~ns~
or it i~ us~d in nonpurlfie~ or only partially purified form;
also, and the forms of t~e enzyme differed, in part prese~ting mul~function~lities.
The goal of the ~nvention there~ore is a GDP-Man-PP that can be obtained for an acceptable expense and doQs not lead to u~ rK~ rA~ n ~c~lr~ c. ~v~

problem~;, particularly becaus~ o~ it~ monofunctional~ty, in contin~ous multiple ~tage processeS over longer peri~ds o~ timo.
The GDP-Man-PP de~eloped for th~s purpose corrQsponds to Clai~ 1. --Other characteristics o~ the invention can be obtainQd ~rom the secondary claim.
GDP-Man-PP is obtained in particular f~m a recombinant ~train of mic~oorganis~ ~uch as yeasts, ~. subtilis, and ~. coli strain~ as well as, po~;sibly, ~rom cell lin~3s of ~n~r~.l origin, which are ~uitable for ~o~i~lcation by ~enetic engineering to ma~e producing strains and into which plasmlds of the known type have been inserted, with these having been manipulated by gen~tic en~ineering to contain the ~ene codi~g for th~ de6ir~d formation Of GDP-~an-PP, where the raw extract of the microorganism8 contain~ the enzyme at a con~iderable concent~ation, so ~hat the expe~se required for the preparation and puri~ication for commercial product~on is entirely acceptable.
The table ~elow shows the enzyme contents o~ the raw extract for different enzyme sources, the specific acti~ity (to the exte~t known) obtained a~ter purification, and the functlonality o~ the enzyme produced.

08/2~/~7 FRI 1~:21 FA~ Ralph llcElro~ A660c. 4~1009 Compar~son of the enzym~ :sourc~ for GDP-mannose pyropho~sphoryla~

~c~lvlt~-n ~Ct1VILt~t n~ch sp-z~ t~t bz~.
P~ n~g~g ~.~ t~t mq Prot~in) (V/-oq Prot~inl (n-.ch 0,0167 ~.~7 ~ c 1-Munch-Pe~ n hocphi~t 1~62 ~5~ n~ cr 0. 00 ~ ~. 25 IGl~leos~
W ~ C~ S:lu~ t ~ Pho:sph~
.g9~J ~V M~rLn~e~l-pho cph~ t P~ .3~ 6,~ unkeionelles r~e~q~ no~a ~ ~n~ us G~
W~ hln;sb~rg~r c~. ~An- PP uo~:l ~1 . 19~1) p;~L ' -nn~7~!1e i~cmer~se ~cherichLa eoli 0, 003 ~) ~Wildstl~mmJ
~1 g~nC MC~ ng mit r~USSA
-kombln~,n~or ~:. O, 370 2, 3 ,~M~ Je-l-~;~ col i S~ m (~ho~ph~c ~ r.d~ .q~

J(~)L~ r~ur~ e~ cr~ (19C21 Mc~h, ~:nzymoloSIy Vol. V, 17~ .7~-5~:umil~ et ~ J, ~io~., Cb~m. 2C~ 9~3-~7gSO.
rqe~ ~t ~ 1) J. ~iol. Chom, .26~, 701~0-aO88.
KQY: 1 Enzyme source 2 Speciric activities Raw extract (U/mg prota~n) 3 Specific a~tivity a~ter purification (U/mg protein) 4 Remarks on the specificity or funct~onality S Yea~t ~according to Munch-Petersen, 1962) 6 Mannose-1-phosphate 7 Porcine liver ~according to S~umilo et al., 1993) 8 (Gluco~e-1-phosphate and ~annose-1-phosphate 9 Pseudomonas ae~ugfnosa ~shinabarger et al., 1991) CA 022l4458 l997-09-02 08/2~/~7 FRI 1~:22 FA~ RalPh ~cElro~ & As60c. ~010 1~ Bifunctional enzyme from GDP-Man~PP and phospho~nnose i~o~erase 11 Escherichia coli (wild strain) indi~ridual mea~urement with N~SSA
12 Recomblnant E. coli strain according to the inventlon 13 Manno~e-l-pho~phate 14 Literatuxe: A. Munch-Petersen (1962), Meth. Enzymology, Vol. V, pp. 171-17~, S~umilo et al. (1993), J. Biol. Chem. Vol. 2~8, pp~
17943-l~g50 Shinab~rger et al. (1991), J. Biol. ~hem., Vol. 266, pp. 2~8~-2088.

One can clearly see the superiority of the strate~y, according to the invention, of preparing a producti~e enzyme source of a monofunctiona} ("manno~e-~pecifi~") enzyme.
This enzyme can be used fo~ the preparation o~ G~P-mannose in larger amounts, and it ~s advantag~ous here to start with the cheaper mannose-6-phosphate, which is first con~er~ed into mannose-1-phosphate using phosphom~nnomt~tase.
Both GDP-Man-PP and phospho~annomutase are obtained, particularl~ starting fro~ pxoducing str~ns ~at contain the corresponding qenes (rfb~ or rfbK) after it has bee~ i~serted into a plasmid and the plasmid has ~een inserted in the corresponding pro~ucing strain, using the following strategy:

. ~ ~ v L~
~ v ~ ~

1. Ampli~ication ~f the gene With PCR (vent [uncon~irmed t~ansla~ion~ po~ymerase) - After 'che chemical ~y~thesis o~ primer based on known gene sequences, the genes are ampl~fied ~y PC~ with the ~ent polymerase.
2. Cloning of the gene in the plasmld pUC13 ~blunt end wlth Sm~I
enzyme) Culti~ating i~ E. coli D~Sa - ~f~er the separation o~ the amplified gene in an ~garose gel ~nd isol~tion of the gene from this gel, the genes are each ligated w~th a Yect~r p~Cl8 (coupled) that ha~ first been hydrolyzed with ~maI ~restriction enzyme) ~or the blunt-end linearization. The ligated vector~ are tr~ns~ormed ~n a strain of E. coll DH5a prepared for DN~ uptake, then the ~ell~ are grown on a solid growth medi ~ .
- The posi~ive tr~ns~onmed colonies (white c~lonies on the agar plate) are isola~ed and again ~rown as above.

3. Cloning o~ the gene in the expression vector pT7-6 using the Eco~I and BarnHI restriction sites Cultivatin~ in E. coli BL21(DE3) ~ From the positi~e transformants, the plasmid ~plas~id pUCl8 ~ inser~ed gene rfbM ox rfbK) is isolated, then hydrolyzed wi~h the enzymes EcoR~ and BarnHI. The expression ~ector pT7-6 i~
al~o hydrolyzed with the en~ymes EcoRI and Ban~HI

.c U ~ U ~ r K l l U ~ 4 v 1~ U u .

1~
- ~ter the ligation of pT7-6 with the isolat~d gene r~bM or r~bK, a s~rain o~ ~ c~li 3121 (DE3), which ha5 been prepared for DNA, i~ transformed with the~e genes. ~he tra~formant3 are grown on a solid nutrient medium.
- A~ter the isolation o~ individual colonles and renewed growin~ on a solid n~trient medium, each plasmid w~th the corre~ponding g~ne is iso~ated and hydrolyzed as a control.
- The poSitive trans~ormants are ~rown again on a solid nutrient mediu~ and are subsequently stored.
Below, the invention is explained in further de~ail and with re~erence to specific embodiment~.
~ he biosynthesis.of ac~i~Ja~ed sugar, particularly GDP-alpha-D-mannose, is, in ~i~o, o~ten carr~ed oUt s~arting with a monosaccharide ~for example, ~annose) that is pho~phorylated at c6. The sugar-6-phosphate ~f~r example, ~annose-~-phosphate) i~
con~erted into a sugar-1-phosphate using a phosphomutase ~EC

5 4), particularly in thl~ instance a pho~pho~omuta~e ~c 5.4.2.8).

Mannose-6-phosphate ~annose-l-phospha~e (I) Pyrophosphorylases ~elonging to the group o~ nucleotidyl trans~era3es (EC 2 . 7 . 7 J, p~rtic~larly in this in-~tance the GDP-alpha-D-mannose pyrophosphorylase ~EC 2.7.7.13), catalyze the transfer of a nucleotidyl group from a nucleoside triphosphate to fdrm a sugar-l-phosphate with the release of inorganic pyrophosp~ate (see Feingold and Barber, 19~0, in Methods in Plant Biochem, Vol, 2, pp. 39-78), particularly the ~ollowing reaction ~II) -Mannose-1-pho~phate ~ GTP ~DP-Mannose + PP~ (II) Pyrophosphoryla~ make sugar nucleotides available as a s~b~trat~ for glycosyl tran~era3eS (EC 2.q)~ which transfer the sugar portion to an accep~or tsee, for example, G$nsberg~ V.
(19~) in Adv. ~nzymol., Vol. 26, pp. 35-88, or for the synthesis o~ other secondary activated sugar~, in thi~ lnstancQ
partic~larly GDP-~-L-~uco~e (Yamamoto, ~., 198~, Agrîc. B~ol.

C~em., Vol. 4~, pp. 823-824 and 19~3, ~rch. Biochem. Biophy3., ~ol. 300, pp. 694-698).

Since the chemic~ ynthesis is often diff~cult and is associat~d with lo~ yi,elds, enzymatic s~nthes~s is increasin~ly being used.
The phosphom~n~o~l~tase 6~C S-4.2.8) and the GDP-mannose-pyrophosphoryla~e (EC 2.7.7.13~ ha~e, so far, been detected in diffe~ent sou~ces.
GDP-mannose pyropho~phorylase wa~ partially i301ated for the ~irst time in 1956 by Mu~ch-Pe~ersen from baker's yeast, with consider~ble ~ariations in the quantity o~ available enzyme dependinq on the yeast lo~d ~nch-Peterse~, 1956, Acta Chem.
Scand., Vol. lO, p. 928). ~h~ enzyme was ~solated in 1962 b~
Preiss and Wood (J. Biol. Che~., ~ol. 239, No. 10, pp. 3119-3126) from Arthoro~acter ~p. However, the authors were unable to rule out that the numerous reacted acti~ated sugars were the result o~
secondary reactions o f other pyrophosphorylases. In P~eudomonas ae~ugi~osa and Rho~ospiril l um ru~rum a bifunctional en~yme has been found, GDP-man~ose-pyrophosphorylase, coupled with phospho~annose-is~merase activity ~Shina~arger et al., 1991, J.
Biol. Chem., Vol. 266, No. 4t pp. 2080-2088 and Ideguchi et al., 1993, Biochimica et ~iophys. Acta, Vol. 1172, pp. 329-331). From the eukaryotic species as well, GDP-mannose-pyroPhosPhorylase has been isolated ~Szumilo et al., 1993, J Biol. Chem., Vol. 268, No. 24, pp. 17943-17950). The acti~rities that wexe determ~ned--conver~ion tc: GDP-gluco3e (10096), IDP-glucose ~72~6), and GDP-mannose (61%)--sug~est that ~his pyxophosphorylase is instead a GDP-glucose-pyrophosphorylase ~EC 2.1.7.34)~
so ~ar, t~e phosph~nn~ I-tase ~EC 5.402.8) has only been con~idered in connection to alginate biosynthesi~ ~Sa~Correia et al., 1987, J. Bacteriol., ~ol. 169, pp. 3224-3231 and Goldberg et al., 1993, J. ~acteriol., ~ol. 175, No. 3, pp. 1605-1611).
The enzymatic synthesis of GDP-m~nnose has until now been descri~ed by Simon et.al., 1~90, in ~. Org. Chem., Vol. 55, pp.
183~-1841, Wong et al., 1993~ in WO 93/082~, Wang et ~1., l9g3, in J. Org. Chem., Vol 58, pp. 3985-39gO, and p;~ k~ and ~urner, 1993, in J. Chem. Soc. Perkion Trans~, Vol. 23, No. l, PF~ 3017-30220 These work gro~ps all use a pro~ein preparation o~tained, according to a method described by Munich-Petersen in 1956, ~ro~
yeast cell~, and they synthesize GDP-man~ose starting with ~annose-1-pho6ph~te prepared by a chemical route.
By cloning in a (production) expression vector (plasmid) (pT7-~ fro~ the Novagen company) and (insertion~ transformation in a ~prod~ction strain) expxession ~trai~ Escherchia coli BL21~DE3)pLysS (fxom the Novagen company), a phosphomannomutase and GDP-mannose pyrophosphorylase were then developed; this, according to the invention, can ~e obtained in larger quantities than ~rom ~he sources known so far ~see Table, page 3). soth enzymes originate fro~ Salmonella enterica, group B (formerly Salmonella t ~ ~imrlrium LT2~. The genes (r~bM coderi for the GDP-mannose pyrophosphorylase; rfb R codes for the phosphomannomuta~e) are located in the rfb gene cluster whose ~Jo, ~ r~~ r~ ~~ 0~ ~ A6~;0C . 4~

.

structure and sequence has been eluc~dated by J~ang et ~1., 1991, in Mol. Microbiol., Vol. 5, No. 3, pp. 695-713.
U~ing the polymerase chain r~cti~ (PCR) the genes rfb M
and r~b ~ are mult~plied (amplifled) and each ~s ~lonQd in ~
vector pUC18 (Nova~en company). Starting wi~h this vector, the genes rfb M and r~b K ~re each clon~d in an expression vector pT7-6 ~rom the ~o~agen Co~pany, and each is i~serted (tran~formed) in an expression strain of Escherichia coli BL21~D~3)pLy~S. The plasmid pT7-6 with the inserted gQnQ rfb M i~
no~ called pERJ-l. The plasmld pT7-6 with the in~erted gene rfb K
wa~ named pERJ-2 (~ee Fig~res 1-3).
The productlon (express~n) o~ the prot~ins (GDP-~anno3e pyrophosphorylase and phospho~n~nmllta3e), coded by the gene~ r~b M and rfb K, was lnduced with 0.4mM isopropyl thioqalaCtosidQ
(IPTG) and amplified with 0.03mM ri~ampicin ~see Figure 4).
By mechanical breakup o~ the cells (~scherichia col~ in 50mM Tris-HCl buff~r, pH 8, a~d 150mN KCl and centrifuga~ion ~2 min at 10,000 rp~), a prot~in-containing raw extract wa~
obtained. This raw extrac~ wa3 loaded tn an anionic ~h~nger (Q-Sepharose FF), and the GDP-mannose pyrophosphorylase was ,o}:~tained by an incremental elution with 150mM Kcl and 400niM KCl.
The eluat~a wi3s reacted with lM a~monium sulfatQ and 209~ glycerin (v/v), and applied t~ ~ phenyl S~pharose FF~ After adsorption, the enz~me i9 elu~ed with a gradient b~twQen lM ~mm~n; um sulf3te and OM a~monium sulfate in 50n~ Tr~ s-Hcl, pH 8, 20 tsic~ glycerin ~etween O.4M and 0.lM ammonium sulfate. A~ter ultrariltration and a buf~er Ghange with 50m~ Trls-HCl, p~ 8, with 150m~ KCl, the GDP-manno3e pyropho~phorylas~ was chromatographed on a gel ~i~tration colum~.

08/2a/~7 FR~ 18:23 FA~ Ralph ~cElro~ ~ A~oc. ~012 A~ter ultrafiltrat~on, the GDP-manno~e pyrophosphoryl~s~ is reactQd with 3M amm~nium sul~ate and s~ored at ~~C.
~ The phospho~ o~-~tase should be partially purif~ed on a Q
Sepharo~ F~ colurLn.
Both in ~che case of pho-Sphom~ ta c ~n-:l $n th~ c~ f the GDP-~anno~e pyrophosphor~lase, t~e enzy~e~ are monofunct~onal and spQcifically cataly~e the re~ctions d~scribod for th~m (see I
and I I abo~e ~ .
Both en~ymes should be used for the enzy~atic synthesi~ 0 GDP-mannose, starting with manno~e, according to the following r~action schemes:

Reaction sche~e 1: (al~o ~ee Figure 14) Manno~ + AT~ ~ M~nno~--6-pho~pha1:e ~ ~DP (1) PEP ~ A~P S Pyruvzlte ~t ~p (2) ~Sanno~e-6-phosphate _ M~nno~ pl~o~ph~te ~3) Mannos~-l-pho3phate 1- GTP $ GD~ n~ e ~ PPI ~4) PP~ ~ 2 Pl (5J

E~eaction 1: Hexokinase R~ction 2: Pyruv~te ki~asQ
React~on 3: Pho~ph~nno~nutase Reaction 4- GDP-mannose pyrophosphoryla-~e ~eaction 5: Pyrophospha~ase Reactions 1 and 2 ha~e already been used ~or the production o~ ma~nos~-6-phosphate by Palanca ~nd Tur~er, 1993, J. Ch~m. SOC.
Perkin Trans~, Vol. 1~ pp. 3017-3022 CA 022l4458 l997-09-02 os/2~/a7 FRI 1~:23 FA~ Ralph ~cElro~ ~ A660c. ~0l3 Th~ GDP-mannosQ formQd in this ~nner ~an be further reacted in sit~ with manno~yl transferase to form oli~osaccharides~
The GDP-mannose pyropho~phorylase acti~ity was demonstrated with a newly developed con~inuou~ ~p~ctrophotometric test for the deter~in~tion o~ pyrophosphate (PPl) producing nucleotidyl transferas~ (EC 2.7). In th~ manner desc~ibed below, lt is po~sible to determine~ by the use o~ the substrates (sugar 1-phosphates or sugar in the ca~ o~ n~ur~minic acid) ~nd ~cleoside triphosphates any pyrophosphorylase acti~ity which p~ese~ts an acti~ity in the te~t mixture o~ >0.2 ~U/m~.
~ he e~zyme test (Nucleot~dyl transferase substrate screening assay 'NUSSA'~ i~ ba~d on thQ ~act that in the nucleotidyl tran~ferasQ reaction (EC 2.7-7), pyroph~sphate is produced with a pyropho~phate-dependent phosphofructokinase (PP1P~K ~rom plants or bacteria ~c 2.7.1.90) with fructose-6-phosphate and ~n the presence of fructose-2,6-dlphosphate to mak~ fr~cto~-1,6-diphosph~tes~ This pro~u~t is cleaved with an aldolase to form dihydroxy~c~tone phosphate ~DH~P) and glycerin-3-pho~hate (GAP).
From glycerinaldehyde-3-phosphate, dihydroxyacetone phosphate i8 produced with the triose phosphate isomerase. Finally, dihy~roxyacetone pho~phate ~s r~du~ed with glyce~in-3-phosphate dehydrogenase to glycer~n-3-phosphate ~G-3-P) with NADH. ~ mol of NAD~ are used per mol of pyrophosphate, this consumption can be monitored by photometry.

CA 022l4458 1997-09-02 v~ ~ ~ r~l 1~ A~ K~lpn Mchlro;sr ~ A1;~OC . 1~ U14 Reaction sche~e 2:

~TP ~ arLnose-1-Phosphat ~- GDP-~ annooe + PP~
PPL ~rU~Se-6-PhOSPhat ~ - - ~~ Fruccose-1,6-P2 + P~ (2) F2:UCtOSe-1, 6-P~ DHAP ~ GAP ~3) DE~AP ~ GAP ~ - 2 GAP ( 4 ) 2 GP~P * 2 NA~H - 2 G- 3 - P ~ 2. N~D ( 5 Key: 1 GTP + Mannose-1-phosphate 2 PPi I fructose-6-phosphate (1) GDP-Mannose-pyroph~sphorylase ~2) Pyrophosphate-dep~nd~nt phosphofruct~lcinase ~3) Aldolase (4) Triose phosphate isomerase ~5) Glycerin-3-phosphate ~ehy~rogena~e The ~ollowing examp~es prQsQnt the protocol ac~ording to the ~nvention in detail. RefQrence is made h~re to the drawing~ in the appendix, where the figures represent:

Figure l; the cloning ~trategy Figure 2: the expressio~ vector pE~J-l Flgure ~: the expression ~rector pERJ-2 Flgure 4: the SDS-gel electrophoresis of the expressed gene products of pERJ-1 and pERJ-2 Fi.gure 5: chromatogram of the gel filtration f~r the determination of the molecular we~ght ~JC~ Kl l~ Z~ P.9~ K~ >ll ~c~:lro;sr ~S AS~;OC. ~I~

~ig~re 6: sta~lity o~ a ~DP-man-pyrophosphoryla~e at 4~C
Figure ~: the su~?strate excess inhibition o~ GTP
Figure 8: the subst~a~e excess lnhibition of ~-l-P
FigurQ 9: the com~?~titlve inhibltion of GDP-Man wi'Ch respect to GTP
~igure 10: the nonco~petitive inhibition Or GDP-Man with respect to M-l-P
Flçrure 11; the ln~luence o~ the p~ on th~ s~r~thesis o~
GDP-mannose Figure 12: th~ dependency o~ the synthesis of ,GDP-mannose on the enzyme concentration Figure 13; the E * t d~agram ~or ~he synthe~ of GDP-man start:1ng With mannose~l-phosphate and GTP
Figure 14: reaction scheme of the biosynthe~is of GDP-ma~nose ~ro~ ~annose Figure 15: s~n~hesi~ o~ GDP-mannose startlng with 5m~
ma~nose Figure 16: capillary electrophoresls chromatogram of the GDP-mannose prepared Exampl~ I

Cloning o~ the genes ~fb N and r~b K from the rfb ge~e cluster ~ Salmonel l a enterica, groUp B.
Using a DNA data bank, the genes rfb M and r~b K were ident~ fied in the r~b gene clust~r, ~nd the readin~ ~ral~le was determined .

08~2~i~7 FRI 1~:25 FA~ Ralph YcElro~ ~ A~oc. ~Ul~

rfb M: codes ~or ~he GDP~alpha-D-mannose pyro~ho~p~orylase ~EC 2.7.7 13~ ~
Lengt~ in ~p: 17386-18831: 1445 base pair~
Start codon: ATG 17386 stop co~on TAA TAA TAG 18a31 Ri~o ome binding. site: AAA AGA GAT A~

~bf K codQs for phospho~nnomu~8e (~C 5.4.2.8) Length in ~p..: 18812~20245: 1433 baso p~irs Start codon; ATG 18812 Stop codon~ TAA 20Z45 Ribosome bin~ng .site: G~A GGA GTG GA

For th~ in vitro ampli~ication, the following oligonucleotide primer~ for both genes were determined.

r~b ~:
Primer 1 . (x~b Ml) 5 ' -CTT GGG TTA CAA ATT AGG CA-3 ' Primer 2: ( r~b M2 ) 31 -~TC TTT TAC AAG ACC GCG AG- 5 r~b K:
~imcr l: (r~b ~l) S' -CCC CCT G~ GTT AAT TG~ ~A-3~
Pri~er 2: (rfb K2~ 3' -CCA TTT AAT CCT C~ CCT CT-5' The length ~f the gene is thus increased for rfb M to 1633 Bp. and for rfb K to 1606 Bp.
The PCR ls carr~ed ou~ as ~ollows:

, 08/2~/~7 FRI 1~:25 FA~ R~lPh ~cElroy ~ A~oc. ~017 Tab1Q I. PCR preparati~n ~or the cloning o~ rfb M and r~b K

r~ M ~ K

n~ - ~o~ Y~ 21 ~V~n~-Pol; ~ rer t~ Oxl _ la ~0 5~
, d~P, dGTr~, ~rrP ~ 1,25 m~ 16 yl Prirn-r 1 ~"~ Ml ~, 2 ,Ul i21 p~ll r~b Kl 6 ,1 ~ 1 /2t c pmoltl~l;
P~lmc~ 2 r~b M2 tl~. ~ pmo~,ul1 r~b K2 ~, ~ yl pmol~
.'. ~l it~hc D~ U5 S~lm~ ella 5 t~ 5~ 100 1~5Cl~ (2~ 10 yl 10 1 Key: 1 Vent pol~mer 2 Ve~t polymer buffer (lOx) 3 dATP, dCTP, dGTP, dTTP, ea~h at 1.25mM
4 Genomic DNA ~rom Sal~onell~

The preparations are covered with 70 ~L of mineral oil eac~, to prevent evaporation.

CA 022l4458 l997-09-02 1.

08/2~ FRI 1~:2~ FA~ R~lph ~cElro~ ~ A~oc. ~018 Vent polymerase bug~fer (BioLab~s, New ~ngland) (lOx) 2~ Tris-HC1, pH ~ . 8, 100~ KCl, lOOmM (~H~) 2SO~, 20 MgSO~, 1% l~riton lOOX ~w/~r) The ~ollowins~ conditions were selected to run the PCR~

5 min ~8~C
Z min 95~C repeat 6 times 30 sec 49~C
~0 sec 72~C

1 min 95~C
5 sec 49~c repeat 25 time~
~0 sec 72~C

.
2 min 72~C

Cool A~ter the PCR, the a~plifled genes, each 1. 6 kB, were isol~e~ accoxdin~ to Lau and Shou, 19g2, Me~h. Mol. C~ll. Biol~, Vol~ 3, pp. 190-192, f~om an agarose gel, also, each was ligated ln an aux~ll ary vector pUCi8, whic~ ha~ fir:3t been "blllht ended"
l~neari~ed using the res~riction enzyme SmaI. The ratio of the v~ctor to the DNA frag~ent wa~ app~oximately l: 4 . The ligatior w~s carri~d out overnight at 14 ~C .

08/2~7 ~I 1~:2~ FA~ Ralph XcElroY & A660c. ~ol~

Table II~ Preparations for the ligation of ~he PCR produ~t5 in pUC18 T4 ligas~ 1 Ligase buffer (lOx) 6 Vector pUC18/Smal 1 20-60 ~g~prepa~ation H20 ~sterile) r~b M 12 rfb K 18 DN~ fragment r~ M 10 80-240 ~g/prepa~ation r~b K 4 Li~ase buffer (lOx): 0.5M Tris-HCl, pH 7.6, lOOmL M~C12, ~OOmM
DTT, 500 ~g/mL ssA

This vector with the inserte~ gene was transformed in compe~ent cell~ o~ Escherichia col i D~Salpha (~ccording to ~ hAn, 1983, J. Mol. 3iol., Vol. 166, pp. 557-580). For this purpose, 5 ~L of the ligation pre~aratlons and 1~ ~L of s~erile H~0 were each reacted with 20~ ~L of competent cells that had been thawed on ic~, then incubat~d for 30 ~in on ic~. Th~
preparations were then heated ror 40 sec at 4~~C, and again placed on ice for 2 min. ~00 ~ of SOC medium were the~ added to th~ preparations; the prepar~tions were then incubated for 1 h at 37~C, then spread on LB~_Ioo agar plates, which were coated with X-Gal.

Kl 1~ A~ ~alpn ~c~lro~ ~ A~OC. ~U~U

SOC madium: pH 7, 20 g tryptone, 5 g yea~t extract, 0.5 g NaCl, 2.5mM KCl, lOIPM ~çrCl2, 20~ (filtration starilized) g~uco~e, ~IzO and l000 m~
oo: 10 g ~ryptone, 5 g yeast extract, 5 g NaCl (and lS g Bacto a~ar) Ampicillin lO~ mg/1 X-Gal: 5-Bromo-4-chloroindolyl-~-D-galactoSe (40 ~g~mL) 70 ~L per a~ar plate The pla~mid pUCl8/~fb ~ or r~b K w~3 isol~ted from the positive coloxless colonies ~according to Bi~nboim and ,I:~oly, 1979 ) .
pUCl8/rfb M was lin~ariliz~d w~ th the restr~ction onzyme6 Eco~ and BamHI; the ~ene rfb M wa~ CUt out o~ the vector. From an agaros~ g~l, thQ gen~s r~b M a~d r~ were isola~ed, then ligated in t~e expression ~ctor (pT7-6).

Table III. ~reparatLons rOr the ~l~ation of the genes rfb M and rfb K ln pT7- 6 11~

T4~1igase 1 ~0.1 ~ei~ unit) T4-ligase bu~fer (lOx~ 6 (See abo~e) ~ect~r pT7-6~Eco ~I-Ba~HI 2 H20 ~terile rfb M 13 rfb K 17 DNA rfb M 8 rfb ~ 3 u~ / v ~ r~ ~ ~L~ r~ C . ~ ~Ll , These ligation preparat~on-~ (pT7-6/rfb M and pT7-6/rfb K) are transformed ln competent cell6 (accordlng to cohen, shr~g ~sic], and Hsu, 197~, Proc. Nat. Acad. Sci. (USA), Vol. 69, No.
8, pp . ~110-2114) with, ~ n 'che case o~ r~b M, Escher~chia col~
BL21(DE3)pLysS fxom the Novagen company, and, in the case of rfb ~f in ~qcherichi~ coli B~21~DE3).
The strain that conta~ns the gen~ rfb M insert~d in pT7-6 will be called E. coli BL21(DE3)pLysSpERJ-~ below. Th~ ~train that conta~ns the gene rfbk inserted in pT7-6 will be called E.
coli BL21~DE3)pERJ-2 b~low.
The expression of the genes rfb M and rfb K ls ~rried out as follows:
From prelim1n~ry cultures of Esche~ichia col7~ ~21(D~3) pLysSpERJ-l ~5 mL LB~C~lor~o (amplcillin ~nd chlora~ph~nico~, e~ch 50 mgJL) o~ernight at 120 rpm and 37~C), main cultures ~10 mLJ were inoculated at 2% and they were cultured ln Erlenmeyer flasks with baf~les at 37~C and 120 rp~ with a shaker until a~
optical deA~ity o~ O.S, at 546 r~n, wa~ reached, 1 ~ of the culture was remo~ed, then cen~ri~uged, with the supernatant being removed and the pellet reacted with 50 ~L of sample ~uffer ~SDS
and cont~;n;ng ~-mercaptoethanol). This samp~e wa~ heated for 3 min at 95~C ~n~ was placed on an SDS polyacrylamide gel (method~
~c~ below) . The rest o~ the c~lt~xe was reacted with 0.4mM IPTG
and incubated ~or 20 min, then 1 mL was again removed and treated as above. ~he rest of the culture was reacted with o. 03~
rifamp~cin ~nd incubated for 60 min; 1 ~ w~s r~moved and treated as described above; 10 ~L of ~ach of these samples was applied onto an SDS-polyacryla~nide gel.

~ ~scherich~a coll BL21tDE3)pERJ-2 was cultured as described above .
An SDS-polyacrylamide gel electrophoresis wa~ carried out accor~ing to Laemmli, 1970, Nature~ Vol. 227, ~p. 680-~85. A
photograph of the Coomassie brllliant blue sta~ned gel is shown in Figure 4.
Isolat~on o~ th~ GDP-mannosQ ~yrophospho~ylas~ ~rom Escherichia col i BL2 1 Culturing o~ ~. coli BL21 and breakup of the cells:
at 37~c $n a shaker at 120 rpm Starting wlth a pr~lim~n~ry culture (overn~ght incubation, 200 mL în a lOOO~m~ Erlen~eyer flask with baffles), 2 ~ o~ LB
~ were placed in e~ch of fi~e 5-L flas~s with ~a~fles ~nd inocula~ed at l~; the cultu~es were grown until the opt~cal density was 0.8 (3.5 h). After a 20-min incubation with 0.4mM
~ IPTG and a 60-min incubation with 0.3~M rifampicin, the cultures were remove~ by centrifug~io~ (Sor~all GS3, 8000 rpm, 10 min, 20~C) and washed twice with 50mM ~ris-HCl, pH 8. The wet weigh~
was th~n dete~mined (approximately ~5 ~) and a 20~ ~w~v) cell ~uspension wa~ prepared. The cell~ were then broken up in a dis$nte~rator s by wet gr~nding. For ~his purpose, 40 g of c~ll suspension were mixed with 80 g of glass beads (0.3 ~m diamete~) ~nd ho~oge~ized fo~ 12 min at 4000 rpm. The cell debris and the glas~ bea~s were separated ~y a 15-~in cen~rifugation ~Sorvall GSA, 10,000 rpm, 20~C), washed in ~OmM Tri~-HCl, pH 8, and centrifuged agaln. The ~upernatants were cleaned: ~hey ~ormed the ~aw extract for the anion exchange chromato5r~aphy on Q-Sepharose FF .

~ V ~ V ~ 1 L ~J . ~ V ~ ' V~ b ~ U L ~

Q-Sepharo~e FF:

400 mL of Q-Sepha~c~se FF were load~ with 226 ml of ra~r extract ~with 11.1 mg~mL of protein). The st~pwise Rlution ~td~tS
with approxima~elY 800 mL 50m~ ~ris-HCl, pH 8, and approxlmately 1400 m~ of 50mM Tris-~Cl, pH 8, with lSOmM ~Cl. The enzyme is eluted wlth approximately gO0 mh o~ 50mM Tri~-HCl, pH 8, with 400~M KCl. This fraction is reacted with lM a~m~u~ sulfate and 20~ glycerin, a~d is loaded o~to phonyl S~pharose FF (66 mL). T~e enzyme is eluted using a gradient that decreases linearly to OM
~mmon~um sulfa~e, with 50~M Tri~-HCl, pH 8, and 20% g~y,cerin (total volume 1000 mL).. The most active fraction~, be~ween 0. 4M
and 0. lM amm~nium sulfa~e, were purified; the buffer was ch~nged after ultrariltrat~on (50mM Trl8-Hcl~ pH ~, 150mM KCl), then chromatographed in a gel filtration colu~n (Superdex G-75) ~see T~ble IV bel~w).
The reco~bina~t GDP-mannose pyrophosphorylase was concen~ra~ed by 6 3 ~ime~ from ~. col ~ . With a yield of 13.5%, a specific activity of 2.34 U~mg could be obtained. Starti~g with 0.37 U~mg, it was possible to achlev~ a puri~icatlon factor ~f 2 usi~y Q-Sepharose, with a yield o~ 85%. The subsequent hydrophobic interaction chromatography o~ phenyl Sepharose led, as a result o~ the combination o~ only the most active fraction~, ~o ~ relati~ely h~gh loss o~ 40%, with an increaSe in the speci~ic ~cti~ity to 2.27 U/my.
The molecular weight of the GDP-mannose pyrophosphorylase was, under denaturing conditions (S~S-p~lyacry~amide gel electrophoresis), 54 kD. ~or the determination of the molecular weight in the native state, with 2 mL (7.5~ mg/~I) of enzyme sample ~rom the puri~ication, see abo~e, a gel filtration was x~ ~ / r~ ~ K~lp~1 ~c~lro;~ oc . 4~ u~,~

performc~d on S~phadex G-200 (115.5 mI.). The clet~ r~t~on of the activity was ca~ried out u:!~in~ the enzym~ t~st, acco3:din~ to th~
invention, ror phosphorylases, as de:3cribed below. Two activity ~-,.x;-n~ were det~rminQd, which correspond to the molecular we~ght~
o~ 208, 700 dalton and 107, 800 dalton. In thQ native state the enzyme wa~ thUs irl the ~orm o;C a dimer or a tetramQr~

Tab1Q IV. Purificat~ on of thQ GD~-mannose py2~opho9phoryl~e~
results of the individ~al purification s~eps Psobc o~a~- Gcs~mt~ ~p~ ch~ Aeinigung - ~u~be~lt~
a ) prot~!in ~I,ct~vit~e A]c~v$e~ f~kto:
~m~ UJ~g~

~ohextr~Xt 250~ gl9, ~ o, 37 1, o loo Q-scpba~ FF 93l 722,1 0,7g 2,1 ~S,~
~IC 153 3~7, 2 2. 77 C, 1 37, ~(~h~nyl~epb. 1 UF (HIC1 126 ~70,~ ~,3.4 5,~ zg,~
C-75 UF S3 lZ3,7 z,3~ C,~ 13.5 ~)~G~ 'Lll:~c~ or~

Key~ 1 S~nple 2 Total protein (mg) 3 Total activity (U) 4 Specif1 c activity (U/mg) 5 Purification factor 6 ~Yield ( % ) 7 Raw ex~ract 8 ~Phenyl Sepharose) 9 (Gel ~1 ltration) ~8/2V~7 ~-Rl 1~ A~ RalPh ~chlroY ~ A~60C. ~uZ5 .

~ h~ following sx~m;n~tiolls wer~ carried out on the method of action and the u~e of GDP-alpha -~o~e pyropho~phorylase:

1) EX~m~n~tio~s o~ ~tability The enzy~e was ~x~mined at 4~C to det~rmino it~ ~tability dur~ng ~torage. For this purpos~, an ~nzyme prepar~tion was reacted with 1.15 U/mg wi~hou~ a sta~ilizer, and with 0.1 mg/m~
BSA, 3~ ammonium sulfate, or 25% glycerin, then reacted fo~ 47 days at 4~C. ~fter 47 days, a r~sidual activity o~ approximately S~ could b~ found in the preparations without a stabilizer and wi~h BSA, ~hereas the preparation~ with glycerin and a~monium 8ul~ate still presented activities o~ 7~ and 65~t r~spectively.
In the prepar~tion w~th am~onium sulfa~e it was possible to determine an activity of 50~ of th~ starting acti~ity even after 4 month~ (Figure 6) FurthQrmo~e, th~ stability at 30~C was f~ ned by incubating a defined stock solution of enzyme with 79 . 3 ~U/mg at 30~C in SOmM Tris-HCl, pH 8, 5~M MgCl2, and an acti~ity detenmination was perfo~med at d~erent ti~e~ (0 h, 2 h, ~ h, and 30 ~.

08/2~7 FRI 1~:30 FA~ RalPh l!lcElroy ~ A~oc. Ie ~2 Table IVa. Temper~ture 3ltability of GDP-man-pyrophosphorylase ~t 30~C

St~n~en ~h~ Aktit~it~t knU/mgl ~cl:-t~Y~ ~c~i~ri~
e~
o 79,3 10~
2 78,4 98,g '!
6 67,~ 85,6 53, 7 6~, 7 sb~s~i~m~g per ~SsA mi~ z nM GTP und 0,08 n~ M-l-P

~ey: 1 Hour~ (h) 2 Acti~ity (mU/mg~L) 3 Rsalat$v~ acti~rity (%) 4 Activity determination by NUSSA wlth 2m~ GTP and 0~08~M M-l-P

2) Determination Or the k,,~ d ~ x ~values for the ~3ubstrate~ GTP
and mannose-i-phosphate ~M l~P) Conditions: 2 . 27 ,ug/~L ~f Gl~P-~ar~se pyrophosphorylase pxepa~ation aft~r th~ gel ~iltrationr with act i vity dete~mination by NUSSP. .

a) In the determina~ion of the ~ ~lue and the v~x value for GTP mannose-l-phosphate, a constant concentrat~on of 0.08~M
was used. GTP was used at concentrations of 0.01-lOmM .~Figure 7~.

/U( rS~ ;J~ ~ n~ c~lro~ ~ A~i~OC. 4~U~I

b) In th~ dete~m~n~tion of the ~ value and the ~ ~alue for M-l-P GTP, a constant concentr~tion of 2mM wa~ used. Mbnnoso-l-phosphate ~as u3ed at a varying co~entration of 0.00~-0.6mM
(~igur~ 8).

Table ~. Kinetic con~tant~ for th~ substrates GTP and M~ P of ~he GDP-mannose pyropho~phoryl~

GTP ~- 1-P

km value O . 2mM O . 01 ~m k~X value 2 . 4 U/msr 1. 8 U/mg K. ~alue (excess su~strate) ~0 3mM 0.7~M

3) Tnfluence of GDP-m~nnose on the ~ynthesi~
~onditions: 2.27 ~g/zE~ (in a) and 5.67 ,ug~mI (in b) GDP-m~nnose py~ophosphorylase preparation a~t~r gel filtration Activity determination by NUSSA

a) Mannose-l-phosphate was used at a constant concentration of 0.08~M.
The concen~ra~ion o~ GTP was ~aried between ~.08mM and 6 ~.
Th~ GDP-mannose was used at O~M, 50~M, and 100~ ~n the ~est~

08/2~/~7 ~I 1~:~1 FA~ Ralph ~cElroYJ~ A660C. 1~1028 The evaluation Or the mea ured aGtiviti~ 3hows a competitive inhibition o~ GDP-~annose with respect to GTP ~ ure 9). The calc~lated ~ value was 14.9~M~
~ ) GTP ~as used at a constan~ ~o~cP~tration of 2mM in th~
t~st.
Mann~se-l-phosph~te wa u~ed at varying conc~ntratl~ns o~
O.003-0.3~.
The evaluatio~ o~ t~ measured activitie~ ~howed a noncompetitive inhibition of GTP-mannoae with r~spect to M-1-P
(Figure 10). The calculated Kl value was 118~M.

~) Substrate ~pect~um of the GDP-manno~e-pyrophosphoryla~e Condi~iohs: The ~DP-m~nose pyrophosphoryla~ was used at concentration of 7.3 mU~mg in the ~USSA enzyme test.

~ Nucleo~ide triphosphat~s: ATP, CTP, GTP, UTP, d~TP, ~ach lmM sug~r-1-phosp~ate: ma~nose-l-pholphate at 2.5mM, b) ~ucleos~de triphospha~e; GTP
Sugar-1-phosphate3: Glucose-l-P, N-acetylglucosamine-l-P-, glucosamine-1-P, g~l~ctose-1-P, galactosamine-l-P, N-acetylgalactosamine-l-P, glucuronic acid-l-P~ galacturonic acid-1-P, xylose-1-P, manno~e-1-P

Both in a) and i~ b~, no re~ction coul~ be d~termined except wi~h the natural substrates GTP and mannose-1-phospha~c.

U~ rKl l~;Jl r~ r~ u ~ lm~;~ ~ A6~0C. ~1~
, 5) Use of the GDP-manno:3e p~rrophosphorylase i~or the synthesis of G~P-~annOSe The synthe~is should be carried out starting with manno~e and us ~ng reaction scheme 1.
Flrst, the s~nthesis o~ ~DP-~annose wa~ ~X~in9d~ starting with mannose-1-phosphate and GTP. For this purpose,. the GDP-mannos~ pyropho~phorylase was used in a for~ coupled to the pyrophosphat~e (l U/m~).
The synthe~ls was carr~ed out at di~ferent pH ~alues ~7, 8r ~) in 50mM Tris-HCl, 5mM MgCl2 with 2mM GTP, ~nd 2mM ma~nose-1-~hosphatQ in a t~tal vo}ume of 2 mL at room temper~tur~. The GDP-mannose pyrophosphorylase was used at 0.04 U/mL and the pyrophosphatase was used at 1 ~ . After di~ferent ti~es, 200 ~L
were remo~ed ~rom the preparation an~ heated ~or 5 m~n at 95~C, ~ollowed by centrifugation (Eppendorr centrifuge, 10,000 rpm, 2 min, room te~perature) and analysis by c~pil~ary electrophoresis.
USins calibration curves and ~ comparlson of the areas undar the cur~es, the content of GTP and GDP-mannose could be dete~mined. The r~actions show a higher yield of GDP-mannos~ at al~aline pH ~alues tFigure 11). SinCQ the o~her auxiliary enzymes (reaction scheme 1: hexokinase and pyru~ate kinase) have optimum pH ~alues of 7-9 (soehringer-mannheim~ 1987, in ~iochemica-In~ormation) a pH of 8 was selected ~or additional synt~eses.

Below, the dependency of the synthesis o* GDP-mannose on t~e enzymQ concentration o~ GDP~ ~ose pyrophosphoryla~e wa~
examined, with the latter being u~ed at 0.Oq U/mL, 0.06 U/mL, O . 08 U/mL, 0 1 IJ/mL, and 0 . 2 U/ml . The react~ on~ show an increased ~ield of GDP-mannose with the same incubation time~ and incxeased enzyme concentratiolls (Figure 12). The multiplication 08~2~/~7 FRl lff~ A~ K~lPh Mc~lro~ & A660C. ~u~u of tha enzym~- concentration by the ~ncubatlon ti~e le~d~; to a reaction constant (E ~ tJ. I~ the enzyme co~c~ntration or t~e incubation time is ch~ng~d, consta~t yields can be obtained the E ~ t product is mai~tainQd con~tant. For an E ~ t o~ 20 (U*~i~/mL~, tho reaction equilibrium is establiqhed u~der selected condit$ons,.reachin~ a yleld of G~P-man~ose of approxi~ately 90~ (Figure 13~.

~xample ~I

~ucleotidyl tr~s~e.rase subs~rate assay (NUSS~) Re~c~ion sc~eme 2 (}) NTP + sugar-I-phosphate NDP sugar I PP~
(2) PP~ I f~ucto~e-~-phosphate Fructose-1,6-Pz ~ P~
(3) Fruc~ose-1,6-Pi DHAP ~ GAP
(4) DHAP ~ GAP Z D HAP
(5) 2 DHA-P ~ 2 NA~H + H~ 2 G-3-P + NAD~

(1) Pyrophosphorylase, (Z) Pyrophosphate-dep~ndent phosphofructokinase, (3) Aldolase, (4) Triose phosphate-isomerase, (5) Glyceri~-3-ph~sphate dehydro~ena~e NTP: Nuclaoside triphosphate~NDP: Nucle~side dipho~ph~te/DHAP~
~ihydroxyacet~ne phosphatQ/GAP: ~lycerin aldehyde-3-phosphate/G~
3-P: Glycerin-3-phosphate~NAD: Nicotinic acid a~ide adenosine diphosp~ate, reduced foxm U8/ZU/~7 PRl 1~ A~ KalPn ~chlro~ & A660C. ~UJl O~Brien, Bo~ien, and Wood degcribed i~ 1975, J. Blol. Ch~., Vol. 250, No. 22, pp. 8690-8695, a coupled photometr~C enzyme te~t for mea~uring a pyropho~pha~e-d~pendent phospho~ructokina (PP,PFKJ, whiCh was disco~ered ~or the flrst time by Reevos e~
al., 1974, J. ~iol. ch~m., YOl. 249~ pp. 7737-77~1, i~ ~ntamoe~a hi~tolytic~. ~n thi~ moa~Ure~ent, the ~PlPFK W~S ~oupled with t~e re~Ction of the aldol~e (reaCtion 3~, thQ trioSe phosphate isomera~3e ( ~eaction 4 ), and the glycerin-3-phosphate de!hydrogenase (reactio~ 5~. The re~ctio~ wa3 monitored by -photom~try at ~40 r~
The following enzymQ test (NUSSA) coupled, according to the in~ention~ the reaction o~ the nucleotldyl tr~ns~erase Wlth th~s test system; it thus makes it po~sible to measure any pyrophosphorylase or any pyrophosphate-rele~sing en~me. Th~
NUSSA test was optimized ~or mea~urement in microtlter p~ates with a total ~olume of 200 ~L.

U~ V ~ V I ~'Kl 10: J v l'A '~ K~lpll 111~ A~ b<J~ . q ~ v v _ Table ~I. Composition o~ the NUSSA enzy~e test 7~Al~n~ r~t~ PlP--K-'re~ t~
~9 ~est UCl . p~ B s o n~M
Mg~ H~O s nM 12t pl 1.00 p - x ,~11 NA~B ~,~S mM 10 ul ao ~il JC C-Phosp2~ ,5 ~ 10 ~ 0 al ~ucto~e-2, 6-P, ~ 0 ~ 0 ,~Ll I~P~ 2,5 ~ al ---~Zuck~r-l-Pho5ph~t v3~riabe~ 10 ~Ll N~l~cleo~idsrll~hosFh~t ~ be~ ,0 ~
i~) P~I~FK-Pr~p~rat~ on ,~rariab~1 20 Itl ' 10. ;~1 x f~l ~ldol~e 0, 0~ U~ '00 ',~
(~r~.O~ p~t-~Jo~er~c 1 ttr200 /~ pl ~G lyce ~ln - 3 - Pho ph~e -Dcl,,.l~ ~. .. ~:.c 0,136 u~2QO ~ ul ~r~PyropS~o~phor~
~r~p~rleion ~ar~bel --- 20 J~l Key: 1 Flnal conce~tration 2 PPIPFK ~est 3 Pyrophospho~yla~e ~est 4 Fruc~ose-6-phosph~te Sugar-1-phosphate ~ Nucleoside triphosphate 7 PPlPFK preparation 8 Triose phosphata-isomerase 9 Glycerin-3-phosphate dahydrogenase Pyrophosphorylase preparatlon 11 Variable u~ / rn~ r~ ~~ ,rL.l-V,y ~Ic ~b~iU~
.

~ 35 The tot~l volume wa~ 200 ~L. The preparations ~ero measured in a Titertek photomete~ ~ lecular devicQ, Munich, by photo~etry.
PP~ was us~d for the start, in the case of the PP~PFg te~ ~d a sugar~1-phosphate or the ~ucleoslde triphosphate was used in the ca8e Or pyropho~phor~lases.
The ~ollowing ~ormula was used to calculate the acti~ity:

U/mL ~ (~ (mOD~time) * sa~ple dilution * measured Yolume)/(1000 ~ sa~pl~ ~olume ~ d ~ * 2) = (10-3 oE/min ~ sample dil~tion * ~00 ~L)/(10~ ~ 20 ~L *
0.67 cm ~ 6.3 ~l~mmol/~l ~ cm-l) * 2) ~ ~E * sample diluti~n ~ 0.0012 ~mol/mL ~ min Use o~ the NUSSA enzyme test 1) Exa~ple see above: Activity measurement of the GDP-m~nnos~ pyrophosphorylase 2J Example: U~e of NUSSA for ~creening pyrophoshorylases ln t~o different enzyme sources: .
a) Esc~erichia co7i BL21(D~3)pLysSpERJ-1 b) Rice (Oryza sativa L. ) For using the test, the PP1PFK has to ~ purified. As a simple and e~sily a~ailable enzyme ~ource, potatoes w~r~ selected ~Solanum tuberosum L~ ~ . Th~ purification was carr~ed out according to the method described by van Schaftingen et ~1./ l9a2 u o ~ v ~ v ~ r ~ ~ cc ~b b ~ v ln Eur. J. Biochem., Vol. 12~, pp. 1~ 5. Th~ enzyme IPP~PFK) was stored in 25~ glycerin ~ -Z0~~.
E~c~erichia coli ~21 (DE3)pLy~SpER~-l wa~ cultuxed ~nd broken up as d~scribQd above. The resulting raw homogenate was contri~uged at lQ,000 rp~ ~or 2 ~in, and at 20~C, then used in the ~zyme Se~t (21.02 ~g/~L). The rice was broken up according to Elllng, 1993, ~erman Patent DE 4,221,5~5 Cl, at 10,000 rpm, 10 mi~, 20~C, and it was used a~ a raw extract (4.2~ mg/m~) in the enzyme scree~ing. The substra~es tested were:
a) Nucleoside tr~phosphate: ATP, CTP, GTP, UTP, CITTP, each at lmM in the test wtth gluco~ pho~phate (2.5mM) b) Nucleoside ~ipbo.~pha~e UTP ~
Suga~ phosphate, each at 2.5mM ln th~ test Ta~le VII ~hows the specific act$v$tie8 Or pyrophosphorylases in a m~ crobial ~nd in a eukaryotic enzyme sou~ce .

~ v ~ ~ v ~ ~ ~

Table VII. Speci~lc act~itles o~ pyropho~phoryl~ses ln E. c~
and rice ~' ~)~-D-7uck~ Ph~ph~e ~P5 ~ col ~12. 5 ~1~ nD/~) lm~/~g3 Cluc~ ph~ 7~
C~P o. ~s ~ . a~
~P 2~ lD ~18~ t~
_ d~ P ~~. 3~ ~ 5' ~)a-~-Glu~os~min-l-~ ~ VJP 2. tS
-D-Glc~A~ TP 2,02 G,27 ~<~-D-G~l~eeo~c~ UsP 1 lo, ~1 ~-D-Cal;leeo-~amin-l-P~ urP - 0,2S
t~D~G~l~tAc-1-P ~ ~P - 0,~.
~-P-Glucur~n~u~e-l-P~ uTP ~,7Z 1 ~)a-D.G~L~uron3~ure-l-PI ;~P 3.~6 .n xylo:~e-l-P . USP 0, 42 a-D-~ o~ -P ~ C~P 3,'11 1,51 B~ zo ~lgJml ~. coll ~L2~ (D~3)pLy~S-pJER-l-~ohex~r~k" ~m ~a~eansa~r, 1,06 ~g~m~ b~-0,14 mg/ml a~a 5~ti~ -P~h-Yr~ Ses~n6a~ ke~,1t~tsb~ imun~ p-t~ ~SS
Key: 1 a-D-Sugar-1-phosphat~s (2.5mM) 2 Rice (mU/~g) 3 ~-D-Glucose-1-phospha~e 4 a-D-Gluc~sami~e-1-P
a ~-Galactosamlne-l-P
6 a-D-Glucuronic a~id-l-P
7 a-D-Gal~cturonic ac~d-1-P
8 20 ~g/mL E. coli BL21(DE3)pLysS~pJER-1 raw extraCt in the test preparation, 1.06 ~g/mL-0.14 mg/~L of Oryza sati~a raw ext~ct in the test preparation. ~cti~ity determination by N~SSA

U8~Z~7 ~Kr 1~:~5 iA~ Pn ~c~ ro~ ~ A~oc. 4~

For the analysis c~ the synthesis of the GDP-mannose, a cap~ llary elect~opho~e~is apparatu~ (~3echna~ company) was used.
The method used was capillary zone elect~ophoresis with a borate buffer system~ For this . purpo~e, 40 mL o$ 0 . ~ boric acid and 20 mI, o~ O.lmM sodium borate ~ere mlxed and the volume wa~ brought up wlth 140 ~ H20 Th~ pH was approximately 8 . 3 ~or t~is mixture ratio. The voltage that was p~r-:select~d was 25 kY. The curren~
established was approximately 35-37 ,~.
To be able to ~etermine l:he conrentrations ~:rom the elect~ophoregramx, di~erent conce~trations, ~. 02-0. q~M, of ~Dp-ma~no~e and ~TP were prepared in 50mh Tris-~Cl, p~ 8, With 5mM
MgCl2, and analy~ed by capillar~ electrophore~i~. By plottln~ the ar~a ~ersus the theoretically ~sQd concentration (mM), a ~traig~t line w~s obtained, so that linear r~gre~ion can be applled:

GTP y ~ b ~ x ~ a with a: 0.05~4 ~: 3.5314 ~ r: 0.991 Total tme~n~ square ~rror: O.qO24 * 10 2 GDP-mannose y ~ b * x + a with a- 0.0552 b~ 3.4742 r: 0.994 Total square e~ror: 0-7178 ~ 10-~

Isolat~on o~ phosphomanno~tase from ~. co~i B~21(DE~) 08/2a~7 FRI 1~:35 FA~ RalPh ~cElro~ & A~oc. ~1~37 C~lturing of E. col f BL~l and br~akup of the cell~:
at ~7~C in a s~aker at 120 rpm, as for G~P-mannos~
pyropho~phor~lase Th~ raw ~xtract obt~ined ~a~ lo~de~ on an anton ~xchangQr;
Q-Sepharose FF;
77 mL of Q-Sepharo~e FF were loaded ~lth 122 mL of raw extrac1: (with a 14 mg/~I protein conte~t). A linearly i~creasing gradi~nt (50~ Tris-~Cl, pH 8, 0.600mM ~C11 was prepared, then the protein was elut~d betwe~n 280 and 460mM KCl. The acti~r~
~rac~ions were co~bined, then the pxotein wa.~ precipit~ted ~ith 3M (NH~)~50q~ A~ter centri~ugati~n (15 m~, lO,000 rp~, 4~C~ th~
pellet wa~ dissolved in 5 m~ Tris-HCl, pH 8. Th~ enzYme could be obtained with a purific~tion factor o~ ~.4 and a yield of 78~.

T~ble VIII. Partial puri~lcat~on o~ the phosphomannomutase G~s~ e:l~m~- ~p~ ;ch~ oiurn--n ~iAis~mg~
pro~:ein ~,~vie5~ viel~ W~lkr.or (~) ~m9] tUl lU~mgl~ %1 (~Jlohcxe~ e 1703 .1 107, 9 0,11 112 ~ lOo ~)Q-ScptUI~r~e 55-;2 3.~6,5 0,2C 24C 2,~ 7c Key: 1 Total protein (~ng) 2 Total activity (U1 3 Spec$ f ic activity (U/mg 4 Volume Imh ) Purification factox U8~ Kl lU:3~ ~A~ Kalpn ~C~lroY ~ A~SOC. ~u~

6 Yield (~) 7 Ra~ extract 8 Q-Sepharo~e Enzymatic synthesis of GDP-a-D-~annose ~tarting with mannose The enzymatiC synthe~is of GDP-mannose (Figure 14) is car~ied out starti~g with m~nnose via the hexo~inase~catalyZed phosphorylation ~t C6, isomer~zation o~ ~annose-6-phosphate to mannose-1-phosph~te by the ph~spho~nom~tase, and the conversion of ma~nose-1-phosphate ~ith GTP to GDP-mannose ~ith th~ GDP-ma~nose pyrophosphoryIase.
The ATP u~3ed i5 recycled, by the conversion of the ADP
produced duxing the hexokinase reaction, with ~osphoenol-pyru~rate and catalysis bY the pyru~rate kinase, to form ATP and pyruvate (Wong et al., 1995, Angew. Chem. t Vol . 107, pp . ~69-593) (Figure 15).
The synthesis on a larger scale Was carried out in the "repetitive batch" pxocedure. The tota~ volu~ne of the synthetic preparation was 80 mI.. The i~ollowing 'cable ~hows the composition of said Yynthetic preparationn ~J O ~ t ~ V~ oc ~b blJ~: . ~ u v V

ng~e~te Mensle 1 GSP 253 ~9 5 ~
M~nno~e 72,1 mg S mM
PtP 125 mg ~ 'J, 5 mM, ~P ~ ~, 2 n~ 2 mM
C1~ , 6 - P~ 0, 25 rx~ U/ml (~)Pyruv~-Klr~ c ~0 V~ml G~PM-P~
P~a~ 2 ~/ol ~ri~-8CL, pH 6 50 ~M
0 m~
XCl ~0 I~M

~)PMM: Pho~ e; ~P--~c~ ,rh~ aDPM-P~: eDP-a-D 1 ---~
p~ ~ph~ .h~ty~

Key: 1 Quantity us~d 2 Final concentra~ion 3 Pyru~ate kinasQ
4 PMM: Phosphomannomutase; PPase: Pyrophosphatase;
GPDM-PP: GDP-a-D-mannose pyrophosphorylase After ~4 h, the prep~ration was reduced using an ~ltrafil~ration module with a YM ~0 me~brane (cutoff 10 kD) from the Ami~on company ~Wit~en) to S mLs the volume was brought up to 50 mL w~th 50mM Tris-HCl, p~ 8, lOmM KCl, and lOmM Mg~l2. It was reduced agaln and the vol~e was again.brought up, ~ollowed by a renewed reductlon to 5 m~. The pr~tein-containing ~etentate was ~ ~ I

reacted with a new synthetic preparation with substxatQ solution ~75 m~ nd ~gain incu~ated i~or 24 h. Thi~3 protoc~l wa~ then repeated one more ~ime.
The filtrates were reacted with alkalin~ pho:~lphat~se ~1 U/mI ) ar~d incubated for 24 h i.n order to dephosphoryla'ce nucleoside ~ono-, di-, and triphosphates or sugar phosphates ~uch as mannosQ-6- or 1-phosphate. The activated sugars ~re not attacked by t~ phosphata~e. .
All in all, 253 mg ~0 . 4 mmol) Or GTP were reacted three ~imes with 216.3 mg of mannose. A determlnation o~ the yields after 24 h, ~or each Feactionl p~od~ced: .

Yield Preparation 1: 4.4mM GDP-~annose 8896 Preparat:ion 2: 4~8mM GDP~mannose 96%
Preparatiorl 3: 2 . 8~rM GDP~mannose 5696 Mean yi~ld in 72 h; 80g6 This co~esponds to 581 mg of ~r~P mannose, with respect to ~ree acid (605.3 g~mol~.

After the lncubation with alkaline phosphatase, ~he preparations were ultrafll~ered and pu~ified, then loaded on an anion e~change~ Dowex~ 1 x 2 Cl-, Serva.
The GDP-mannose was elut~d u~ing a linear gr~dient between 0 and 0. 5M LiCl (500 m~) with 1~ LiCl. The GDP-mannos~ containing solution (~00 mL with 0.92mM GD~annose~ wa~ reduced using a rotatory ~raporator. Thi~ fraction was subj~cted to gel 08~2~7 FRI 1~:45 FA~ RalPh ~cElroy & A~oc. ~058 filtration using Sephadex G-10, then the ~DP ~ ~~nose-co~t~n~ng fraction~ ~rere lyophilized. Th~a lyoph~ l~zate wa~ d$ssolved in small amoun~ o~ water, then reacted wi~h lce cold ac~tone. ~he precipitated GDP-manno~3e Was removed ~y filtration, di~:ol~ed ~ n water, lyophilized, and analyzed by capillary electrophore~is wlth a comparison o~.the area~ to a st~n~rcl cur~ . Fi~uro 16 shows the olectrophoregram 01' the nondiluted samplR ( 1 ~g of the lyophilizate/mL water).
A total of 199 mg of GDP-~anno~ in 500 mg o~ lyophil~ z~te was obtsIned.

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CA 022l4458 l997-09-02

Claims (21)

Claims

1. Method for the preparation of GDP-mannose, in which the gene expression of phosphomannomutase or GDP-mannose pyrophosphorylase (GDP-Man-PP) in a microorganism is increased.

2. Method according to Claim 1, characterized in that the gene expression of phosphomannomutase (rfbK) or GDP-Man-PP (rfbM) is increased by increasing the number of copies of the .rfbK or rfbM genes.

3. Method according to Claim 2, characterized in that, to increase the number of the copies of the rfbK or rfbM genes, a gene construct is incorporated.

4. Method according to Claim 3, characterized in that a microorganism is transformed with the gene construct that contains the rfbK or rfbM gene.

5. Method according to Claim 4, characterized in that an Escherichia coli strain is transformed with the gene construct that contains the rfbK or rfbM gene.

6. Method according to Claim 5, characterized in that E.
coli BL21(DE3) is transformed with the gene construct.

7. Method according to one of the preceding claims, characterized in that the genes are isolated from a microorganism.

8. Method according to Claim 7, characterized in that the genes are isolated from Salmonella enterica, group B,

9. Method according to one of the preceding claims, characterized in that, after increasing the gene expression, the phosphomannomutase or GDP-Man-PP is isolated.

10. Method according to Claim 9, characterized in that, for the isolation of the enzymes, the raw extract of the recombinant strain is loaded on an anionic exchanger.

11. Method according to Claim 10, characterized in that, for the isolation of the GDP-Man-PP, the ion exchanger is subjected to a stepwise gradient elution, from whose enzyme-enriched fraction the GDP-Man-PP is obtained by hydrophobic interaction chromatography (HIC) with a linearly decreasing (NH4)2SO4 gradient.

12. Method according to one of the preceding claims, characterized in that the phosphomannomutase formed after the increase in the gene expression is used for the reaction of mannose-6-phosphate to form mannose-1-phosphate.

13. Method according to one of the preceding claims, characterized in that the GDP-Man-PP formed after the increase in the gene expression is used with GTP for converting mannose-1-phosphate to GDP-mannose.

14. Mannose- or mannose-derivative-specific GDP-Man-PP, which can be isolated from recombinant cells, having a specific activity ~2 U/mg.

15. Phosphomannomutase, which can be obtained by the method according to one of Claims 1 to 10.

16. Transformed cell, containing phosphomannomutase or GDP-Man-PP in overexpressed form.

17. Transformed cell according to Claim 16, characterized in that it is Escherichia coli.

18. Transformed cell according to Claim 17, characterized in that it is Escherichia coli BL21(DE3).

19. Photometric test in which the pyrophosphate obtained by means of a pyrophosphate releasing enzyme is converted by means of a pyrophosphate-dependent phosphofructokinase, an aldolase, a triose phosphate isomerase, and a glycerin-3-phosphate dehydrogenase, with the reduction that occurs as a result of the dehydrogenase being photometrically determined.

20. Photometric test according to Claim 19 for the determination of pyrophosphate-releasing nucleotidyl transferases.

21. Photometric test according to Claim 20 for the determination of the GDP-mannose pyrophosphorylase.