gdp-mannose-3', 5' -epimerase (arabidopsis thaliana),k178r, with gdp-beta-l-gulose and gdp-4-keto-beta-l-gulose bound in active site.

4-(2-HYDROXYETHYL)-1-PIPERAZINE ETHANESULFONIC ACID

GUANOSINE 5'-DIPHOSPHATE-4-KETO-BETA-L-GULOSE

Structure and function of GDP-mannose-3',5'-epimerase: an enzyme which performs three chemical reactions at the same active site.

GDP-BETA-L-GULOSE WAS REFINED USING GMP (DEFINED AS GDP, MODIFIED IN THE CIF FILE TO REMOVE THE SECOND PHOSPHATE GROUP) AND GULOSE-MONOPHOSPHATE (GUL), LINKING THE GDP O3A TO THE GUL PB. GDP-BETA-L-4-KETO-GULOSE WAS REFINED USING GMP (DEFINED AS GDP, MODIFIED IN THE CIF FILE TO REMOVE THE SECOND PHOSPHATE GROUP) AND 4-KETO-GULOSE- MONOPHOSPHATE (4KG), LINKING THE GDP O3A TO THE 4KG PB NAD WAS REFINED USING ADP AND A NICOTINAMIDE RING (NI3), LINKING THE ADP PB TO THE NI3 O5* NADH WAS REFINED USING ADP AND A SKEWED REDUCED NICOTINAMIDE RING (NDH), LINKING THE ADP PB TO THE NDH O5*

GAMGTTNGTDYGAYTYKELEREQYWPSENLKISITGAGGFIASHIARRLKHEGHYVIASDWKKNEHMTEDMFCDEFHLVD
LRVMENCLKVTEGVDHVFNLAADMGGMGFIQSNHSVIMYNNTMISFNMIEAARINGIKRFFYASSACIYPEFKQLETTNV
SLKESDAWPAEPQDAYGLERLATEELCKHYNKDFGIECRIGRFHNIYGPFGTWKGGREKAPAAFCRKAQTSTDRFEMWGD
GLQTRSFTFIDECVEGVLRLTKSDFREPVNIGSDEMVSMNEMAEMVLSFEEKKLPIHHIPGPEGVRGRNSDNNLIKEKLG
WAPNMRLKEGLRITYFWIKEQIEKEKAKGSDVSLYGSSKVVGTQAPVQLGSLRAADGKE

INITIAL MODEL FOR MOLECULAR REPLACEMENT BY PHASER DETERMINED BY MAD OF SELENO-METHIONINE PROTEIN. DATA COLLECTED ON BM14, ANALYSED WITH SOLVE AND RESOLVE.

PROTEIN CRYSTALISED IN 100MM HEPES PH 7.4, 2.16 M AMMONIUM SULPHATE, VAPOUR DIFFUSION, SITTING DROP. CRYOPROTECTED WITH 4M SODIUM FORMATE.

GDP-MANNOSE-3,5-EPIMERASE (ARABIDOPSIS THALIANA), WITH GDP-ALPHA-D-MANNOSE AND GDP-BETA-L-GALACTOSE BOUND IN THE ACTIVE SITE

GDP-MANNOSE-3,5-EPIMERASE (ARABIDOPSIS THALIANA), Y174F, WITH GDP-BETA-L-GALACTOSE BOUND IN THE ACTIVE SITE

GDP-MANNOSE-3, 5-EPIMERASE (ARABIDOPSIS THALIANA), K217A, WITH GDP-ALPHA-D-MANNOSE BOUND IN THE ACTIVE SITE

ENGINEERED RESIDUE IN CHAIN A, LYS 178 TO ARG ENGINEERED RESIDUE IN CHAIN B, LYS 178 TO ARG
THE FIRST TWO RESIDUES (GA) IN THE SPECIFIED SEQUENCE ARE A REMNANT FROM A CLEAVED HIS-TAG (AND ARE NOT IN THE UNIPROT SEQUENCE), THE RESIDUE NUMBERING IN THE STRUCTURE USES THE THIRD RESIDUE (M) AS RESIDUE 1.

HYDROGENS HAVE BEEN ADDED IN THE RIDING POSITIONS. EACH MONOMER OF GME K178R CONTAINS A MIXTURE OF COMPOUNDS IN THE ACTIVE SITE -NAD AND NADH IN THE NUCLEOTIDE BINDING SITE, GDP-BETA-L-GULOSE AND GDP-BETA-L-4- KETO-GULOSE IN THE NUCLEOTIDE SUGAR BINDING SITE. DURING REFINEMENT THE CONSTANT PORTION OF EACH MOLECULE (ADP FOR NAD, GMP FOR THE GDP-SUGARS, AS THE SECOND PHOSPHATE GROUP MOVES WITH DIFFERENT LIGANDS) WAS FIXED WITH OCCUPANCY OF 1 AND LINKED TO TWO VARIABLE PORTIONS WITH PARTIAL OCCUPANCY.

3' 5'-EPIMERASE, SHORT CHAIN DEHYDRATASE/REDUCTASE, GDP-MANNOSE, GDP-GULOSE, GDP-GALACTOSE, KETO INTERMEDIATE, VITAMIN C, SDR, ISOMERASE, ASCORBATE BIOSYNTHESIS, NAD