AGAA_GEOSE
ID AGAA_GEOSE Reviewed; 729 AA.
AC Q9ALJ4;
DT 12-APR-2017, integrated into UniProtKB/Swiss-Prot.
DT 01-JUN-2001, sequence version 1.
DT 03-AUG-2022, entry version 68.
DE RecName: Full=Alpha-galactosidase AgaA {ECO:0000303|Ref.1, ECO:0000312|EMBL:AAG49420.1};
DE EC=3.2.1.22 {ECO:0000269|PubMed:16511274, ECO:0000269|PubMed:23012371, ECO:0000269|Ref.1};
GN Name=agaA {ECO:0000303|Ref.1, ECO:0000312|EMBL:AAG49420.1};
OS Geobacillus stearothermophilus (Bacillus stearothermophilus).
OC Bacteria; Firmicutes; Bacilli; Bacillales; Bacillaceae; Geobacillus.
OX NCBI_TaxID=1422 {ECO:0000312|EMBL:AAG49420.1};
RN [1] {ECO:0000312|EMBL:AAG49420.1}
RP NUCLEOTIDE SEQUENCE [GENOMIC DNA], CATALYTIC ACTIVITY, ACTIVITY REGULATION,
RP BIOPHYSICOCHEMICAL PROPERTIES, AND BIOTECHNOLOGY.
RC STRAIN=KVE39 {ECO:0000303|Ref.1, ECO:0000312|EMBL:AAG49420.1};
RA Ganter C., Boeck A., Buckel P., Mattes R.;
RT "Production of thermostable, recombinant alpha-galactosidase suitable for
RT raffinose elimination from sugar beet syrup.";
RL J. Biotechnol. 8:301-310(1988).
RN [2]
RP CRYSTALLIZATION OF MUTANT GLU-355, AND CATALYTIC ACTIVITY.
RC STRAIN=KVE39 {ECO:0000303|PubMed:16511274};
RX PubMed=16511274; DOI=10.1107/s1744309105042582;
RA Foucault M., Watzlawick H., Mattes R., Haser R., Gouet P.;
RT "Crystallization and preliminary X-ray diffraction studies of two
RT thermostable alpha-galactosidases from glycoside hydrolase family 36.";
RL Acta Crystallogr. F 62:100-103(2006).
RN [3] {ECO:0007744|PDB:4FNP, ECO:0007744|PDB:4FNR, ECO:0007744|PDB:4FNS}
RP X-RAY CRYSTALLOGRAPHY (2.60 ANGSTROMS) OF SUBSTRATE-FREE WILD-TYPE AND
RP MUTANT GLU-355 AND IN COMPLEX WITH RAFFINOSE; STACHYOSE AND INHIBITOR,
RP FUNCTION, CATALYTIC ACTIVITY, ACTIVITY REGULATION, BIOPHYSICOCHEMICAL
RP PROPERTIES, ACTIVE SITE, AND MUTAGENESIS OF TRP-336; ASP-478 AND ASP-548.
RX PubMed=23012371; DOI=10.1074/jbc.m112.394114;
RA Merceron R., Foucault M., Haser R., Mattes R., Watzlawick H., Gouet P.;
RT "The molecular mechanism of thermostable alpha-galactosidases AgaA and AgaB
RT explained by x-ray crystallography and mutational studies.";
RL J. Biol. Chem. 287:39642-39652(2012).
CC -!- FUNCTION: Hydrolyzes the short-chain alpha-galactosaccharides raffinose
CC and stachyose. {ECO:0000269|PubMed:23012371}.
CC -!- CATALYTIC ACTIVITY:
CC Reaction=Hydrolysis of terminal, non-reducing alpha-D-galactose
CC residues in alpha-D-galactosides, including galactose
CC oligosaccharides, galactomannans and galactolipids.; EC=3.2.1.22;
CC Evidence={ECO:0000269|PubMed:16511274, ECO:0000269|PubMed:23012371,
CC ECO:0000269|Ref.1};
CC -!- ACTIVITY REGULATION: Not inhibited by D-galactose or sucrose (Ref.1).
CC Inhibited by pharmaceutical drug 1-deoxygalactonojirimycin
CC (PubMed:23012371). {ECO:0000269|PubMed:23012371, ECO:0000269|Ref.1}.
CC -!- BIOPHYSICOCHEMICAL PROPERTIES:
CC Kinetic parameters:
CC KM=0.21 mM for p-nitrophenyl-alpha-galactopyranoside (at 25 degrees
CC Celsius and pH 6.5) {ECO:0000269|PubMed:23012371};
CC KM=3.8 mM for raffinose (at 25 degrees Celsius and pH 6.5)
CC {ECO:0000269|PubMed:23012371};
CC Note=kcat is 105 sec(-1) for p-nitrophenyl-alpha-galactopyranoside
CC (at 25 degrees Celsius and pH 6.5). kcat is 180 sec(-1) for raffinose
CC (at 25 degrees Celsius and pH 6.5). {ECO:0000269|PubMed:23012371};
CC pH dependence:
CC Optimum pH is 6.5. {ECO:0000269|Ref.1};
CC Temperature dependence:
CC Optimum temperature is 65 degrees Celsius. {ECO:0000269|Ref.1};
CC -!- SUBUNIT: Homotetramer. {ECO:0000250|UniProtKB:G1UB44}.
CC -!- BIOTECHNOLOGY: Has properties that make it suitable for elimination of
CC D-raffinose from low green syrup in sugar manufacturing process from
CC beets in order to improve the yield of sucrose. {ECO:0000269|Ref.1}.
CC -!- SIMILARITY: Belongs to the glycosyl hydrolase 36 family. {ECO:0000305}.
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DR EMBL; AY013286; AAG49420.1; -; Genomic_DNA.
DR PDB; 4FNP; X-ray; 2.80 A; A/B/C/D=1-729.
DR PDB; 4FNR; X-ray; 3.20 A; A/B/C/D=1-729.
DR PDB; 4FNS; X-ray; 2.60 A; A/B/C/D=1-729.
DR PDB; 4FNT; X-ray; 2.60 A; A/B/C/D=1-729.
DR PDB; 4FNU; X-ray; 3.60 A; A/B/C/D=1-729.
DR PDBsum; 4FNP; -.
DR PDBsum; 4FNR; -.
DR PDBsum; 4FNS; -.
DR PDBsum; 4FNT; -.
DR PDBsum; 4FNU; -.
DR AlphaFoldDB; Q9ALJ4; -.
DR SMR; Q9ALJ4; -.
DR CAZy; GH36; Glycoside Hydrolase Family 36.
DR BRENDA; 3.2.1.22; 623.
DR GO; GO:0004557; F:alpha-galactosidase activity; IDA:UniProtKB.
DR GO; GO:0052692; F:raffinose alpha-galactosidase activity; IEA:UniProtKB-EC.
DR GO; GO:0051289; P:protein homotetramerization; ISS:UniProtKB.
DR GO; GO:0034484; P:raffinose catabolic process; IDA:UniProtKB.
DR GO; GO:0033531; P:stachyose metabolic process; IDA:UniProtKB.
DR CDD; cd14791; GH36; 1.
DR Gene3D; 2.60.40.1180; -; 1.
DR Gene3D; 2.70.98.60; -; 1.
DR Gene3D; 3.20.20.70; -; 1.
DR InterPro; IPR013785; Aldolase_TIM.
DR InterPro; IPR038417; Alpga-gal_N_sf.
DR InterPro; IPR000111; Glyco_hydro_27/36_CS.
DR InterPro; IPR002252; Glyco_hydro_36.
DR InterPro; IPR031705; Glyco_hydro_36_C.
DR InterPro; IPR031704; Glyco_hydro_36_N.
DR InterPro; IPR013780; Glyco_hydro_b.
DR InterPro; IPR017853; Glycoside_hydrolase_SF.
DR Pfam; PF16874; Glyco_hydro_36C; 1.
DR Pfam; PF16875; Glyco_hydro_36N; 1.
DR PIRSF; PIRSF005536; Agal; 1.
DR PRINTS; PR00743; GLHYDRLASE36.
DR SUPFAM; SSF51445; SSF51445; 1.
DR PROSITE; PS00512; ALPHA_GALACTOSIDASE; 1.
PE 1: Evidence at protein level;
KW 3D-structure; Carbohydrate metabolism; Glycosidase; Hydrolase.
FT CHAIN 1..729
FT /note="Alpha-galactosidase AgaA"
FT /id="PRO_0000439585"
FT ACT_SITE 478
FT /note="Nucleophile"
FT /evidence="ECO:0000269|PubMed:23012371"
FT ACT_SITE 548
FT /note="Proton donor"
FT /evidence="ECO:0000269|PubMed:23012371"
FT BINDING 53
FT /ligand="substrate"
FT /evidence="ECO:0000269|PubMed:23012371,
FT ECO:0007744|PDB:4FNT"
FT BINDING 199
FT /ligand="substrate"
FT /evidence="ECO:0000269|PubMed:23012371,
FT ECO:0007744|PDB:4FNT"
FT BINDING 366..367
FT /ligand="substrate"
FT /evidence="ECO:0000269|PubMed:23012371,
FT ECO:0007744|PDB:4FNT"
FT BINDING 443
FT /ligand="substrate"
FT /evidence="ECO:0000269|PubMed:23012371,
FT ECO:0007744|PDB:4FNT"
FT BINDING 476..480
FT /ligand="substrate"
FT /evidence="ECO:0000269|PubMed:23012371,
FT ECO:0007744|PDB:4FNT"
FT BINDING 526
FT /ligand="substrate"
FT /evidence="ECO:0000269|PubMed:23012371,
FT ECO:0007744|PDB:4FNT"
FT BINDING 548
FT /ligand="substrate"
FT /evidence="ECO:0000269|PubMed:23012371,
FT ECO:0007744|PDB:4FNT"
FT MUTAGEN 336
FT /note="W->A: Very strongly reduced hydrolytic efficiency
FT against raffinose, but displays medium level of
FT transglycosylation activity compared to none with wild-type
FT enzyme."
FT /evidence="ECO:0000269|PubMed:23012371"
FT MUTAGEN 336
FT /note="W->F,S: Strongly reduced hydrolytic efficiency
FT against raffinose, but displays high level of
FT transglycosylation activity compared to none with wild-type
FT enzyme."
FT /evidence="ECO:0000269|PubMed:23012371"
FT MUTAGEN 336
FT /note="W->N: Very strongly reduced hydrolytic efficiency
FT against raffinose, but displays low level of
FT transglycosylation activity compared to none with wild-type
FT enzyme."
FT /evidence="ECO:0000269|PubMed:23012371"
FT MUTAGEN 478
FT /note="D->A: Loss of activity."
FT /evidence="ECO:0000269|PubMed:23012371"
FT MUTAGEN 548
FT /note="D->N: Loss of activity."
FT /evidence="ECO:0000269|PubMed:23012371"
FT STRAND 11..16
FT /evidence="ECO:0007829|PDB:4FNS"
FT STRAND 19..26
FT /evidence="ECO:0007829|PDB:4FNS"
FT TURN 27..29
FT /evidence="ECO:0007829|PDB:4FNS"
FT STRAND 30..38
FT /evidence="ECO:0007829|PDB:4FNS"
FT STRAND 45..47
FT /evidence="ECO:0007829|PDB:4FNR"
FT STRAND 59..61
FT /evidence="ECO:0007829|PDB:4FNS"
FT HELIX 69..71
FT /evidence="ECO:0007829|PDB:4FNS"
FT STRAND 74..76
FT /evidence="ECO:0007829|PDB:4FNS"
FT STRAND 79..82
FT /evidence="ECO:0007829|PDB:4FNS"
FT STRAND 88..92
FT /evidence="ECO:0007829|PDB:4FNS"
FT STRAND 103..112
FT /evidence="ECO:0007829|PDB:4FNS"
FT HELIX 127..129
FT /evidence="ECO:0007829|PDB:4FNS"
FT STRAND 130..139
FT /evidence="ECO:0007829|PDB:4FNS"
FT TURN 140..143
FT /evidence="ECO:0007829|PDB:4FNS"
FT STRAND 144..156
FT /evidence="ECO:0007829|PDB:4FNS"
FT STRAND 158..167
FT /evidence="ECO:0007829|PDB:4FNS"
FT STRAND 169..171
FT /evidence="ECO:0007829|PDB:4FNS"
FT STRAND 173..186
FT /evidence="ECO:0007829|PDB:4FNS"
FT STRAND 190..196
FT /evidence="ECO:0007829|PDB:4FNS"
FT STRAND 206..209
FT /evidence="ECO:0007829|PDB:4FNS"
FT STRAND 212..219
FT /evidence="ECO:0007829|PDB:4FNS"
FT STRAND 221..223
FT /evidence="ECO:0007829|PDB:4FNS"
FT STRAND 226..236
FT /evidence="ECO:0007829|PDB:4FNS"
FT STRAND 241..244
FT /evidence="ECO:0007829|PDB:4FNS"
FT STRAND 246..251
FT /evidence="ECO:0007829|PDB:4FNS"
FT STRAND 257..263
FT /evidence="ECO:0007829|PDB:4FNS"
FT STRAND 269..275
FT /evidence="ECO:0007829|PDB:4FNS"
FT STRAND 282..284
FT /evidence="ECO:0007829|PDB:4FNS"
FT STRAND 289..291
FT /evidence="ECO:0007829|PDB:4FNS"
FT STRAND 295..302
FT /evidence="ECO:0007829|PDB:4FNS"
FT HELIX 303..318
FT /evidence="ECO:0007829|PDB:4FNS"
FT STRAND 325..327
FT /evidence="ECO:0007829|PDB:4FNS"
FT STRAND 332..334
FT /evidence="ECO:0007829|PDB:4FNS"
FT HELIX 336..339
FT /evidence="ECO:0007829|PDB:4FNS"
FT HELIX 345..358
FT /evidence="ECO:0007829|PDB:4FNS"
FT STRAND 362..365
FT /evidence="ECO:0007829|PDB:4FNS"
FT STRAND 367..370
FT /evidence="ECO:0007829|PDB:4FNS"
FT STRAND 374..378
FT /evidence="ECO:0007829|PDB:4FNP"
FT TURN 387..389
FT /evidence="ECO:0007829|PDB:4FNS"
FT HELIX 393..402
FT /evidence="ECO:0007829|PDB:4FNS"
FT TURN 403..405
FT /evidence="ECO:0007829|PDB:4FNS"
FT STRAND 407..412
FT /evidence="ECO:0007829|PDB:4FNS"
FT STRAND 419..421
FT /evidence="ECO:0007829|PDB:4FNS"
FT HELIX 422..426
FT /evidence="ECO:0007829|PDB:4FNS"
FT HELIX 428..430
FT /evidence="ECO:0007829|PDB:4FNS"
FT STRAND 441..444
FT /evidence="ECO:0007829|PDB:4FNS"
FT STRAND 446..448
FT /evidence="ECO:0007829|PDB:4FNS"
FT HELIX 453..468
FT /evidence="ECO:0007829|PDB:4FNS"
FT TURN 469..471
FT /evidence="ECO:0007829|PDB:4FNS"
FT STRAND 474..477
FT /evidence="ECO:0007829|PDB:4FNS"
FT HELIX 493..498
FT /evidence="ECO:0007829|PDB:4FNS"
FT HELIX 499..517
FT /evidence="ECO:0007829|PDB:4FNS"
FT STRAND 522..525
FT /evidence="ECO:0007829|PDB:4FNS"
FT HELIX 535..540
FT /evidence="ECO:0007829|PDB:4FNS"
FT STRAND 541..545
FT /evidence="ECO:0007829|PDB:4FNS"
FT HELIX 554..562
FT /evidence="ECO:0007829|PDB:4FNS"
FT TURN 563..565
FT /evidence="ECO:0007829|PDB:4FNS"
FT HELIX 568..570
FT /evidence="ECO:0007829|PDB:4FNS"
FT STRAND 571..575
FT /evidence="ECO:0007829|PDB:4FNS"
FT TURN 581..583
FT /evidence="ECO:0007829|PDB:4FNS"
FT HELIX 589..596
FT /evidence="ECO:0007829|PDB:4FNS"
FT STRAND 599..603
FT /evidence="ECO:0007829|PDB:4FNS"
FT HELIX 607..609
FT /evidence="ECO:0007829|PDB:4FNS"
FT HELIX 612..634
FT /evidence="ECO:0007829|PDB:4FNS"
FT STRAND 635..641
FT /evidence="ECO:0007829|PDB:4FNS"
FT TURN 643..645
FT /evidence="ECO:0007829|PDB:4FNS"
FT STRAND 646..654
FT /evidence="ECO:0007829|PDB:4FNS"
FT STRAND 658..667
FT /evidence="ECO:0007829|PDB:4FNS"
FT STRAND 677..679
FT /evidence="ECO:0007829|PDB:4FNT"
FT STRAND 688..692
FT /evidence="ECO:0007829|PDB:4FNS"
FT TURN 693..695
FT /evidence="ECO:0007829|PDB:4FNS"
FT STRAND 696..699
FT /evidence="ECO:0007829|PDB:4FNS"
FT HELIX 700..705
FT /evidence="ECO:0007829|PDB:4FNS"
FT STRAND 708..710
FT /evidence="ECO:0007829|PDB:4FNT"
FT STRAND 715..726
FT /evidence="ECO:0007829|PDB:4FNS"
SQ SEQUENCE 729 AA; 83211 MW; 6E59129E1C3502A2 CRC64;
MSVAYNPQTK QFHLRAGKAS YVMQLFRSGY LAHVYWGKAV RDVRGARAFP RLDRAFSPNP
DPSDRTFSLD TLLQEYPAYG NTDFRAPAYQ VQLENGSTVT DLRYKTHRIY KGKPRLNGLP
ATYVEHEQEA ETLEIVLGDA LIGLEVTLQY TAYEKWNVIT RSARFENKGG ERLKLLRALS
MSVDFPTADY DWIHLPGAWG RERWIERRPL VTGVQAAESR RGASSHQQNP FIALVAKNAD
EHQGEVYGFS FVYSGNFLAQ IEVDQFGTAR VSMGINPFDF TWLLQPGESF QTPEVVMVYS
DQGLNGMSQT YHELYRTRLA RGAFRDRERP ILINNWEATY FDFNEEKIVN IARTAAELGI
ELVVLDDGWF GERDDDRRSL GDWIVNRRKL PNGLDGLAKQ VNELGLQFGL WVEPEMVSPN
SELYRKHPDW CLHVPNRPRS EGRNQLVLDY SREDVCDYII ETISNVLASA PITYVKWDMN
RHMTEIGSSA LPPERQRETA HRYMLGLYRV MDEITSRFPH ILFESCSGGG GRFDPGMLYY
MPQTWTSDNT DAVSRLKIQY GTSLVYPISA MGAHVSAVPN HQVGRVASLK TRGHVAMSGN
FGYELDITKL TETEKQMMKQ QVAFYKDVRR LVQFGTFYRL LSPFEGNEAA WMFVSADRSE
ALVAYFRVLA EANAPLSYLR LKGLDSNQDY EIEGLGVYGG DELMYAGVAL PYRSSDFISM
MWRLKAVQQ
