The Cellvibrio japonicus mannanase CjMan26C displays a unique exo-mode of action that is conferred by subtle changes to the distal region of the active site.
The microbial degradation of the plant cell wall is a pivotal biological process that is of increasing industrial significance. One of the major plant structural polysaccharides is mannan, a beta-1,4-linked d-mannose polymer, which is hydrolyzed by endo- and exo-acting mannanases. The mechanisms by which the exo-acting enzymes target the chain ends of mannan and how galactose decorations influence activity are poorly understood. Here we report the crystal structure and biochemical properties of CjMan26C, a Cellvibrio japonicus GH26 mannanase. The exo-acting enzyme releases the disaccharide mannobiose from the nonreducing end of mannan and mannooligosaccharides, harnessing four mannose-binding subsites extending from -2 to +2. The structure of CjMan26C is very similar to that of the endo-acting C. japonicus mannanase CjMan26A. The exo-activity displayed by CjMan26C, however, reflects a subtle change in surface topography in which a four-residue extension of surface loop creates a steric block at the distal glycone -2 subsite. endo-Activity can be introduced into enzyme variants through truncation of an aspartate side chain, a component of a surface loop, or by removing both the aspartate and its flanking residues. The structure of catalytically competent CjMan26C, in complex with a decorated manno-oligosaccharide, reveals a predominantly unhydrolyzed substrate in an approximate (1)S(5) conformation. The complex structure helps to explain how the substrate "side chain" decorations greatly reduce the activity of the enzyme; the galactose side chain at the -1 subsite makes polar interactions with the aglycone mannose, possibly leading to suboptimal binding and impaired leaving group departure. This report reveals how subtle differences in the loops surrounding the active site of a glycoside hydrolase can lead to a change in the mode of action of the enzyme.
Cartmell
Alan
A
Institute for Cell and Molecular Biosciences, The Medical School, Newcastle University, Framlington Place, Newcastle upon Tyne NE2 4HH, United Kingdom.
Topakas
Evangelos
E
Ducros
Valérie M-A
VM
Suits
Michael D L
MD
Davies
Gideon J
GJ
Gilbert
Harry J
HJ
D002384:Catalysis; D020134:Catalytic Domain; D020633:Cellvibrio / Q000201:enzymology*; D003001:Cloning, Molecular; D007700:Kinetics; D008351:Mannans / Q000737:chemistry; D008956:Models, Chemical; D008958:Models, Molecular; D008968:Molecular Conformation; D016296:Mutagenesis; D009154:Mutation; D009844:Oligosaccharides / Q000737:chemistry; D011485:Protein Binding; D013379:Substrate Specificity; D044902:beta-Mannosidase / Q000737:chemistry*
D008351:Mannans; D009844:Oligosaccharides; D044902:beta-Mannosidase
Acta Crystallogr D Biol Crystallogr. 2004 Dec;60(Pt 12 Pt 1):2256-68
15572779
J Biol Chem. 2004 Jun 11;279(24):25517-26
15014076
Angew Chem Int Ed Engl. 2006 Aug 4;45(31):5136-40
16823793
Biochem J. 1997 Jan 15;321 ( Pt 2):557-9
9020895
Chembiochem. 2004 Nov 5;5(11):1596-9
15515081
Adv Microb Physiol. 1995;37:1-81
8540419
Acta Crystallogr D Biol Crystallogr. 2004 Dec;60(Pt 12 Pt 1):2126-32
15572765
FEMS Microbiol Lett. 2006 Aug;261(1):123-32
16842369
J Bacteriol. 2002 Aug;184(15):4124-33
12107129
J Biol Chem. 2001 Sep 7;276(36):33652-6
11445590
J Biol Chem. 2001 Aug 17;276(33):31186-92
11382747
J Biol Chem. 2004 Mar 19;279(12):11777-88
14670951
Acta Crystallogr D Biol Crystallogr. 1997 May 1;53(Pt 3):240-55
15299926
Angew Chem Int Ed Engl. 2002 Aug 2;41(15):2824-7
12203498
Nat Biotechnol. 2008 Feb;26(2):169-72
18259168
Methods Enzymol. 1997;276:307-26
27754618
Biosci Biotechnol Biochem. 2001 Mar;65(3):548-54
11330667
Biochem J. 1999 Apr 1;339 ( Pt 1):43-53
10085226
Annu Rev Microbiol. 1996;50:183-212
8905079
Biochem J. 2003 May 1;371(Pt 3):1027-43
12523937
Proc Natl Acad Sci U S A. 2005 Feb 22;102(8):2697-702
15708971
J Biotechnol. 2003 Feb 27;101(1):37-48
12523968
Science. 2006 Jan 27;311(5760):484-9
16439654
Biochem J. 1991 Dec 1;280 ( Pt 2):309-16
1747104
Biochemistry. 2000 Aug 8;39(31):9099-107
10924103
Biochem J. 2005 Jun 15;388(Pt 3):949-57
15743273
Microbiology (Reading). 1999 Nov;145 ( Pt 11):3101-3108
10589717
J Biol Chem. 1995 Mar 3;270(9):4383-6
7876202
J Bacteriol. 2008 Aug;190(15):5455-63
18556790
Nat Chem Biol. 2008 May;4(5):306-12
18408714
J Biochem. 1991 Apr;109(4):566-9
1869509
J Mol Graph Model. 1997 Apr;15(2):132-4, 112-3
9385560
J Biol Chem. 2005 Sep 23;280(38):32761-7
15987675
Appl Environ Microbiol. 1999 Jun;65(6):2598-605
10347049
Biochemistry. 1999 Jun 29;38(26):8385-92
10387084
Biochim Biophys Acta. 2004 Nov 1;1674(3):239-50
15541293
Biochem J. 1995 Feb 1;305 ( Pt 3):1005-10
7848261
Nat Struct Biol. 1996 Jul;3(7):638-48
8673609
Proc Natl Acad Sci U S A. 1995 Jul 18;92(15):7090-4
7624375