Syn/anti lateral protonation

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• Author: ^^^Wim Nerinckx^^^
• Responsible Curator: ^^^Spencer Williams^^^

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Overview

This page provides a table that summarizes the spatial positioning of the catalytic general acid residue in the active sites of glycoside hydrolases, relative to the substrate. The table below updates those found in the seminal paper on this concept by Heightman and Vasella [1], and a following paper by Nerinckx et al. [2].

Background

The "not from above, but from the side" concept of semi-lateral glycosidic oxygen protonation by glycoside hydrolases was introduced by Heightman and Vasella [1]. It was originally only described for beta-equatorial glycoside hydrolases, but appears to be equally applicable to enzymes acting on an alpha-axial glycosidic bond [2]. When dividing subsite -1 into half-spaces by a plane defined by the glycosidic oxygen and C1' and H1' of the –1 glycoside, many ligand-complexed structures reveal that the proton donor is positioned either in the syn half-space (near the ring-oxygen of the –1 glycoside), or in the anti half-space (on the opposite side of the ring-oxygen). Members of the same GH family appear to share a common syn or anti protonator arrangement and further, this specificity appears to be preserved within Clans of families. This page's compilation of subsite -1 occupied complexes shows that about 70% of all GH families are anti protonators.

Closer inspection of crystal structures of –1/+1 subsite-spanning substrates, or substrate-analogue ligands, in complex with enzymes reveals a further intriguing corollary [2, 3]. In substrate-bound complexes with anti protonating GH enzymes, the scissile anomeric bond (often studied using the thio-analogue) shows a dihedral angle φ (O5'-C1'-[O,S]x-Cx) that is in the lowest-energy synclinal (gauche) conformation. The rationale for this is that a minus synclinal dihedral angle φ for an equatorial glycosidic bond, or plus synclinal for an axial glycosidic bond [4], allows for hyperconjugative overlap of the C1'-O5' antibonding orbital with an antiperiplanar-oriented lone pair orbital lobe of the glycosidic oxygen, thereby creating partial double bond character and stabilization of the glycosidic bond by 4–5 kcal/mol; this ground-state stabilizing phenomenon is known as the ‘exo-anomeric effect’ [5, 6]. Anti protonation occurs on the glycosidic oxygen’s antiperiplanar lone pair, thereby removing the stabilizing exo-anomeric effect. This suggests that anti protonation is an enzymic approach for lowering the activation barrier leading to the transition state (Figure 1 centre).

Syn protonating glycoside hydrolases apparently make use of a different approach [2, 3]. In many –1/+1 subsite-spanning ligand complexes, the dihedral angle φ of the scissile anomeric bond has been rotated away from its lowest-energy synclinal position: clockwise to minus-anticlinal or antiperiplanar for beta-equatorial; counterclockwise to plus-anticlinal or antiperiplanar for alpha-axial anomeric bonds. This removes the hyperconjugative overlap and thus also the stabilizing exo-anomeric effect. And because of this rotation, a lone pair of the glycosidic oxygen is directed into the syn half-space, allowing it to be protonated by the syn-positioned proton donor (Figure 1 right).

Figure 1. Newman projections, with the glycosidic oxygen as proximal atom and the anomeric carbon as distal atom, showing anti (centre) versus syn (right) semi-lateral protonation in beta-equatorial (top) and alpha-axial (bottom) glycoside hydrolases. The indicated φ is the dihedral angle for O5'-C1'-O4-C4.

Table of syn/anti protonation examples

This table contains only one example per GH family of a ligand-complexed protein structure where the syn or anti positioning of the proton donor can be clearly observed; other examples may be available on a family-by-family basis. The reader is thus advised to consult the CAZy database for a current, comprehensive list of CAZyme structures. Where available, the selected examples are Michaelis-type complexes with the ligand spanning the -1/+1 subsites, since these have an intact glycosidic or thioglycosidic bond, or are N-analogs of the substrate (e.g. acarbose). In some examples, the proton donor has been mutated (e.g., to the corresponding amide or to an alanine), and in those cases one may wish to look at a superposition of the given PDB example with the structure of the native enzyme. If a Michaelis-type complex is not yet available, the second and third example choices, respectively, are trapped glycosyl-enzyme intermediates and product complexes where subsite -1 is occupied.

Please also be aware that this is a large table with many data. Please contact the page Author or Responsible Curator with corrections.

Table

This table can be re-sorted by clicking on the icons in the header (javascript must be turned on in your browser). To reset the page to be sorted by GH family, click the page tab at the very top of the page.

Family	Clan	Structure fold	Anomeric specificity	Mechanism	Syn/anti protonator	Example PDB ID	Enzyme	Organism	Ligand	General acid	Nucleophile or General base	Reference
GH1	A	(β/α)8	beta	retaining	anti	2cer	β-glycosidase S	Sulfolobus solfataricus P2	phenethyl glucoimidazole	Glu206	Glu387	[7]
GH2	A	(β/α)8	beta	retaining	anti	2vzu	exo-β-glucosaminidase	Amicolatopsis orientalis	PNP-β-d-glucosamine	Glu469	Glu541	[8]
GH3	none	(β/α)8	beta	retaining	anti	1iex	exo-1,3-1,4-glucanase	Hordeum vulgare	thiocellobiose	Glu491	Asp285	[9]
GH5	A	(β/α)8	beta	retaining	anti	1h2j	endo-β-1,4-glucanase	Bacillus agaradhaerens	2',4'-DNP-2-F-cellobioside	Glu129	Glu228	[10]
GH6	none	(β/α)8	beta	inverting	syn	1qjw	cellobiohydrolase 2	Hypocrea jecorina	(Glc)2-S-(Glc)2	Asp221	debated	[11]
GH7	B	β-jelly roll	beta	retaining	syn	1ovw	endo-1,4-glucanase	Fusarium oxysporum	thio-(Glc)5	Glu202	Glu197	[12]
GH8	M	(α/α)6	beta	inverting	anti	1kwf	endo-1,4-glucanase	Clostridium thermocellum	cellopentaose	Glu95	Asp278	[13]
GH9	none	(α/α)6	beta	inverting	syn	1rq5	cellobiohydrolase	Clostridium thermocellum	cellotetraose	Glu795	Asp383	[14]
GH10	A	(β/α)8	beta	retaining	anti	2d24	β-1,4-xylanase	Streptomyces olivaceoviridis E-86	xylopentaose	Glu128	Glu236	[15]
GH11	C	β-jelly roll	beta	retaining	syn	1bvv	xylanase	Bacillus circulans	Xyl-2-F-xylosyl	Glu172	Glu78	[16]
GH12	C	β-jelly roll	beta	retaining	syn	1w2u	endoglucanase	Humicola grisea	thiocellotetraose	Glu205	Glu120	[17]
GH13	H	(β/α)8	alpha	retaining	anti	1cxk	β-cyclodextrin glucanotransferase	Bacillus circulans	maltononaose	Glu257	Asp229	[18]
GH14	none	(β/α)8	alpha	inverting	syn	1itc	β-amylase	Bacillus cereus	maltopentaose	Glu172	Glu367	[19]
GH15	L	(α/α)6	alpha	inverting	anti	1dog	glucoamylase	Aspergillus awamori	1-deoxynojirimycin	Glu179	Glu400	[20]
GH16	B	β-jelly roll	beta	retaining	syn	1urx	β-agarase A	Zobellia galactanivorans	oligoagarose	Glu152	Glu147	[21]
GH17	A	(β/α)8	beta	retaining	predicted anti by clan	see e.g. at GH1
GH18	K	(β/α)8	beta	retaining	anti	1ffr	chitinase A	Serratia marcescens	(NAG)6	Glu315	internal	[22]
GH19	none	lysozyme type	beta	inverting	syn	3wh1	chitinase	Bryum coronatum	(GlcNAc)4	Glu61	Glu70	[23]
GH20	K	(β/α)8	beta	retaining	anti	1c7s	chitobiase	Serratia marcescens	chitobiose	Glu540	internal	[24]
GH22	none	lysozyme type	beta	retaining	syn	1h6m	lysozyme C	Gallus gallus	Chit-2-F-chitosyl	Glu35	Asp52	[25]
GH23	none	lysozyme type	beta	inverting	syn	1lsp	lysozyme G	Cygnus atratus	Bulgecin A	Glu73	internal	[26]
GH24	I	α + β	beta	inverting	syn	148l	lysozyme E	Bacteriophage T4	chitobiosyl	Glu11	Glu26	[27]
GH26	A	(β/α)8	beta	retaining	anti	1gw1	mannanase A	Cellvibrio japonicus	2',4'-DNP-2-F-cellotrioside	Glu212	Glu320	[28]
GH27	D	(β/α)8	alpha	retaining	anti	3lrm	α-galactosidase	Saccharomyces cerevisiae	raffinose	Asp209	Asp141	[29]
GH28	N	β-helix	alpha	inverting	anti	2uvf	exo-polygalacturonosidase	Yersinia enterocolitica ATCC9610D	digalacturonic acid	Asp402	Asp381 Asp403	[30]
GH29	none	(β/α)8	alpha	retaining	syn	3uet	α-1,3/4-fucosidase	Bifidobacterium longum subsp. infantis	lacto-N-fucopentaose II	Glu217	Asp172	[31]
GH30	A	(β/α)8	beta	retaining	anti	2v3d	glucocerebrosidase 1	Homo sapiens	N-butyl-deoxynojirimycin	Glu235	Glu340	[32]
GH31	D	(β/α)8	alpha	retaining	anti	2qmj	maltase-glucoamylase	Homo sapiens	acarbose	Asp542	Asp443	[33]
GH32	J	5-fold β-propeller	beta	retaining	anti	2add	fructan β-(2,1)-fructosidase	Cichorium intybus	sucrose	Glu201	Asp22	[34]
GH33	E	6-fold β-propeller	alpha	retaining	anti	1s0i	transsialidase	Trypanosoma cruzi	sialyl-lactose	Asp59	Tyr342	[35]
GH34	E	6-fold β-propeller	alpha	retaining	anti	4gzw	N2 neuraminidase	Influenza A Tanzania/205/2010 H3N2	α-d-Neup5Ac-(2,3)-β-d-Galp-(1,4)-β-d-GlcpNAc	Asp151	Tyr406	[36]
GH35	A	(β/α)8	beta	retaining	anti	1xc6	β-galactosidase	Penicillium sp.	d-galactose	Glu200	Glu299	[37]
GH36	D	(β/α)8	alpha	retaining	anti	4fns	β-galactosidase	Geobacillus stearothermophilus	1-deoxy galactonojirimycin	Asp584	Asp478	[38]
GH37	G	(α/α)6	alpha	inverting	anti	2jf4	trehalase	Escherechia coli	validoxylamine	Asp312	Glu496	[39]
GH38	none	(β/α)7	alpha	retaining	anti	3czn	Golgi α-mannosidase II	Drosophila melanogaster	GlcNAcMan(5)GlcNAc(2)	Asp341	Asp204	[40]
GH39	A	(β/α)8	beta	retaining	anti	2bfg	β-xylosidase	Geobacillus stearothermophilus	2,5-dinitrophenyl-β-d-xyloside	Glu160	Glu278	[41]
GH42	A	(β/α)8	beta	retaining	anti	4ucf	β-galactosidase	Bifidobacterium bifidum	d-galactose	Glu161	Glu320	[42]
GH44	none	(β/α)8	beta	retaining	anti	2eqd	endoglucanase	Clostridium thermocellum	cellooctaose	Glu186	Glu359	[43]
GH45	none	6-strand. β-barrel	beta	inverting	syn	4eng	endo-1,4-glucanase	Humicola insolens	cellohexaose	Asp121	Asp10	[44]
GH46	I	lysozyme type	beta	inverting	syn	4olt	chitosanase	Microbacterium sp. OU01	hexa-glucosamine	Glu25	Asp43	[45]
GH47	none	(α/α)7	alpha	inverting	anti	1x9d	α-mannosidase I	Homo sapiens	Me-2-S-(α-Man)-2-thio-α-Man	Asp463	Glu599	[46], [47]
GH48	M	(α/α)6	beta	inverting	predicted anti by clan	see at GH8
GH49	N	β-helix	alpha	inverting	predicted anti by clan	see at GH28
GH50	A	(β/α)8	beta	retaining	anti	4bq5	exo-β-agarase	Saccharophagus degradans	neoagarotetraose	Glu535	Glu695	[48]
GH51	A	(β/α)8	alpha	retaining	anti	1qw9	α-l-arabinofuranosidase	Geobacillus stearothermophilus	PNP-l-arabino-furanoside	Glu175	Glu294	[49]
GH52	O	(α/α)6	beta	retaining	anti	4c1p	β-xylosidase	Geobacillus thermoglucosidasius	xylobiose	Asp517	Glu537	[50]
GH53	A	(β/α)8	beta	retaining	anti	2ccr	β-1,4-galactanase	Bacillus licheniformis	galactotriose	Glu165	Glu263	[51]
GH54	none	β-sandwich	alpha	retaining	anti	1wd4	α-l-arabinofuranosidase B	Aspergillus kawachii	l-arabinofuranose	Asp297	Glu221	[52]
GH55	none	β-helix	beta	inverting	syn	4tz5	exo-β-1,3-glucanase	Streptomyces sp. SirexAA-E	laminarihexaose	Glu502	unknown	[53]
GH56	none	(β/α)7	beta	retaining	anti	1fcv	hyaluronidase	Apis mellifera	(hyaluron.)4	Glu113	internal	[54]
GH57	none	(β/α)7	alpha	retaining	anti	1k1y	glucanotransferase	Thermococcus litoralis	acarbose	Asp214	Glu123	[55]
GH59	A	(β/α)8	beta	retaining	anti	4ccc	β-galactocerebrosidase	Mus musculus	PNP-β-d-galactoside	Glu182	Glu258	[56]
GH63	G	(α/α)6	alpha	inverting	predicted anti by clan	see at GH37
GH65	L	(α/α)6	alpha	inverting	anti	4ktr	2-O-α-glucosylglycerol phosphorylase	Bacillus selenitireducens	isofagomine	Glu475	phosphate	[57]
GH66	L	(β/α)8	alpha	retaining	anti	5axh	dextranase	Thermoanaerobacter pseudethanolicus	isomaltohexaose	Glu374	Asp312	[58]
GH67	none	(β/α)8	alpha	inverting	syn	1gql	α-glucuronidase	Cellvibrio japonicus Ueda107	d-glucuronic acid	Glu292	unknown	[59]
GH68	J	5-fold β-propeller	beta	retaining	anti	1pt2	levansucrase	Bacillus subtilis	sucrose	Glu342	Asp86	[60]
GH70	H	(β/α)8	alpha	retaining	anti	3aic	glucansucrase	Streptococcus mutans	α-acarbose	Glu515	Asp477	[61]
GH72	A	(β/α)8	beta	retaining	anti	2w62	β-1,3-glucanotransferase	Saccharomyces cerevisiae S288C	laminaripentaose	Glu176	Glu275	[62]
GH74	none	7-fold β-propeller	beta	inverting	syn	2ebs	cellobiohydrolase (OXG-RCBH)	Geotrichum sp. m128	xyloglucan heptasaccharide	Asp465	Asp35	[63]
GH76	none	(α/α)6	alpha	retaining	anti	5agd	endo-α-1,6-mannanase	Bacillus circulans	α-1,6-mannopentaose	Asp125	Asp124	[64]
GH77	H	(β/α)8	alpha	retaining	anti	1esw	amylomaltase	Thermus aquaticus	acarbose	Asp395	Asp293	[65]
GH78	H	(α/α)6	alpha	inverting	anti	3w5n	α-l-rhamnosidase	Streptomyces avermitilis	l-rhamnose	Glu636	Glu895	[66]
GH79	A	(β/α)8	beta	retaining	anti	5e9c	heparanase	Homo sapiens	heparin tetrasaccharide	Glu225	Glu343	[67]
GH80	I	α + β	beta	inverting	predicted syn by clan	see at GH24
GH83	E	6-fold β-propeller	alpha	retaining	predicted anti by clan	see e.g. at GH33
GH84	none	(β/α)8	beta	retaining	anti	2chn	β-N-acetyl- glucosaminidase	Bacteroides thetaiota- omicron VPI-5482	NAG-thiazoline	Glu242	internal	[68]
GH85	K	(β/α)8	beta	retaining	anti	2w92	endo-β-N-acetyl- glucosaminidase D	Streptococcus pneumoniae TIGR4	NAG-thiazoline	Glu337	internal	[69]
GH86	A	(β/α)8	beta	retaining	anti	4aw7	β-porphyranase	Bacteroides plebeius	porphyran fragment	Glu152	Glu279	[70]
GH89	none	(β/α)8	alpha	retaining	anti	2vcb	α-N-acetyl- glucosaminidase	Clostridium perfringens	PUGNAc	Glu483	Glu601	[71]
GH92	none	(α/α)6 + β-sandw.	alpha	inverting	anti	2ww1	α-1,2-mannosidase	Bacteroides thetaiota- omicron VPI-5482	thiomannobioside	Glu533	Asp644 Asp642	[72]
GH93	E	6-bladed β-propeller	alpha	retaining	anti	3a72	exo-arabinanase	Penicillium chrysogenum	arabinobiose	Glu246	Glu174	[73]
GH94	none	(α/α)6	beta	inverting	syn	1v7x	chitobiose phosphorylase	Vibrio proteolyticus	GlcNAc	Asp492	phosphate	[74]
GH95	none	(α/α)6	alpha	inverting	anti	2ead	α-1,2-l-fucosidase	Bifidobacterium bifidum	Fuc-α-1,2-Gal	Glu566	Asn423 Asp766	[75]
GH97	none	(β/α)8	alpha	retaining + inverting	anti	2zq0	α-glucosidase	Bacteroides thetaiota- omicron VPI-5482	acarbose	Glu532	Glu508	[76]
GH99	none	(β/α)8	alpha	retaining	anti	4ad4	endo-α-mannosidase	Bacteroides xylanisolvens	glucose-1,3-isofagomine and α-1,2- mannobiose	Glu336	debated	[77]
GH100	none	(α/α)6 core	beta	inverting	anti	5gop	invertase	Anabaena (Nostoc) sp. pcc7120	sucrose	Asp188	Glu414	[78]
GH102	none	double-ψ β-barrel	beta	retaining	syn	2pi8	lytic transglycosylase A	Escherichia coli	chitohexaose	Asp308	none	[79]
GH113	A	(β/α)8	beta	retaining	anti	4cd8	β-mannanase	Alicyclobacillus acidocaldarius	mannobioimidazole	Glu151	Glu231	[80]
GH116	O	β-sandwich + (α/α)6	beta	retaining	predicted anti by clan	see at GH52
GH117	none	five-bladed β-propeller	alpha	inverting	anti	4ak7	α-1,3-3,6-anhydro-l-galactosidase	Bacteroides plebeius	neoagarobiose	His302	Asp90	[81]
GH123	none	(β/α)8 + β-sandwich	beta	retaining	anti	5fr0	exo-β-N-acetylgalactosaminidase	Clostridium perfringens	N-difluoroacetyl-d-galactosamine	Glu345	internal	[82]
GH125	L	(α/α)6	alpha	inverting	anti	5m7y	exo-α-1,6-mannosidase	Clostridium perfringens	1,6-α-mannotriose	Asp220	Glu393	[83]
GH127	none	(α/α)6 + β-sandwich	beta	retaining	anti	3wrg	β-l-arabinofuranosidase	Bifidobacterium longum	l-arabinose	Glu322	Cys417	[84]
GH128	A	(β/α)8	beta	retaining	predicted anti by clan	see e.g. at GH1
GH130	none	five-bladed β-propeller	beta	inverting	anti	5b0s	β-1,2-mannobiose phosphorylase	Listeria innocua	β-1,2-mannotriose	Asp141-relay	phosphate	[85]
GH134	none	β + α	beta	inverting	syn	5jug	β-mannanase	Streptomyces sp.	mannopentaose	Glu45	Asp57	[86]

References

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83. To be published

84. Huang CH, Zhu Z, Cheng YS, Chan HC, Ko TP, Chen CC, Wang I, Ho MR, Hsu ST, Zeng YF, Huang YN, Liu JR, Guo RT. Structure and Catalytic Mechanism of a Glycoside Hydrolase Family-127 β-L-Arabinofuranosidase (HypBA1). J. Bioprocess Biotech. 2014 4:171 [DOI:10.4172/2155-9821.1000171]

85. Tsuda T, Nihira T, Chiku K, Suzuki E, Arakawa T, Nishimoto M, Kitaoka M, Nakai H, and Fushinobu S. (2015). Characterization and crystal structure determination of β-1,2-mannobiose phosphorylase from Listeria innocua. FEBS Lett. 2015;589(24 Pt B):3816-21. DOI:10.1016/j.febslet.2015.11.034 |

86. Jin Y, Petricevic M, John A, Raich L, Jenkins H, Portela De Souza L, Cuskin F, Gilbert HJ, Rovira C, Goddard-Borger ED, Williams SJ, and Davies GJ. A β-Mannanase with a Lysozyme-like Fold and a Novel Molecular Catalytic Mechanism. ACS Cent. Sci. 2016 Nov DOI:10.1021/acscentsci.6b00232