Structural and kinetic analysis of Bacillus subtilis N-acetylglucosaminidase reveals a unique Asp-His dyad mechanism.
Three-dimensional structures of NagZ of Bacillus subtilis, the first structures of a two-domain β-N-acetylglucosaminidase of family 3 of glycosidases, were determined with and without the transition state mimicking inhibitor PUGNAc bound to the active site, at 1.84- and 1.40-Å resolution, respectively. The structures together with kinetic analyses of mutants revealed an Asp-His dyad involved in catalysis: His(234) of BsNagZ acts as general acid/base catalyst and is hydrogen bonded by Asp(232) for proper function. Replacement of both His(234) and Asp(232) with glycine reduced the rate of hydrolysis of the fluorogenic substrate 4'-methylumbelliferyl N-acetyl-β-D-glucosaminide 1900- and 4500-fold, respectively, and rendered activity pH-independent in the alkaline range consistent with a role of these residues in acid/base catalysis. N-Acetylglucosaminyl enzyme intermediate accumulated in the H234G mutant and β-azide product was formed in the presence of sodium azide in both mutants. The Asp-His dyad is conserved within β-N-acetylglucosaminidases but otherwise absent in β-glycosidases of family 3, which instead carry a "classical" glutamate acid/base catalyst. The acid/base glutamate of Hordeum vulgare exoglucanase (Exo1) superimposes with His(234) of the dyad of BsNagZ and, in contrast to the latter, protrudes from a second domain of the enzyme into the active site. This is the first report of an Asp-His catalytic dyad involved in hydrolysis of glycosides resembling in function the Asp-His-Ser triad of serine proteases. Our findings will facilitate the development of mechanism-based inhibitors that selectively target family 3 β-N-acetylglucosaminidases, which are involved in bacterial cell wall turnover, spore germination, and induction of β-lactamase.
Litzinger
Silke
S
Department of Molecular Microbiology, University of Konstanz, 78457 Konstanz, Germany.
Fischer
Stefanie
S
Polzer
Patrick
P
Diederichs
Kay
K
Welte
Wolfram
W
Mayer
Christoph
C
D000117:Acetylglucosamine / Q000737:chemistry / Q000378:metabolism; D000118:Acetylglucosaminidase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D000595:Amino Acid Sequence; D001224:Aspartic Acid / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D001412:Bacillus subtilis / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D001665:Binding Sites / Q000235:genetics; D055162:Biocatalysis / Q000187:drug effects; D020134:Catalytic Domain; D018360:Crystallography, X-Ray; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006639:Histidine / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D006860:Hydrogen Bonding; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D015394:Molecular Structure; D009154:Mutation; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D017386:Sequence Homology, Amino Acid; D019810:Sodium Azide / Q000494:pharmacology; D013379:Substrate Specificity
D001426:Bacterial Proteins; D004791:Enzyme Inhibitors; D001224:Aspartic Acid; D006639:Histidine; D019810:Sodium Azide; D000118:Acetylglucosaminidase; D000117:Acetylglucosamine
Biosci Biotechnol Biochem. 2006 May;70(5):1127-33
16717412
J Bacteriol. 2000 Sep;182(17):4836-40
10940025
Proteins. 2003 Feb 15;50(3):437-50
12557186
Acta Crystallogr D Biol Crystallogr. 2002 Jun;58(Pt 6 Pt 2):968-75
12037299
Curr Opin Struct Biol. 1997 Oct;7(5):637-44
9345621
J Biol Chem. 2000 Feb 18;275(7):4973-80
10671536
FEBS J. 2006 Jul;273(13):2929-41
16762038
Nature. 2001 Aug 23;412(6849):835-8
11518970
Acta Crystallogr D Biol Crystallogr. 2006 Sep;62(Pt 9):1002-11
16929101
Acta Crystallogr D Biol Crystallogr. 2004 Dec;60(Pt 12 Pt 1):2126-32
15572765
FEBS Lett. 1995 Apr 10;362(3):281-5
7729513
Curr Opin Chem Biol. 2000 Oct;4(5):573-80
11006547
Biochemistry. 2002 Feb 26;41(8):2751-9
11851422
Structure. 1999 Feb 15;7(2):179-90
10368285
Cell. 1997 Mar 21;88(6):823-32
9118225
Chem Biol. 2007 Feb;14(2):121-9
17317566
Cell Mol Life Sci. 2008 Mar;65(6):928-39
18049859
Biochemistry. 2007 May 29;46(21):6288-98
17487984
Chem Rev. 2002 Dec;102(12):4501-24
12475199
Biochemistry. 1998 Jun 23;37(25):8886-98
9636030
Biochem J. 2002 Aug 1;365(Pt 3):857-63
11978178
Cell. 2008 Oct 31;135(3):486-96
18984160
J Biol Chem. 2005 Aug 26;280(34):30100-6
15983044
J Am Chem Soc. 2003 Jun 25;125(25):7532-3
12812490
Annu Rev Biophys Bioeng. 1976;5:271-306
7991
Acta Crystallogr D Biol Crystallogr. 1999 Jan;55(Pt 1):191-205
10089410
Angew Chem Int Ed Engl. 2001 May 18;40(10):1808-1827
11385651
Biochemistry. 2000 Jan 11;39(1):117-26
10625486
Biophys J. 1999 Mar;76(3):1571-9
10049337
Science. 1997 Nov 7;278(5340):1128-32
9353195
Biochemistry. 2002 Sep 3;41(35):10834-48
12196023
J Bacteriol. 2008 Oct;190(20):6598-608
18723630
Biochem J. 2004 Feb 15;378(Pt 1):141-9
14561218
J Biol Chem. 2000 Dec 15;275(50):39385-93
10978344
J Bacteriol. 2010 Jun;192(12):3132-43
20400549
J Mol Biol. 2004 Feb 27;336(4):943-55
15095871
Acta Crystallogr D Biol Crystallogr. 2002 Nov;58(Pt 11):1937-40
12393925
J Bacteriol. 2006 Feb;188(4):1660-2
16452451
Structure. 2001 Nov;9(11):1005-16
11709165
J Mol Microbiol Biotechnol. 2003;5(3):190-8
12766348
Nature. 1990 Feb 22;343(6260):767-70
2304552
Acta Crystallogr D Biol Crystallogr. 2002 Nov;58(Pt 11):1948-54
12393927
J Biol Chem. 2007 Jul 20;282(29):21382-91
17439950
Acta Crystallogr D Biol Crystallogr. 2004 Mar;60(Pt 3):432-8
14993666
J Biol Chem. 2006 Feb 17;281(7):4149-55
16298994
Acc Chem Res. 2000 Jan;33(1):11-8
10639071
Biochemistry. 2009 Dec 1;48(47):11220-30
19824695
J Bacteriol. 2004 Apr;186(8):2385-92
15060041
J Biol Chem. 2004 Feb 6;279(6):4970-80
14597633