Crystal structure of full-length Mycobacterium tuberculosis H37Rv glycogen branching enzyme: insights of N-terminal beta-sandwich in substrate specificity and enzymatic activity.
The open reading frame Rv1326c of Mycobacterium tuberculosis (Mtb) H37Rv encodes for an alpha-1,4-glucan branching enzyme (MtbGlgB, EC 2.4.1.18, Uniprot entry Q10625). This enzyme belongs to glycoside hydrolase (GH) family 13 and catalyzes the branching of a linear glucose chain during glycogenesis by cleaving a 1-->4 bond and making a new 1-->6 bond. Here, we show the crystal structure of full-length MtbGlgB (MtbGlgBWT) at 2.33-A resolution. MtbGlgBWT contains four domains: N1 beta-sandwich, N2 beta-sandwich, a central (beta/alpha)(8) domain that houses the catalytic site, and a C-terminal beta-sandwich. We have assayed the amylase activity with amylose and starch as substrates and the glycogen branching activity using amylose as a substrate for MtbGlgBWT and the N1 domain-deleted (the first 108 residues deleted) MtbDelta108GlgB protein. The N1 beta-sandwich, which is formed by the first 105 amino acids and superimposes well with the N2 beta-sandwich, is shown to have an influence in substrate binding in the amylase assay. Also, we have checked and shown that several GH13 family inhibitors are ineffective against MtbGlgBWT and MtbDelta108GlgB. We propose a two-step reaction mechanism, for the amylase activity (1-->4 bond breakage) and isomerization (1-->6 bond formation), which occurs in the same catalytic pocket. The structural and functional properties of MtbGlgB and MtbDelta108GlgB are compared with those of the N-terminal 112-amino acid-deleted Escherichia coli GlgB (ECDelta112GlgB).
Pal
Kuntal
K
Department of Biological Sciences, National University of Singapore, Singapore 117543.
Kumar
Shiva
S
Sharma
Shikha
S
Garg
Saurabh Kumar
SK
Alam
Mohammad Suhail
MS
Xu
H Eric
HE
Agrawal
Pushpa
P
Swaminathan
Kunchithapadam
K
D002236:Carbohydrate Conformation; D018360:Crystallography, X-Ray; D017931:DNA Primers; D006003:Glycogen / Q000737:chemistry; D006867:Hydrolases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D007700:Kinetics; D008958:Models, Molecular; D009169:Mycobacterium tuberculosis / Q000201:enzymology*; D011487:Protein Conformation; D017384:Sequence Deletion; D013379:Substrate Specificity
D017931:DNA Primers; D006003:Glycogen; D006867:Hydrolases; C527647:HsaD protein, Mycobacterium tuberculosis
Infect Immun. 2004 Oct;72(10):5676-86
15385466
J Mol Biol. 2008 May 9;378(4):852-70
18387632
Methods Enzymol. 1997;276:307-26
27754618
Cell Microbiol. 2007 Aug;9(8):2081-92
17441985
Acta Crystallogr A. 1991 Mar 1;47 ( Pt 2):110-9
2025413
Biochemistry. 1997 Sep 9;36(36):10837-45
9283074
Arch Biochem Biophys. 2000 Feb 1;374(1):73-8
10640398
Infect Immun. 1997 Oct;65(10):4258-66
9317035
Lab Invest. 1956 Jan-Feb;5(1):11-20
13279125
Microbiology (Reading). 1996 Jun;142 ( Pt 6):1513-1520
8704991
Nat Struct Biol. 1999 May;6(5):458-63
10331874
Acta Crystallogr D Biol Crystallogr. 2000 Dec;56(Pt 12):1622-4
11092928
Mol Microbiol. 2003 Apr;48(1):77-84
12657046
Microbiology (Reading). 1995 Jul;141 ( Pt 7):1609-20
7551029
J Biol Chem. 2003 Sep 12;278(37):35428-34
12819210
J Vet Intern Med. 2001 Nov-Dec;15(6):572-80
11817063
Acta Crystallogr D Biol Crystallogr. 1998 Sep 1;54(Pt 5):905-21
9757107
J Bacteriol. 1959 Feb;77(2):205-11
13630872
Appl Environ Microbiol. 2009 Mar;75(5):1355-62
19139240
Mol Microbiol. 2008 Nov;70(3):762-74
18808383
Nucleic Acids Res. 2009 Jan;37(Database issue):D233-8
18838391
Bioinformatics. 2008 Dec 1;24(23):2780-1
18818215
Int J Lepr. 1961 Jan-Mar;29:74-83
13711245
Adv Microb Physiol. 1998;39:131-203
9328647
Nat Struct Biol. 1999 May;6(5):432-6
10331869
Bioinformatics. 2002 Jan;18(1):213-4
11836238
Methods Enzymol. 1997;277:505-24
18488322
Nature. 1998 Jun 11;393(6685):537-44
9634230
Arch Biochem Biophys. 2003 Oct 1;418(1):34-8
13679080
Protein Expr Purif. 2007 Feb;51(2):198-208
17005418
J Biol Chem. 2002 Nov 1;277(44):42164-70
12196524
J Biol Chem. 2002 Dec 6;277(49):47741-7
12364331
J Biol Chem. 2004 Mar 26;279(13):12369-78
14715664
Cell Mol Life Sci. 2006 Dec;63(23):2710-24
17013558
Biochemistry. 2001 Jul 31;40(30):9032-9
11467966
Proc Natl Acad Sci U S A. 1966 Aug;56(2):725-9
5229990
Chembiochem. 2008 Feb 15;9(3):433-8
18214874
Biochem J. 1994 Jan 15;297 ( Pt 2):351-7
8297342
FEBS Lett. 2006 May 15;580(11):2646-52
16647060