title: Crystal structure of full-length Mycobacterium tuberculosis H37Rv glycogen branching enzyme: insights of N-terminal beta-sandwich in substrate specificity and enzymatic activity.
abstract: 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).
authors.affiliation: Department of Biological Sciences, National University of Singapore, Singapore 117543.
authors.forename: Kuntal
authors.initials: K
authors.lastname: Pal
authors.forename: Shiva
authors.initials: S
authors.lastname: Kumar
authors.forename: Shikha
authors.initials: S
authors.lastname: Sharma
authors.forename: Saurabh Kumar
authors.initials: SK
authors.lastname: Garg
authors.forename: Mohammad Suhail
authors.initials: MS
authors.lastname: Alam
authors.forename: H Eric
authors.initials: HE
authors.lastname: Xu
authors.forename: Pushpa
authors.initials: P
authors.lastname: Agrawal
authors.forename: Kunchithapadam
authors.initials: K
authors.lastname: Swaminathan
mesh_terms: 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
chemical_list: D017931:DNA Primers; D006003:Glycogen; D006867:Hydrolases; C527647:HsaD protein, Mycobacterium tuberculosis
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