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Structure-based catalytic mechanisms of galacto-N-biose/lacto-N-biose I phosphorylase and N-acetylhexosamine 1-kinase in the LNB/GNB pathway of Bifidobacterium longum
Date Issued
2014
Date
2014
Author(s)
Wang, Kuei-Chen
Abstract
Bifidobacteria, gram-positive anaerobic bacteria, feature a unique lacto-N-biose I (LNB)/galacto-N-biose (GNB) pathway to utilize LNB and GNB for colonization in the gastrointestinal tract of humans. The LNB/GNB pathway is encoded in a four-gene lnpABCD operon. Galacto-N-biose/lacto-N-biose I phosphorylase (GLNBP) encoded by the gene lnpA catalyzes phosphorolysis of LNB and GNB into galactose-1-phosphate (Gal-1P) and N-acetyl-D-hexoamine (GlcNAc/GalNAc) without ATP consumption. This maximizes the effectiveness of ATP production under anaerobic conditions. A novel N-acetylhexosamine 1-kinase (NahK) encoded by the gene lnpB then phosphorylates N-acetyl-D-hexoamine to N-acetylhexoamine 1-phosphate (GlcNAc-1P/GalNAc-1P). NahK has been extensively used in chemoenzymatic synthesis of carbohydrates. However, the molecular mechanisms for both GLNBP and NahK remain unclear. In this study, we wanted to determine the crystal structures of GLNBP and NahK in complex with substrates/products to gain insight into the catalytic mechanisms of these two enzymes. For GLNBP, crystal structures of GLNBP in complex with LNB/GNB were solved. Based on the structural information, GLNBP was proposed to proceed through an inverting phosphorolytic reaction by the direct nucleophilic attack of an inorganic phosphate at the anomeric carbon. For NahK, seven 3-D structures of NahK in complex with GlcNAc, GalNAc, GlcNAc-1P, GlcNAc/AMPPNP and GlcNAc-1P/ADP were solved. Based on these snapshot structures, a direct in-line phosphoryl transfer mechanism was proposed. The NahK structures presented here represent the first multiple-reaction complexes of the enzyme. The demonstration of the protein crystal structures and the elucidation of the catalytic mechanisms would provide useful information for expanding the utilizations of both GLNBP and NahK to a great extent.
Subjects
LNB
GNB
磷酸解酶
激酶
Type
thesis
File(s)
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Name
ntu-103-D97b46016-1.pdf
Size
23.32 KB
Format
Adobe PDF
Checksum
(MD5):1104227fa435dc2fc0279863e3e6e69e