Producing glucose 6-phosphate from cellulosic biomass: structural insights into levoglucosan bioconversion.
The most abundant carbohydrate product of cellulosic biomass pyrolysis is the anhydrosugar levoglucosan (1,6-anhydro-β-d-glucopyranose), which can be converted to glucose 6-phosphate by levoglucosan kinase (LGK). In addition to the canonical kinase phosphotransfer reaction, the conversion requires cleavage of the 1,6-anhydro ring to allow ATP-dependent phosphorylation of the sugar O6 atom. Using x-ray crystallography, we show that LGK binds two magnesium ions in the active site that are additionally coordinated with the nucleotide and water molecules to result in ideal octahedral coordination. To further verify the metal binding sites, we co-crystallized LGK in the presence of manganese instead of magnesium and solved the structure de novo using the anomalous signal from four manganese atoms in the dimeric structure. The first metal is required for catalysis, whereas our work suggests that the second is either required or significantly promotes the catalytic rate. Although the enzyme binds its sugar substrate in a similar orientation to the structurally related 1,6-anhydro-N-acetylmuramic acid kinase (AnmK), it forms markedly fewer bonding interactions with the substrate. In this orientation, the sugar is in an optimal position to couple phosphorylation with ring cleavage. We also observed a second alternate binding orientation for levoglucosan, and in these structures, ADP was found to bind with lower affinity. These combined observations provide an explanation for the high Km of LGK for levoglucosan. Greater knowledge of the factors that contribute to the catalytic efficiency of LGK can be used to improve applications of this enzyme for levoglucosan-derived biofuel production.
From the Bioscience Division, Los Alamos National Laboratory, Los Alamos, New Mexico 87545, jbacik@lanl.gov.
John-Paul
JP
Bacik
the Departments of Biochemistry and Molecular Biology.
Justin R
http://orcid.org/0000-0003-2908-9355
JR
Klesmith
Chemical Engineering and Materials Science, and Biosystems and Agricultural Engineering, Michigan State University, East Lansing, Michigan 48824.
Timothy A
TA
Whitehead
the Department of Chemical and Biological Engineering, Iowa State University, Ames, Iowa 50011, and.
Laura R
LR
Jarboe
From the Bioscience Division, Los Alamos National Laboratory, Los Alamos, New Mexico 87545.
Clifford J
CJ
Unkefer
the Department of Microbiology, University of Manitoba, Winnipeg, Manitoba R3T 2N2, Canada.
Brian L
BL
Mark
From the Bioscience Division, Los Alamos National Laboratory, Los Alamos, New Mexico 87545.
Ryszard
R
Michalczyk
D055162:Biocatalysis; D018533:Biomass; D002482:Cellulose / Q000737:chemistry* / Q000378:metabolism; D018360:Crystallography, X-Ray; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005656:Fungal Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D005947:Glucose / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism; D019298:Glucose-6-Phosphate / Q000737:chemistry* / Q000378:metabolism; D007700:Kinetics; D055160:Lipomyces / Q000737:chemistry* / Q000201:enzymology; D008274:Magnesium / Q000737:chemistry / Q000378:metabolism; D008345:Manganese / Q000737:chemistry / Q000378:metabolism; D008958:Models, Molecular; D010766:Phosphorylation; D010770:Phosphotransferases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding; D011487:Protein Conformation; D055503:Protein Multimerization; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
D005656:Fungal Proteins; D011994:Recombinant Proteins; D008345:Manganese; C014989:1,6-anhydro-beta-glucopyranose; D019298:Glucose-6-Phosphate; D002482:Cellulose; D010770:Phosphotransferases; D008274:Magnesium; D005947:Glucose
anhydrosugar; biofuel; carbohydrate processing; crystal structure; hexokinase; levoglucosan; structural biology