5tkr
Crystal structure of a Lipomyces starkeyi levoglucosan kinase G359R mutant
Structural highlights
FunctionLGK_LIPST Levoglucosan kinase that catalyzes the transfer of a phosphate group from ATP to levoglucosan (1,6-anhydro-beta-D-glucopyranose, LG) to yield glucose 6-phosphate in the presence of magnesium ion and ATP (Ref.1, PubMed:21719279, PubMed:26354439). 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 O-6 atom (Ref.1, PubMed:21719279, PubMed:26354439).[1] [2] [3] Publication Abstract from PubMedProteins are marginally stable, and an understanding of the sequence determinants for improved protein solubility is highly desired. For enzymes, it is well known that many mutations that increase protein solubility decrease catalytic activity. These competing effects frustrate efforts to design and engineer stable, active enzymes without laborious high-throughput activity screens. To address the trade-off between enzyme solubility and activity, we performed deep mutational scanning using two different screens/selections that purport to gauge protein solubility for two full-length enzymes. We assayed a TEM-1 beta-lactamase variant and levoglucosan kinase (LGK) using yeast surface display (YSD) screening and a twin-arginine translocation pathway selection. We then compared these scans with published experimental fitness landscapes. Results from the YSD screen could explain 37% of the variance in the fitness landscapes for one enzyme. Five percent to 10% of all single missense mutations improve solubility, matching theoretical predictions of global protein stability. For a given solubility-enhancing mutation, the probability that it would retain wild-type fitness was correlated with evolutionary conservation and distance to active site, and anticorrelated with contact number. Hybrid classification models were developed that could predict solubility-enhancing mutations that maintain wild-type fitness with an accuracy of 90%. The downside of using such classification models is the removal of rare mutations that improve both fitness and solubility. To reveal the biophysical basis of enhanced protein solubility and function, we determined the crystallographic structure of one such LGK mutant. Beyond fundamental insights into trade-offs between stability and activity, these results have potential biotechnological applications. Trade-offs between enzyme fitness and solubility illuminated by deep mutational scanning.,Klesmith JR, Bacik JP, Wrenbeck EE, Michalczyk R, Whitehead TA Proc Natl Acad Sci U S A. 2017 Feb 28;114(9):2265-2270. doi:, 10.1073/pnas.1614437114. Epub 2017 Feb 14. PMID:28196882[4] From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine. References
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