The combination of molecular modeling and X-ray crystallography has failed to yield a consensus model of the mechanism for selective binding of inhibitors to the phosphoinositide 3-kinase (PI3K) p110 ␣-isoform. Here we have used kinetic analysis to determine that the p110␣-selective inhibitor 2-methyl-5-nitro-2-[(6-bromoimidazo[1,2-␣]pyridin-3-yl)methylene]-1-methylhydrazide-benzenesulfonic acid (PIK-75) is a competitive inhibitor with respect to a substrate, phosphatidylinositol (PI) in contrast to most other PI3K inhibitors, which bind at or near the ATP site. Using sequence analysis and the existing crystal structures of inhibitor complexes with the p110␥ and -␦ isoforms, we have identified a new region of nonconserved amino acids (region 2) that was postulated to be involved in PIK-75 p110␣ selectivity. Analysis of region 2, using in vitro mutation of identified nonconserved amino acids to alanine, showed that Ser773 was a critical amino acid involved in PIK-75 binding, with an 8-fold-increase in the IC 50 compared with wild-type. Kinetic analysis showed that, with respect to PI, the PIK-75 K i for the isoform mutant S773D increased 64-fold compared with wild-type enzyme. In addition, a nonconserved amino acid, His855, from the previously identified region 1 of nonconserved amino acids, was found to be involved in PIK-75 binding. These results show that these two regions of nonconserved amino acids that are close to the substrate binding site could be targeted to produce p110␣ isoformselective inhibitors.
The binding mechanism of a new class of lipid-competitive, ATP non-competitive, p110α isoform-selective PI3K (phosphoinositide 3-kinase) inhibitors has been elucidated. Using the novel technique of isoform reciprocal mutagenesis of non-conserved amino acids in the p110α and p110β isoforms, we have identified three unique binding mechanisms for the p110α-selective inhibitors PIK-75, A-66S and J-32. Each of the inhibitor’s p110α-isoform-selective binding was found to be due to interactions with different amino acids within p110. The PIK-75 interaction bound the non-conserved region 2 amino acid p110α Ser773, A-66S bound the region 1 non-conserved amino acid p110α Gln859, and J-32 binding had an indirect interaction with Lys776 and Ile771. The isoform reciprocal mutagenesis technique is shown to be an important analytical tool for the rational design of isoform-selective inhibitors.
Figure S1. The superposition of predicted docking poses of LipKV1 onto Mut-LipKV1 at (A) pH 8.0 (B) pH 9.0 (C) pH 10.0 (D) pH 11.0 (E) pH 12.0 and, (F) all Mut-LipKV1 proteins at different pH when in complex with tributyrin. Docked poses of tributyrin are shown in stick (by atom type) and colored in turqoise to present the poses for LipKV1 and green for Mut-LipKV1. Amino acid residues are shown in stick and colored according to their inspected pHs. All structures are generated using PyMOL.
Starch and pullulan degrading enzymes are essential industrial biocatalysts. Pullulan-degrading enzymes are grouped into pullulanases (types I and type II) and pullulan hydrolase (types I, II and III). Generally, these enzymes hydrolyse the α-1,6 glucosidic bonds (and α-1,4 for certain enzyme groups) of substrates and form reducing sugars such as glucose, maltose, maltotriose, panose or isopanose. This review covers two main aspects: (i) bibliometric analysis of publications and patents related to pullulan-degrading enzymes and (ii) biological aspects of free and immobilised pullulan-degrading enzymes and protein engineering. The collective data suggest that most publications involved researchers within the same institution or country in the past and current practice. Multi-national interaction shall be improved, especially in tapping the enzymes from unculturable prokaryotes. While the understanding of pullulanases may reach a certain extend of saturation, the discovery of pullulan hydrolases is still limited. In this report, we suggest readers consider using the next-generation sequencing technique to fill the gaps of finding more new sequences encoding pullulan-degrading enzymes to expand the knowledge body of this topic.
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