Snapshots during the catalytic cycle of a histidine acid phytase reveal an induced-fit structural mechanism

Highly engineered phytases, which sequentially hydrolyze the hexakisphosphate ester of inositol known as phytic acid, are routinely added to the feeds of monogastric animals to improve phosphate bioavailability. New phytases are sought as starting points to further optimize the rate and extent of dephosphorylation of phytate in the animal digestive tract. Multiple inositol polyphosphate phosphatases (MINPPs) are clade 2 histidine phosphatases (HP2P) able to carry out the stepwise hydrolysis of phytate. MINPPs are not restricted by a strong positional specificity making them attractive targets for development as feed enzymes. Here, we describe the characterization of a MINPP from the Gram-positive bacterium Bifidobacterium longum (BlMINPP). BlMINPP has a typical HP2P-fold but, unusually, possesses a large α-domain polypeptide insertion relative to other MINPPs. This insertion, termed the U-loop, spans the active site and contributes to substrate specificity pockets underpopulated in other HP2Ps. Mutagenesis of U-loop residues reveals its contribution to enzyme kinetics and thermostability. Moreover, four crystal structures of the protein along the catalytic cycle capture, for the first time in an HP2P, a large ligand-driven α-domain motion essential to allow substrate access to the active site. This motion recruits residues both downstream of a molecular hinge and on the U-loop to participate in specificity subsites, and mutagenesis identified a mobile lysine residue as a key determinant of positional specificity of the enzyme. Taken together, these data provide important new insights to the factors determining stability, substrate recognition, and the structural mechanism of hydrolysis in this industrially important group of enzymes.

A thraustochytrid-specific lipase/phospholipase with unique positional specificity contributes to microbial competition and fatty acid acquisition from the environment

Thraustochytrids are heterotrophic marine protists that are considered as important decomposers in the marine ecosystem; however, how they digest and uptake lipid nutrients from the environment is largely unknown. Genomic clustering analysis using thraustochytrid draft genome databases revealed that novel proteins with a Lipase_3 domain are commonly present in thraustochytrids, including Aurantiochytrium limacinum. After heterologous expression and His tag-based purification, protein ID: 145138 was identified as lipase/phospholipase capable of hydrolyzing triacylglycerol (TG) and phosphatidylcholine (PC). 145138 was secreted into the medium, and deletion of the 145138 gene in A. limacinum reduced the degradation of extracellular lipids. Fatty acids generated by 145138 were reused for the biosynthesis of PC and TG, and 145138 allowed A. limacinum to survive in the medium containing TG as a sole carbon source. 145138 hydrolyzed all the acyl-ester linkages of TG; however, the enzyme showed strict positional specificity toward phospholipids, generating 2-acyl lysophospholipids. The 2-acyl lysophospholipids showed stronger antimicrobial activity compared with 1-acyl lysophospholipids. These results suggested that 145138 is a bifunctional enzyme that contributes to the acquisition of lipid nutrients from the environment, as well as to generate antimicrobial lysophospholipids that are beneficial for competition with bacteria over lipid nutrients in the marine environment.

Enzymatic Reetherification in the Production of Butterfat Substitutes

Enzymatic reetherification of fats has numerous technological and economic advantages, which makes its large-scale implementation highly efficient. Unlike chemical modification, enzymatic reetherification demonstrates a greater specificity, typical of the catalytic action of lipase, and a higher controllability. Lipases with positional specificity cause redistribution of fatty acids to occur only in extreme provisions of triglycerides. In addition, this method is 1.5 times lower than hydrogenation of fats. The authors used the facilities of an innovative laboratory provided by JSC Eurasian Foods Corporation to conduct practical research on reetherification of fatty mixes. The main objective was to study the effect of the fats obtained by fermental reetherification on the quality indicators of butterfat substitutes. The research featured the input products to be used in the formula of reetherified fat and prepared fat mixes for butterfat substitutes. The paper describes the process of enzymatic reetherification of mixes of oils and fats, prepared reesterified fats, and buttermilk substitutes obtained from reetherified fats. The process involved a sequence of reactors filled with Lipozyme TL IM, a granulated substance of a microbic 1.3-specific lipase. The lipase was obtained from Thermomyces Lanuginosus, which had been immobilized with silica gel. The obtained products conformed to the butterfat standards in that they contained 16–2% of polynonsaturated fatty acids, no transisomers of fatty acids, ≤ 38% of palmitiny acid, and ≤ 5% of solid triglycerides at 35 of °C. The melting temperature was under body heat. The resulting characteristics of butterfat substitutes make them high-quality dairy products.

Positional specificity of different transcription factor classes within enhancers

Gene expression is controlled by sequence-specific transcription factors (TFs), which bind to regulatory sequences in DNA. TF binding occurs in nucleosome-depleted regions of DNA (NDRs), which generally encompass regions with lengths similar to those protected by nucleosomes. However, less is known about where within these regions specific TFs tend to be found. Here, we characterize the positional bias of inferred binding sites for 103 TFs within ∼500,000 NDRs across 47 cell types. We find that distinct classes of TFs display different binding preferences: Some tend to have binding sites toward the edges, some toward the center, and some at other positions within the NDR. These patterns are highly consistent across cell types, suggesting that they may reflect TF-specific intrinsic structural or functional characteristics. In particular, TF classes with binding sites at NDR edges are enriched for those known to interact with histones and chromatin remodelers, whereas TFs with central enrichment interact with other TFs and cofactors such as p300. Our results suggest distinct regiospecific binding patterns and functions of TF classes within enhancers.

Computational insight into the catalytic implication of head/tail-first orientation of arachidonic acid in human 5-lipoxygenase: consequences for the positional specificity of oxygenation

Using a multi-scale approach to search for the arachidonic acid binding modes that determine the catalytic specificity of human 5-LOX.