Structure and biochemical characterization of l-2-hydroxyglutarate dehydrogenase and its role in the pathogenesis of l-2-hydroxyglutaric aciduria.

l-2-hydroxyglutarate dehydrogenase (L2HGDH) is a mitochondrial membrane-associated metabolic enzyme, which catalyzes the oxidation of l-2-hydroxyglutarate (l-2-HG) to 2-oxoglutarate (2-OG). Mutations in human L2HGDH lead to abnormal accumulation of l-2-HG, which causes a neurometabolic disorder named l-2-hydroxyglutaric aciduria (l-2-HGA). Here, we report the crystal structures of Drosophila melanogaster L2HGDH (dmL2HGDH) in FAD-bound form and in complex with FAD and 2-OG and show that dmL2HGDH exhibits high activity and substrate specificity for l-2-HG. dmL2HGDH consists of an FAD-binding domain and a substrate-binding domain, and the active site is located at the interface of the two domains with 2-OG binding to the re-face of the isoalloxazine moiety of FAD. Mutagenesis and activity assay confirmed the functional roles of key residues involved in the substrate binding and catalytic reaction and showed that most of the mutations of dmL2HGDH equivalent to l-2-HGA-associated mutations of human L2HGDH led to complete loss of the activity. The structural and biochemical data together reveal the molecular basis for the substrate specificity and catalytic mechanism of L2HGDH and provide insights into the functional roles of human L2HGDH mutations in the pathogeneses of l-2-HGA.

The energetic and allosteric landscape for KRAS inhibition.

Thousands of proteins have been validated genetically as therapeutic targets for human diseases1. However, very few have been successfully targeted, and many are considered 'undruggable'. This is particularly true for proteins that function via protein-protein interactions-direct inhibition of binding interfaces is difficult and requires the identification of allosteric sites. However, most proteins have no known allosteric sites, and a comprehensive allosteric map does not exist for any protein. Here we address this shortcoming by charting multiple global atlases of inhibitory allosteric communication in KRAS. We quantified the effects of more than 26,000 mutations on the folding of KRAS and its binding to six interaction partners. Genetic interactions in double mutants enabled us to perform biophysical measurements at scale, inferring more than 22,000 causal free energy changes. These energy landscapes quantify how mutations tune the binding specificity of a signalling protein and map the inhibitory allosteric sites for an important therapeutic target. Allosteric propagation is particularly effective across the central ß-sheet of KRAS, and multiple surface pockets are genetically validated as allosterically active, including a distal pocket in the C-terminal lobe of the protein. Allosteric mutations typically inhibit binding to all tested effectors, but they can also change the binding specificity, revealing the regulatory, evolutionary and therapeutic potential to tune pathway activation. Using the approach described here, it should be possible to rapidly and comprehensively identify allosteric target sites in many proteins.

Ubiquitin-specific protease 11 structure in complex with an engineered substrate mimetic reveals a molecular feature for deubiquitination selectivity.

Ubiquitin-specific proteases (USPs) are crucial for controlling cellular proteostasis and signaling pathways but how deubiquitination is selective remains poorly understood, in particular between paralogues. Here, we developed a fusion tag method by mining the Protein Data Bank and trapped USP11, a key regulator of DNA double-strand break repair, in complex with a novel engineered substrate mimetic. Together, this enabled structure determination of USP11 as a Michaelis-like complex that revealed key S1 and S1' binding site interactions with a substrate. Combined mutational, enzymatic, and binding experiments identified Met77 in linear diubiquitin as a significant residue that leads to substrate discrimination. We identified an aspartate "gatekeeper" residue in the S1' site of USP11 as a contributing feature for discriminating against linear diubiquitin. When mutated to a glycine, the corresponding residue in paralog USP15, USP11 acquired elevated activity toward linear diubiquitin in-gel shift assays, but not controls. The reverse mutation in USP15 confirmed that this position confers paralog-specific differences impacting diubiquitin cleavage rates. The results advance our understanding of the molecular basis for the higher selectivity of USP11 compared to USP15 and may aid targeted inhibitor development. Moreover, the reported carrier-based crystallization strategy may be applicable to other challenging targets.

Enhancing the Catalytic Activity of Thermo-Asparaginase from Thermococcus sibiricus by a Double Mesophilic-like Mutation in the Substrate-Binding Region.

L-asparaginases (L-ASNases) of microbial origin are the mainstay of blood cancer treatment. Numerous attempts have been performed for genetic improvement of the main properties of these enzymes. The substrate-binding Ser residue is highly conserved in L-ASNases regardless of their origin or type. However, the residues adjacent to the substrate-binding Ser differ between mesophilic and thermophilic L-ASNases. Based on our suggestion that the triad, including substrate-binding Ser, either GSQ for meso-ASNase or DST for thermo-ASNase, is tuned for efficient substrate binding, we constructed a double mutant of thermophilic L-ASNase from Thermococcus sibiricus (TsA) with a mesophilic-like GSQ combination. In this study, the conjoint substitution of two residues adjacent to the substrate-binding Ser55 resulted in a significant increase in the activity of the double mutant, reaching 240% of the wild-type enzyme activity at the optimum temperature of 90 °C. The mesophilic-like GSQ combination in the rigid structure of the thermophilic L-ASNase appears to be more efficient in balancing substrate binding and conformational flexibility of the enzyme. Along with increased activity, the TsA D54G/T56Q double mutant exhibited enhanced cytotoxic activity against cancer cell lines with IC90 values from 2.8- to 7.4-fold lower than that of the wild-type enzyme.

Structural basis of odorant recognition by a human odorant receptor.

Our sense of smell enables us to navigate a vast space of chemically diverse odour molecules. This task is accomplished by the combinatorial activation of approximately 400 odorant G protein-coupled receptors encoded in the human genome1-3. How odorants are recognized by odorant receptors remains unclear. Here we provide mechanistic insight into how an odorant binds to a human odorant receptor. Using cryo-electron microscopy, we determined the structure of the active human odorant receptor OR51E2 bound to the fatty acid propionate. Propionate is bound within an occluded pocket in OR51E2 and makes specific contacts critical to receptor activation. Mutation of the odorant-binding pocket in OR51E2 alters the recognition spectrum for fatty acids of varying chain length, suggesting that odorant selectivity is controlled by tight packing interactions between an odorant and an odorant receptor. Molecular dynamics simulations demonstrate that propionate-induced conformational changes in extracellular loop 3 activate OR51E2. Together, our studies provide a high-resolution view of chemical recognition of an odorant by a vertebrate odorant receptor, providing insight into how this large family of G protein-coupled receptors enables our olfactory sense.

Structural basis for replicase polyprotein cleavage and substrate specificity of main protease from SARS-CoV-2.

The main protease (Mpro) of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a key enzyme, which extensively digests CoV replicase polyproteins essential for viral replication and transcription, making it an attractive target for antiviral drug development. However, the molecular mechanism of how Mpro of SARS-CoV-2 digests replicase polyproteins, releasing the nonstructural proteins (nsps), and its substrate specificity remain largely unknown. Here, we determine the high-resolution structures of SARS-CoV-2 Mpro in its resting state, precleavage state, and postcleavage state, constituting a full cycle of substrate cleavage. The structures show the delicate conformational changes that occur during polyprotein processing. Further, we solve the structures of the SARS-CoV-2 Mpro mutant (H41A) in complex with six native cleavage substrates from replicase polyproteins, and demonstrate that SARS-CoV-2 Mpro can recognize sequences as long as 10 residues but only have special selectivity for four subsites. These structural data provide a basis to develop potent new inhibitors against SARS-CoV-2.

Identification of an acetyl esterase in the supernatant of the environmental strain Bacillus sp. HR21-6.

Bacillus sp. HR21-6 is capable of the chemo- and regioselective synthesis of lipophilic partially acetylated phenolic compounds derived from olive polyphenols, which are powerful antioxidants important in the formulation of functional foods. In this work, an acetyl esterase was identified in the secretome of this strain by non-targeted proteomics, and classified in the GDSL family (superfamily SGNH). The recombinant protein was expressed and purified from Escherichia coli in the soluble form, and biochemically characterized. Site-directed mutagenesis was performed to understand the role of different amino acids that are conserved among GDSL superfamily of esterases. Mutation of Ser-10, Gly-45 or His-185 abolished the enzyme activity, while mutation of Asn-77 or Thr-184 altered the substrate specificity of the enzyme. This new enzyme is able to perform chemoselective conversions of olive phenolic compounds with great interest in the food industry, such as hydroxytyrosol, 3,4-dihydroxyphenylglycol, and oleuropein.

A distinct RNA recognition mechanism governs Np4 decapping by RppH.

Dinucleoside tetraphosphates, often described as alarmones because their cellular concentration increases in response to stress, have recently been shown to function in bacteria as precursors to nucleoside tetraphosphate (Np4) RNA caps. Removal of this cap is critical for initiating 5' end-dependent degradation of those RNAs, potentially affecting bacterial adaptability to stress; however, the predominant Np4 decapping enzyme in proteobacteria, ApaH, is inactivated by the very conditions of disulfide stress that enable Np4-capped RNAs to accumulate to high levels. Here, we show that, in Escherichia coli cells experiencing such stress, the RNA pyrophosphohydrolase RppH assumes a leading role in decapping those transcripts, preferring them as substrates over their triphosphorylated and diphosphorylated counterparts. Unexpectedly, this enzyme recognizes Np4-capped 5' ends by a mechanism distinct from the one it uses to recognize other 5' termini, resulting in a one-nucleotide shift in substrate specificity. The unique manner in which capped substrates of this kind bind to the active site of RppH positions the δ-phosphate, rather than the ß-phosphate, for hydrolytic attack, generating triphosphorylated RNA as the primary product of decapping. Consequently, a second RppH-catalyzed deprotection step is required to produce the monophosphorylated 5' terminus needed to stimulate rapid RNA decay. The unconventional manner in which RppH recognizes Np4-capped 5' ends and its differential impact on the rates at which such termini are deprotected as a prelude to RNA degradation could have major consequences for reprogramming gene expression during disulfide stress.

Accessible, fast and easy fabrication of hydrophilic-in-hydrophobic microdroplet arrays.

Microdroplet arrays (MDAs) are powerful tools for digital immunoassays, high-throughput screening and single cell analysis. However, MDAs are usually produced with cleanroom processes, which are associated with high costs and low availability. Furthermore, in order to obtain robust and stable MDAs based on hydrophilic spots surrounded by a hydrophobic background, the chemistry must be strictly controlled, which is challenging using shared equipment. Here, we developed a new method to fabricate MDA substrates independently from the cleanroom. A small and low-cost in-house built system to collimate the light source was assembled for photopatterning a negative resist, and spots with diameters down to 4 µm were obtained, with only 3% to 5% spot-to-spot variation across the same sample and high batch-to-batch reproducibility. The use of a negative photoresist enabled the formation of a hydrophobic coating in solution which yielded high-quality MDAs. The feasibility for carrying out digital assays was demonstrated by measuring anti-Tau antibody in sample buffers containing bovine serum albumin, with no noticeable surface fouling. The reported, robust, cost-effective, and fast process could hence lower the threshold to fabricate and use MDAs for digital immunoassays and other microcompartmentalization-based applications.

UbiBrowser 2.0: a comprehensive resource for proteome-wide known and predicted ubiquitin ligase/deubiquitinase-substrate interactions in eukaryotic species.

As an important post-translational modification, ubiquitination mediates ∼80% of protein degradation in eukaryotes. The degree of protein ubiquitination is tightly determined by the delicate balance between specific ubiquitin ligase (E3)-mediated ubiquitination and deubiquitinase-mediated deubiquitination. In 2017, we developed UbiBrowser 1.0, which is an integrated database for predicted human proteome-wide E3-substrate interactions. Here, to meet the urgent requirement of proteome-wide E3/deubiquitinase-substrate interactions (ESIs/DSIs) in multiple organisms, we updated UbiBrowser to version 2.0 (http://ubibrowser.ncpsb.org.cn). Using an improved protocol, we collected 4068/967 known ESIs/DSIs by manual curation, and we predicted about 2.2 million highly confident ESIs/DSIs in 39 organisms, with >210-fold increase in total data volume. In addition, we made several new features in the updated version: (i) it allows exploring proteins' upstream E3 ligases and deubiquitinases simultaneously; (ii) it has significantly increased species coverage; (iii) it presents a uniform confidence scoring system to rank predicted ESIs/DSIs. To facilitate the usage of UbiBrowser 2.0, we also redesigned the web interface for exploring these known and predicted ESIs/DSIs, and added functions of 'Browse', 'Download' and 'Application Programming Interface'. We believe that UbiBrowser 2.0, as a discovery tool, will contribute to the study of protein ubiquitination and the development of drug targets for complex diseases.

The catalytic core of Leishmania donovani RECQ helicase unwinds a wide spectrum of DNA substrates and is stimulated by replication protein A.

RecQ helicases are superfamily 2 (SF2) DNA helicases that unwind a wide spectrum of complex DNA structures in a 3' to 5' direction and are involved in maintaining genome stability. RecQ helicases from protozoan parasites have gained significant interest in recent times because of their involvement in cellular DNA repair pathways, making them important targets for drug development. In this study, we report biophysical and biochemical characterization of the catalytic core of a RecQ helicase from hemoflagellate protozoan parasite Leishmania donovani. Among the two putative RecQ helicases identified in L. donovani, we cloned, overexpressed and purified the catalytic core of LdRECQb. The catalytic core was found to be very efficient in unwinding a wide variety of DNA substrates like forked duplex, 3' tailed duplex and Holliday junction DNA. Interestingly, the helicase core also unwound blunt duplex with slightly less efficiency. The enzyme exhibited high level of DNA-stimulated ATPase activity with preferential stimulation by forked duplex, Holliday junction and 3' tailed duplex. Walker A motif lysine mutation severely affected the ATPase activity and significantly affected unwinding activity. Like many other RecQ helicases, L. donovani RECQb also possesses strand annealing activity. Unwinding of longer DNA substrates by LdRECQb catalytic core was found to be stimulated in the presence of replication protein A (LdRPA-1) from L. donovani. Detailed biochemical characterization and comparison of kinetic parameters indicate that L. donovani RECQb shares considerable functional similarity with human Bloom syndrome helicase.

Bacterial flavoprotein monooxygenase YxeK salvages toxic S-(2-succino)-adducts via oxygenolytic C-S bond cleavage.

Thiol-containing nucleophiles such as cysteine react spontaneously with the citric acid cycle intermediate fumarate to form S-(2-succino)-adducts. In Bacillus subtilis, a salvaging pathway encoded by the yxe operon has recently been identified for the detoxification and exploitation of these compounds as sulfur sources. This route involves acetylation of S-(2-succino)cysteine to N-acetyl-2-succinocysteine, which is presumably converted to oxaloacetate and N-acetylcysteine, before a final deacetylation step affords cysteine. The critical oxidative cleavage of the C-S bond of N-acetyl-S-(2-succino)cysteine was proposed to depend on the predicted flavoprotein monooxygenase YxeK. Here, we characterize YxeK and verify its role in S-(2-succino)-adduct detoxification and sulfur metabolism. Detailed biochemical and mechanistic investigation of YxeK including 18 O-isotope-labeling experiments, homology modeling, substrate specificity tests, site-directed mutagenesis, and (pre-)steady-state kinetics provides insight into the enzyme's mechanism of action, which may involve a noncanonical flavin-N5-peroxide species for C-S bond oxygenolysis.

Each phospholipase A2 type exhibits distinct selectivity toward sn-1 ester, alkyl ether, and vinyl ether phospholipids.

Glycerophospholipids are major components of cell membranes and have enormous variation in the composition of fatty acyl chains esterified on the sn-1 and sn-2 position as well as the polar head groups on the sn-3 position of the glycerol backbone. Phospholipase A2 (PLA2) enzymes constitute a superfamily of enzymes which play a critical role in metabolism and signal transduction by hydrolyzing the sn-2 acyl chains of glycerophospholipids. In human cell membranes, in addition to the conventional diester phospholipids, a significant amount is the sn-1 ether-linked phospholipids which play a critical role in numerous biological activities. However, precisely how PLA2s distinguish the sn-1 acyl chain linkage is not understood. In the present study, we expanded the technique of lipidomics to determine the unique in vitro specificity of three major human PLA2s, including Group IVA cytosolic cPLA2, Group VIA calcium-independent iPLA2, and Group V secreted sPLA2 toward the linkage at the sn-1 position. Interestingly, cPLA2 prefers sn-1 vinyl ether phospholipids known as plasmalogens over conventional ester phospholipids and the sn-1 alkyl ether phospholipids. iPLA2 showed similar activity toward vinyl ether and ester phospholipids at the sn-1 position. Surprisingly, sPLA2 preferred ester phospholipids over alkyl and vinyl ether phospholipids. By taking advantage of molecular dynamics simulations, we found that Trp30 in the sPLA2 active site dominates its specificity for diester phospholipids.

Structure, Function, and Thermodynamics of Lactate Dehydrogenases from Humans and the Malaria Parasite P. falciparum.

Temperature adaptation is ubiquitous among all living organisms, yet the molecular basis for this process remains poorly understood. It can be assumed that for parasite-host systems, the same enzymes found in both organisms respond to the same selection factor (human body temperature) with similar structural changes. Herein, we report the existence of a reversible temperature-dependent structural transition for the glycolytic enzyme lactate dehydrogenase (LDH) from the malaria parasite Plasmodium falciparum (pfLDH) and human heart (hhLDH) occurring in the temperature range of human fever. This transition is observed for LDHs from psychrophiles, mesophiles, and moderate thermophiles in their operating temperature range. Thermodynamic analysis reveals unique thermodynamic signatures of the LDH-substrate complexes defining a specific temperature range to which human LDH is adapted and parasite LDH is not, despite their common mesophilic nature. The results of spectroscopic analysis combined with the available crystallographic data reveal the existence of an active center within pfLDH that imparts psychrophilic structural properties to the enzyme. This center consists of two pockets, one formed by the five amino acids (5AA insert) within the substrate specificity loop and the other by the active site, that mutually regulate one another in response to temperature and induce structural and functional changes in the Michaelis complex. Our findings pave the way toward a new strategy for malaria treatments and drug design using therapeutic agents that inactivate malarial LDH selectively at a specific temperature range of the cyclic malaria paroxysm.

Cloning, biochemical characterization and molecular docking of novel thermostable ß-glucosidase BglA9 from Anoxybacillus ayderensis A9 and its application in de-glycosylation of Polydatin.

This study reports a novel BglA9 gene of 1345 bp encoding ß-glucosidase from Anoxybacillus ayderensis A9, which was amplified and expressed in E. coli BL21 (DE3): pLysS cells, purified with Ni-NTA column having molecular weight of 52.6 kDa and was used in the bioconversion of polydatin to resveratrol. The kinetic parameters values using pNPG as substrate were Km (0.28 mM), Vmax (43.8 µmol/min/mg), kcat (38.43 s-1) and kcat/Km (135.5 s-1 mM-1). The BglA9 was active in a broad pH range and had an activity half-life around 24 h at 50 °C. The de-glycosylation efficiency of BglA9 for polydatin was determined by estimating the amount of glucose released after enzymatic reaction by a dinitrosalicylic acid (DNS) assay. The kinetic parameters of BglA9 for polydatin were 5.5 mM, 20.84 µmol/min/mg, 18.28 s-1and 3.27 s-1 mM-1 for Km, Vmax, kcat, and kcat/Km values, respectively. The Ki value for glucose was determined to be 1.7 M. The residues Gln19, His120, Glu355, Glu409, Glu178, Asn222 may play a crucial role in the deglycosylation as revealed by the 3D structure of enzyme docked with polydatin.

The Crystal Structure of Cysteamine Dioxygenase Reveals the Origin of the Large Substrate Scope of This Vital Mammalian Enzyme.

We report the crystal structure of the mammalian non-heme iron enzyme cysteamine dioxygenase (ADO) at 1.9 Šresolution, which shows an Fe and three-histidine (3-His) active site situated at the end of a wide substrate access channel. The open approach to the active site is consistent with the recent discovery that ADO catalyzes not only the conversion of cysteamine to hypotaurine but also the oxidation of N-terminal cysteine (Nt-Cys) peptides to their corresponding sulfinic acids as part of the eukaryotic N-degron pathway. Whole-protein models of ADO in complex with either cysteamine or an Nt-Cys peptide, generated using molecular dynamics and quantum mechanics/molecular mechanics calculations, suggest occlusion of access to the active site by peptide substrate binding. This finding highlights the importance of a small tunnel that leads from the opposite face of the enzyme into the active site, providing a path through which co-substrate O2 could access the Fe center. Intriguingly, the entrance to this tunnel is guarded by two Cys residues that may form a disulfide bond to regulate O2 delivery in response to changes in the intracellular redox potential. Notably, the Cys and tyrosine residues shown to be capable of forming a cross-link in human ADO reside ∼7 Šfrom the iron center. As such, cross-link formation may not be structurally or functionally significant in ADO.

Identification of New Specificity Determinants in Bacterial Purine Nucleobase Transporters based on an Ancestral Sequence Reconstruction Approach.

The relation of sequence with specificity in membrane transporters is challenging to explore. Most relevant studies until now rely on comparisons of present-day homologs. In this work, we study a set of closely related transporters by employing an evolutionary, ancestral-reconstruction approach and reveal unexpected new specificity determinants. We analyze a monophyletic group represented by the xanthine-specific XanQ of Escherichia coli in the Nucleobase-Ascorbate Transporter/Nucleobase-Cation Symporter-2 (NAT/NCS2) family. We reconstructed AncXanQ, the putative common ancestor of this clade, expressed it in E. coli K-12, and found that, in contrast to XanQ, it encodes a high-affinity permease for both xanthine and guanine, which also recognizes adenine, hypoxanthine, and a range of analogs. AncXanQ conserves all binding-site residues of XanQ and differs substantially in only five intramembrane residues outside the binding site. We subjected both homologs to rationally designed mutagenesis and present evidence that these five residues are linked with the specificity change. In particular, we reveal Ser377 of XanQ (Gly in AncXanQ) as a major determinant. Replacement of this Ser with Gly enlarges the specificity of XanQ towards an AncXanQ-phenotype. The ortholog from Neisseria meningitidis retaining Gly at this position is also a xanthine/guanine transporter with extended substrate profile like AncXanQ. Molecular Dynamics shows that the S377G replacement tilts transmembrane helix 12 resulting in rearrangement of Phe376 relative to Phe94 in the XanQ binding pocket. This effect may rationalize the enlarged specificity. On the other hand, the specificity effect of S377G can be masked by G27S or other mutations through epistatic interactions.

A novel thermophilic ß-carotene 15,15'-monooxygenase with broad substrate specificity from the marine bacterium Candidatus Pelagibacter sp. HTCC7211.

To characterize a novel thermophilic ß-carotene 15,15'-monooxygenase BCMO7211 isolated from the marine bacterium Candidatus Pelagibacter sp. HTCC7211. BCMO7211 was functionally overexpressed in Escherichia coli and purified to homogeneity by Ni-NTA affinity chromatography and Superdex-200 gel filtration chromatography. Labeling experiments with H218O demonstrated that the oxygen atom in the terminal aldehyde group of the produced retinal molecules was provided from both molecular oxygen and water, indicating that BCMO7211 is the first characterized bacterial ß-carotene 15,15'-monooxygenase. BCMO7211 exhibited broad carotenoid substrate specificity toward α-carotene, ß-cryptoxanthin, ß-carotene, zeaxanthin, and lutein. The optimum temperature, pH, and concentrations of the substrate and enzyme for retinal production were 60 °C, 9.0, 500 mg ß-carotene/L, and 2.5 U/ml, respectively. Under optimum conditions, 888.3 mg/L retinal was produced in 60 min with a conversion rate of 89.0% (w/w). BCMO7211 is a potential candidate for the enzymatic synthesis of retinal in biotechnological applications.

Substrate Specificity of Human Long-Chain Acyl-CoA Synthetase ACSL6 Variants.

Long-chain acyl-CoA synthetases (ACSLs) are a family of enzymes that convert long-chain free fatty acids into their active form, acyl-CoAs. Recent knock-out mouse studies revealed that among ACSL isoenzymes, ACSL6 plays an important role in the maintenance of docosahexaenoic acid (DHA)-containing glycerophospholipids. Several transcript variants of the human ACSL6 gene have been found; the two major ACSL6 variants, ACSL6V1 and V2, encode slightly different short motifs that both contain a conserved structural domain, the fatty acid Gate domain. In the present study, we expressed recombinant human ACSL6V1 and V2 in Spodoptera frugiperda 9 (Sf9) cells using the baculovirus expression system, and then, using our novel ACSL assay system with liquid chromatography-tandem mass spectrometry (LC-MS/MS), we examined the substrate specificities of the recombinant human ACSL6V1 and V2 proteins. The results showed that both ACSL6V1 and V2 could convert various kinds of long-chain fatty acids into their acyl-CoAs. Oleic acid was a good common substrate and eicosapolyenoic acids were poor common substrates for both variants. However, ACSL6V1 and V2 differed considerably in their preferences for octadecapolyenoic acids, such as linoleic acid, and docosapolyenoic acids, such as DHA and docosapentaenoic acid (DPA): ACSL6V1 preferred octadecapolyenoic acids, whereas V2 strongly preferred docosapolyenoic acids. Moreover, our kinetic studies revealed that ACSL6V2 had a much higher affinity for DHA than ACSL6V1. Our results suggested that ACSL6V1 and V2 might exert different physiological functions and indicated that ACSL6V2 might be critical for the maintenance of membrane phospholipids bearing docosapolyenoic acids such as DHA.

Enzyme modification using mutation site prediction method for enhancing the regioselectivity of substrate reaction sites.

Enzymes with low regioselectivity of substrate reaction sites may produce multiple products from a single substrate. When a target product is produced industrially using these enzymes, the production of non-target products (byproducts) causes adverse effects such as increased processing costs for purification and the amount of raw material. Thus it is required the development of modified enzymes to reduce the amount of byproducts' production. In this paper, we report a method called mutation site prediction for enhancing the regioselectivity of substrate reaction sites (MSPER). MSPER takes conformational data for docking poses of an enzyme and a substrate as input and automatically generates a ranked list of mutation sites to destabilize docking poses for byproducts while maintaining those for target products in silico. We applied MSPER to the enzyme cytochrome P450 CYP102A1 (BM3) and the two substrates to enhance the regioselectivity for four target products with different reaction sites. The 13 of the total 14 top-ranked mutation sites predicted by MSPER for the four target products succeeded in selectively enhancing the regioselectivity up to 6.4-fold. The results indicate that MSPER can distinguish differences of substrate structures and the reaction sites, and can accurately predict mutation sites to enhance regioselectivity without selection by directed evolution screening.