Escherichia coli expression and characterization of α-amylase from Geobacillus thermodenitrificans DSM-465

Alpha amylase, catalyzing the hydrolysis of starch is a ubiquitous enzyme with tremendous industrial applications. A 1698 bp gene coding for 565 amino acid amylase was PCR amplified from Geobacillus thermodenitrificans DSM-465, cloned in pET21a (+) plasmid, expressed in BL21 (DE3) strain of E. coli and characterized. The recombinant enzyme exhibited molecular weight of 63 kDa, optimum pH 8, optimum temperature 70°C, and KM value of 157.7µM. On pilot scale, the purified enzyme efficiently removed up to 95% starch from the cotton fabric indicating its desizing ability at high temperature. 3D model of enzyme built by Raptor-X and validated by Ramachandran plot appeared as a monomer having 31% α-helices, 15% β-sheets, and 52% loops. Docking studies have shown the best binding affinity of enzyme with amylopectin (∆G -10.59). According to our results, Asp 232, Glu274, Arg448, Glu385, Asp34, Asn276, and Arg175 constitute the potential active site of enzyme.

STUDIES OF RADIO REFRACTIVE INDEX IN ASSOCIATION WITH ONSET OF MONSOON

Variations of radio refractive index (RRI) in the lower troposphere over Bombay and Thiru-vananthapuram have been studied for the months from April to July for the years 1979 to 1987. It is seen that in most of the years, the radio refractive index at 900 hPa over Bombay increased significantly prior to onset of monsoon over Kerala (Thiruvananthapuram) and well in advance of the arrival monsoon over Bom-bay. Variation of RRI at 850 hPa level over Bombay also shows significant increase a few days later than at 900 liPa level. At higher levels no such significant changes are observed. The KM value over Thinivanantha-purain at 900 hPa or at 850 hPa does not show significant rise from pre-monsoon to monsoon months. The results show a slight decreasing trend during the same period.

NUDT18 catalyzes hydrolysis of active metabolites of the antivirals Remdesivir and Ribavirin

Remdesivir (GS-5734) has gained considerable interest due to its activity against replication of SARS-CoV2. Remdesivir is a broad-spectrum antiviral prodrug that is hydrolyzed intracellularly and phosphorylated by cellular kinases to its active triphosphate form (Remdesivir-TP). Here we tested Remdesivir-TP as a substrate for a panel of human hydrolases and found that NUDIX hydrolase 18 (NUDT18) catalyzes the hydrolysis of Remdesivir-TP. NUDT18 is expressed in respiratory epithelial cells suggesting that NUDT18 may limit the antiviral efficacy of Remdesivir by decreasing the intracellular concentration of its active metabolite at its intended site of action. The kcat of NUDT18 for Remdesivir-TP was determined to 2.6 s-1 and the Km value was 156 μM, suggesting that NUDT18 catalyzed hydrolysis occurs in cells. In addition, we found that the triphosphate of the antiviral Ribavirin, with broad-spectrum activity against several RNA and DNA viruses, was hydrolyzed by NUDT18, albeit with a lower efficiency compared to Remdesivir-TP. NUDT18 activity was also tested with the triphosphates of the antivirals Sofosbuvir and Aciclovir for which low activity, in comparison to activities with Remdesivir-TP and Ribavirin-TP, was detected. These results suggest that NUDT18 can act as a cellular sanitizer and may influence the antiviral efficacy of Remdesivir and Ribavirin.

1390. Durlobactam, a Diazabicyclooctane (DBO) β-lactamase Inhibitor (BLI), Inhibits BlaC and Peptidoglycan (PG) Transpeptidases of Mycobacterium tuberculosis (Mtb): A Novel Approach to Therapeutics for Tuberculosis (TB)?

Background Novel therapies for multidrug-resistant TB are needed and new BLIs could answer this call. Mtb encodes for BlaC, a class A β-lactamase. BlaC is inhibited by clavulanate (CLA) while the DBO avibactam (AVI) is an inefficient inhibitor (low k2/K value). Carbapenems are hydrolyzed slowly by BlaC (low kcat/Km value) making them “dual action” compounds that inhibit both BlaC and PG transpeptidases, the intended β-lactam targets. DBOs inhibit PG transpeptidases in other bacteria. To explore the therapeutic potential of new DBOs against Mtb, we compared the inhibitor activity of AVI, relebactam (REL), and durlobactam (DUR, formerly ETX2514) against BlaC and Mtb PG transpeptidases using a biochemical approach. We also investigated the ability of DUR to lower minimum inhibitory concentrations (MICs) of β-lactams against Mtb H37Rv. Methods Mass spectrometry was performed to capture acyl-enzyme complexes (AECs) of purified BlaC and PG transpeptidases (PonA1, LdtMt1, LdtMt2, LdtMt3, and LdtMt5) with β-lactams and BLIs. Steady-state enzyme kinetics were determined using nitrocefin as a substrate. MICs with amoxicillin (AMX), meropenem (MER), CLA, and DUR alone and in combination against Mtb H37Rv were assessed using a microdilution method. Results DUR alone had a MIC of 2 µg/mL with Mtb H37Rv (Table 1). BlaC formed AECs with all carbapenems and BLIs. BlaC had lower Ki app and higher k2/K with DUR than those with AVI and REL and comparable to those with CLA; however, with a period of pre-incubation, AVI fully inhibits BlaC (Table 2). The carbapenems and DUR formed the most AECs with PG transpeptidases of the β-lactams and BLIs respectively; PG transpeptidases had lower Ki app values with DUR than those with AVI (Table 3). Table 1. Minimum Inhibitory Concetrations for Mycobacterium tuberculosis H37Rv Conclusion DUR alone has some antimicrobial activity against Mtb H37Rv. The likely mechanism that underlies this activity is inhibition of BlaC and several PG transpeptidases. Inhibition of enzyme targets with DUR was more potent and efficient than AVI and REL. DUR in combination with β-lactams lowered MICs but the DUR concentration used was higher than its MIC. Our findings support the exploration of novel BLIs against BlaC and PG transpeptidases with the ultimate goal of repurposing these drugs for the treatment of TB. Disclosures Robert A. Bonomo, MD, entasis (Research Grant or Support)Merck (Grant/Research Support)NIH (Grant/Research Support)VA Merit Award (Grant/Research Support)VenatoRx (Grant/Research Support)

Use of various keratometry data (ring and zone) in trifocal IOL calculation

Purpose. To compare the results of trifocal IOL calculation using various corneal tomographic data (ring and zone). Methods. This retrospective study involved 46 patients (46 eyes), underwent cataract surgery with trifocal IOL implantation (AcrySof IQ PanOptix). The calculation was performed using Tomey OA-2000 according to 2 formulas (Barrett II Universal, Olsen). Keratometry values included Km (the average of two main meridians of a cornea) provided by Pentacam HR Power Distribution Apex map, which describes total corneal refractive power (TCRP) with diameter of 3.0, 4.0 and 5.0 mm on a ring and zone. Mean (MAE) and median (MedAE) predicted postoperative refraction errors were assessed after surgery. Results. Mean Km value on 3 mm zone and ring was: 42.75±1,46 D and 42,91±1,43 D, respectively (p<0,0001). Mean Km on 4 mm zone and ring was: 42.6±1.5 D and 43.3 ± 1.5 D, respectively (p <0.005). Mean Km value on 5 mm zone and ring was: 43,09±1,5 D and 43,55±1,48 D, respectively (p<0,0001). Calculations using the Barrett II Universal formula revealed significant difference between MAE and MedAE of the predicted postoperative refraction on 5mm zone and ring (p=0.045). When using the Olsen formula in the calculations, significant difference was revealed using the Km data with a diameter of 3 mm and 5 mm (p=0.001 и p=0.009, respectively). The calculation on 3 mm ring was more accurate than for 3 mm zone. With a 5 mm diameter, the calculation is more accurate according to the zone data. Conclusion. Mean Km value on Power Distribution Apex map according to ring is significantly greater then according to zone. 1) The calculation of the trifocal IOL based on the TCRP zone data is reliably more accurate than the ring data according to both formulas (Barrett II Universal and Olsen) with a diameter of 5 mm. 2) According to the Olsen formula with a diameter of 3 mm, the calculation of the optical power of trifocal IOL based on TCRP ring data is more accurate. Key words: IOL calculation, Trifocal IOL, corneal topography

Phenylalanine, Tyrosine, and DOPA Are Bona Fide Substrates for Bambusa oldhamii BoPAL4

Phenylalanine ammonia-lyase (PAL) links the plant primary and secondary metabolisms, and its product, trans-cinnamic acid, is derived into thousands of diverse phenylpropanoids. Bambusa oldhamii BoPAL4 has broad substrate specificity using L-phenylalanine, L-tyrosine, and L-3,4-dihydroxy phenylalanine (L-DOPA) as substrates to yield trans-cinnamic acid, p-coumaric acid, and caffeic acid, respectively. The optimum reaction pH of BoPAL4 for three substrates was measured at 9.0, 8.5, and 9.0, respectively. The optimum reaction temperatures of BoPAL4 for three substrates were obtained at 50, 60, and 40 °C, respectively. The Km values of BoPAL4 for three substrates were 2084, 98, and 956 μM, respectively. The kcat values of BoPAL4 for three substrates were 1.44, 0.18, and 0.06 σ-1, respectively. The major substrate specificity site mutant, BoPAL4-H123F, showed better affinity toward L-phenylalanine by decreasing its Km value to 640 μM and increasing its kcat value to 1.87 s-1. In comparison to wild-type BoPAL4, the specific activities of BoPAL4-H123F using L-tyrosine and L-DOPA as substrates retained 5.4% and 17.8% residual activities. Therefore, L-phenylalanine, L-tyrosine, and L-DOPA are bona fide substrates for BoPAL4.

Perfluoroalkyl Carboxylic Acids Interact with the Human Bile Acid Transporter NTCP

Na+/taurocholate cotransporting polypeptide (NTCP) is important for the enterohepatic circulation of bile acids, which has been suggested to contribute to the long serum elimination half-lives of perfluoroalkyl substances in humans. We demonstrated that some perfluoroalkyl sulfonates are transported by NTCP; however, little was known about carboxylates. The purpose of this study was to determine if perfluoroalkyl carboxylates would interact with NTCP and potentially act as substrates. Sodium-dependent transport of [3H]-taurocholate was measured in human embryonic kidney cells (HEK293) stably expressing NTCP in the absence or presence of perfluoroalkyl carboxylates with varying chain lengths. PFCAs with 8 (PFOA), 9 (PFNA), and 10 (PFDA) carbons were the strongest inhibitors. Inhibition kinetics demonstrated competitive inhibition and indicated that PFNA was the strongest inhibitor followed by PFDA and PFOA. All three compounds are transported by NTCP, and kinetics experiments revealed that PFOA had the highest affinity for NTCP with a Km value of 1.8 ± 0.4 mM. The Km value PFNA was estimated to be 5.3 ± 3.5 mM and the value for PFDA could not be determined due to limited solubility. In conclusion, our results suggest that, in addition to sulfonates, perfluorinated carboxylates are substrates of NTCP and have the potential to interact with NTCP-mediated transport.

Biochemical Characterization of 13-lipoxygenases of Arabidopsis thaliana

13-Lipoxygenases (13-LOX) catalyze the dioxygenation of various polyunsaturated fatty acids (PUFAs), of which α-linolenic acid (LeA) is converted to 13-S-hydroperoxyoctadeca-9, 11, 15-trienoic acid (13-HPOT), the precursor for the prostaglandin-like plant hormones cis-(+)-12-oxophytodienoic acid (12-OPDA) and methyl jasmonate (MJ). This study aimed for characterizing the four annotated A. thaliana 13-LOX enzymes (LOX2, LOX3, LOX4, and LOX6) focusing on synthesis of 12-OPDA and 4Z,7Z,10Z)-12-[[-(1S,5S)-4-oxo-5-(2Z)-pent-2-en-1yl] cyclopent-2-en-1yl] dodeca-4,7,10-trienoic acid (OCPD). In addition, we performed interaction studies of 13-LOXs with ions and molecules to advance our understanding of 13-LOX. Cell imaging indicated plastid targeting of fluorescent proteins fused to 13-LOXs-N-terminal extensions, supporting the prediction of 13-LOX localization to plastids. The apparent maximal velocity (Vmaxapp) values for LOX-catalyzed LeA oxidation were highest for LOX4 (128 nmol.s−1.mg protein−1), with a Km value of 5.8 µM. A. thaliana 13-LOXs, in cascade with 12-OPDA pathway enzymes, synthesized 12-OPDA and OCPD from LeA and docosahexaenoic acid, previously shown only for LOX6. The activities of the four isoforms were differently affected by physiologically relevant chemicals, such as Mg2+, Ca2+, Cu2+ and Cd2+, and by 12-OPDA and MJ. As demonstrated for LOX4, 12-OPDA inhibited enzymatic LeA hydroperoxidation, with half-maximal enzyme inhibition at 48 µM. Biochemical interactions, such as the sensitivity of LOX toward thiol-reactive agents belonging to cyclopentenone prostaglandins, are suggested to occur in human LOX homologs. Furthermore, we conclude that 13-LOXs are isoforms with rather specific functional and regulatory enzymatic features.

First Dye-Decolorizing Peroxidase from an Ascomycetous Fungus Secreted by Xylaria grammica

Background: Fungal DyP-type peroxidases have so far been described exclusively for basidiomycetes. Moreover, peroxidases from ascomycetes that oxidize Mn2+ ions are yet not known. Methods: We describe here the physicochemical, biocatalytic, and molecular characterization of a DyP-type peroxidase (DyP, EC 1.11.1.19) from an ascomycetous fungus. Results: The enzyme oxidizes classic peroxidase substrates such as 2,6-DMP but also veratryl alcohol and notably Mn2+ to Mn3+ ions, suggesting a physiological function of this DyP in lignin modification. The KM value (49 µM) indicates that Mn2+ ions bind with high affinity to the XgrDyP protein but their subsequent oxidation into reactive Mn3+ proceeds with moderate efficiency compared to MnPs and VPs. Mn2+ oxidation was most effective at an acidic pH (between 4.0 and 5.0) and a hypothetical surface exposed an Mn2+ binding site comprising three acidic amino acids (two aspartates and one glutamate) could be localized within the hypothetical XgrDyP structure. The oxidation of Mn2+ ions is seemingly supported by four aromatic amino acids that mediate an electron transfer from the surface to the heme center. Conclusions: Our findings shed new light on the possible involvement of DyP-type peroxidases in lignocellulose degradation, especially by fungi that lack prototypical ligninolytic class II peroxidases.

Thermal stability of purified superoxide dismutase from liver of commercially valuable fish, Labeo rohita Exposed to Pb+Cr mixture

In this experiment, effect of lead (Pb) + chromium (Cr) mixture on superoxide dismutase (SOD) in the liver of Labeo rohita at a concentration of 11.1 mgL-1 was observed. The ammonium sulphate precipitation and ion exchange chromatography techniques were successfully used to purify SOD. After purification, SOD activity of control and Pb+Cr treated fish was noted as 581.00 and 645.45 UmL-1, respectively while the specific activity was 1383.33 and 1613.62 Umg-1, respectively. The fold purification value of SOD was 2.75 and 2.45 for control and stressed fish, respectively. The recovery was calculated as 77.06 and 57.43% for control and stressed fish, respectively. The results of kinetic characterization showed that SOD form control and exposed fish had maximum activity at pH 6.5 and 7.0. Temperature also had a significant effect on activity of SOD. The SOD activity was measured maximum at 30°C for both control and Pb+Cr exposed fish. The Km value of liver SOD for control and Pb+Cr treated L. rohita was calculated as 1.48 and 0.62 mM, respectively. The value of Vmax for SOD from liver of control and Pb+Cr exposed fish was 1000 and 570 U mL-1, respectively. The enthalpy of denaturation (∆H*) for liver SOD from control and Pb+Cr exposed L. rohita was computed as 3.492 and 2.802 KJ mol-1 at 40°C, respectively and these values were dropped off with increasing the temperature until it remains 3.251 and 2.561 KJ mol-1 at 70°C, respectively. The free energy of thermal denaturation (ΔGº) of liver SOD was slightly increased with increasing temperature until 75°C which shows its resistance against heat. The values of ΔGº was observed as 58.03 and 57.95 KJ mol-1 for control and exposed fish at 40°C, respectively while the same was increased upto 62.37 and 62.00 KJ mol-1 at 70°C, respectively. It was concluded from negative value of ΔS* (entropy of inactivation) that the SOD is stable thermodynamically.