2.2-Diphenic Acid: A Reliable Biomarker Of Phenanthrene Biodegradation

Phenanthrene is among the 16 priority pollutant and its mitigation in the environment has been a global concern. It serves as a model compound when it comes to biodgradation study of polyaromatic hydrocarbons (PAHs) because it has both the Bay- and K-region found in most PAH pollutants. Like other PAH pollutants, different means are available for its remediation in the environment, including microbial biodegradation. Diverse species of bacteria and fungi metabolize phenanthrenes as their sole source of carbon and energy. However, bacteria are more diverse in comparison to fungi. This has been shown in published pathways of phenanthrene biodegradation implicating various intermediary metabolites, including 2,2-diphenic acid, which is a downline metabolite of 9,10-dihydroxyphenanthrene. Though the 2,2-diphenic acid has been widely demonstrated to produce carbon (iv) oxide and linked to phthalate, only few has traced salicylic acid as its downstream molecule. 2,2-diphenic acid mounts equivalent position to 1-hydroxy-2-naphthoic acid, metabolite that ends the phenanthrene metabolic pathway. This is because they both produce phthalic acid and salicylic acid. As a product of bacteria and fungi during phenanthrene degradation, 2,2-diphenic acid can serve as a dependable biomarker of phenanthrene metabolism in a polluted habitat, where microbial community exist freely.

Transpressional Deformation Of Bukit C, Guar Sanai: Lithostratigraphic Implication

The upper part of the Setul Formation and the lower part of the Kubang Pasu Formation are well exposed in Guar Sanai, Perlis due to the earth quarry activities. However, compared to the Hill A and Hill B, the outcrops in Hill C clearly illustrates the influence of structure on the lithostratigraphy of the area. The boundary between the Setul Formation and the Kubang Pasu Formation is marked by the thrust fault generally trending north-south to northeast-southwest and mylonite. The thrust belt associated with folding and uplift clearly developed in the zone between parallel lateral faults (trending north-south and southeast-northwest) and is interpreted to accommodate slip along the transpression zone. The sinistral faults are then deformed by transpression movement of dextral faults (trending east-southwest and east-west) which amplified the earlier structures. The combination of lateral and thrust movements formed flower structure that associated with folding and uplift, commonly found in transpressional zone of the strike-slip region. Deformation has caused the older Setul Formation being uplifted to almost equivalent position to the younger Kubang Pasu Formation and led to the displacement of original bed position and repetition of similar sequences. The formation of folding and reverse faulting associated with left lateral strike-slip fault are interpreted to cause by movement of major fault in the northwest Peninsular Malaysia, known as the Bok Bak Fault.

Aurora A regulation by reversible cysteine oxidation reveals evolutionarily conserved redox control of Ser/Thr protein kinase activity

Reactive oxygen species (ROS) are physiological mediators of cellular signaling and play potentially damaging roles in human diseases. In this study, we found that the catalytic activity of the Ser/Thr kinase Aurora A was inhibited by the oxidation of a conserved cysteine residue (Cys290) that lies adjacent to Thr288, a critical phosphorylation site in the activation segment. Cys is present at the equivalent position in ~100 human Ser/Thr kinases, a residue that we found was important not only for the activity of human Aurora A but also for that of fission yeast MAPK-activated kinase (Srk1) and PKA (Pka1). Moreover, the presence of this conserved Cys predicted biochemical redox sensitivity among a cohort of human CAMK, AGC, and AGC-like kinases. Thus, we predict that redox modulation of the conserved Cys290 of Aurora A may be an underappreciated regulatory mechanism that is widespread in eukaryotic Ser/Thr kinases. Given the key biological roles of these enzymes, these findings have implications for understanding physiological and pathological responses to ROS and highlight the importance of protein kinase regulation through multivalent modification of the activation segment.

Natural Variation in the ‘Control Loop’ of BVMOAFL210 and Its Influence on Regioselectivity and Sulfoxidation

Baeyer-Villiger monooxygenases (BVMOs) are flavin-dependent enzymes that primarily convert ketones to esters, but can also catalyze heteroatom oxidation. Several structural studies have highlighted the importance of the ‘control loop’ in BVMOs, which adopts different conformations during catalysis. Central to the ‘control loop’ is a conserved tryptophan that has been implicated in NADP(H) binding. BVMOAFL210 from Aspergillus flavus, however, contains a threonine in the equivalent position. Here, we report the structure of BVMOAFL210 in complex with NADP+ in both the ‘open’ and ‘closed’ conformations. In neither conformation does Thr513 contact the NADP+. Although mutagenesis of Thr513 did not significantly alter the substrate scope, changes in peroxyflavin stability and reaction rates were observed. Mutation of this position also brought about changes in the regio- and enantioselectivity of the enzyme. Moreover, lower rates of overoxidation during sulfoxidation of thioanisole were also observed.

Characterization of l-2-keto-3-deoxyfuconate aldolases in a nonphosphorylating l-fucose metabolism pathway in anaerobic bacteria

The genetic context in bacterial genomes and screening for potential substrates can help identify the biochemical functions of bacterial enzymes. The Gram-negative, strictly anaerobic bacterium Veillonella ratti possesses a gene cluster that appears to be related to l-fucose metabolism and contains a putative dihydrodipicolinate synthase/N-acetylneuraminate lyase protein (FucH). Here, screening of a library of 2-keto-3-deoxysugar acids with this protein and biochemical characterization of neighboring genes revealed that this gene cluster encodes enzymes in a previously unknown “route I” nonphosphorylating l-fucose pathway. Previous studies of other aldolases in the dihydrodipicolinate synthase/N-acetylneuraminate lyase protein superfamily used only limited numbers of compounds, and the approach reported here enabled elucidation of the substrate specificities and stereochemical selectivities of these aldolases and comparison of them with those of FucH. According to the aldol cleavage reaction, the aldolases were specific for (R)- and (S)-stereospecific groups at the C4 position of 2-keto-3-deoxysugar acid but had no structural specificity or preference of methyl groups at the C5 and C6 positions, respectively. This categorization corresponded to the (Re)- or (Si)-facial selectivity of the pyruvate enamine on the (glycer)aldehyde carbonyl in the aldol-condensation reaction. These properties are commonly determined by whether a serine or threonine residue is positioned at the equivalent position close to the active site(s), and site-directed mutagenesis markedly modified C4-OH preference and selective formation of a diastereomer. I propose that substrate specificity of 2-keto-3-deoxysugar acid aldolases was convergently acquired during evolution and report the discovery of another l-2-keto-3-deoxyfuconate aldolase involved in the same nonphosphorylating l-fucose pathway in Campylobacter jejuni.

Insertion sequence 1 from calpain-3 is functional in calpain-2 as an internal propeptide

Calpains are intracellular, calcium-activated cysteine proteases. Calpain-3 is abundant in skeletal muscle, where its mutation-induced loss of function causes limb-girdle muscular dystrophy type 2A. Unlike the small subunit–containing calpain-1 and -2, the calpain-3 isoform homodimerizes through pairing of its C-terminal penta-EF-hand domain. It also has two unique insertion sequences (ISs) not found in the other calpains: IS1 within calpain-3's protease core and IS2 just prior to the penta-EF-hand domain. Production of either native or recombinant full-length calpain-3 to characterize the function of these ISs is challenging. Therefore, here we used recombinant rat calpain-2 as a stable surrogate and inserted IS1 into its equivalent position in the protease core. As it does in calpain-3, IS1 occupied the catalytic cleft and restricted the enzyme's access to substrate and inhibitors. Following activation by Ca2+, IS1 was rapidly cleaved by intramolecular autolysis, permitting the enzyme to freely accept substrate and inhibitors. The surrogate remained functional until extensive intermolecular autoproteolysis inactivated the enzyme, as is typical of calpain-2. Although the small-molecule inhibitors E-64 and leupeptin limited intermolecular autolysis of the surrogate, they did not block the initial intramolecular cleavage of IS1, establishing its role as a propeptide. Surprisingly, the large-molecule calpain inhibitor, calpastatin, completely blocked enzyme activity, even with IS1 intact. We suggest that calpastatin is large enough to oust IS1 from the catalytic cleft and take its place. We propose an explanation for why calpastatin can inhibit calpain-2 bearing the IS1 insertion but cannot inhibit WT calpain-3.

decryst: an efficient software suite for structure determination from powder diffraction

Presented here is decryst, a software suite for structure determination from powder diffraction, which uses the direct-space method, and is able to apply anti-bump constraints automatically and efficiently during the procedure of global optimization using the crystallographic collision detection algorithm by Liu [Acta Cryst. (2017), A73, 414–422]. decryst employs incremental computation in its global-optimization cycles, which results in dramatic performance enhancement. It is also designed with parallel and distributed computing in mind, allowing for even better performance by simultaneous use of multiple processors. Owing to the parallelized usage of the equivalent position combination method [Deng & Dong (2009). J. Appl. Cryst. 42, 953–958] in decryst, it is particularly suitable for determination of structures with mostly unknown bonding relations, and offers some unprecedented opportunities for these structures. decryst is free and open-source software, and can be obtained at https://gitlab.com/CasperVector/decryst/; it strives to be simple yet flexible, in the hope that the underlying techniques could be adopted in more crystallographic applications.

Cantu syndrome-associated SUR2 (ABCC9) mutations in distinct structural domains result in KATP channel gain-of-function by differential mechanisms

The complex cardiovascular disorder Cantu Syndrome arises from gain-of-function mutations in either KCNJ8 or ABCC9, the genes encoding the Kir6.1 and SUR2 subunits of ATP-sensitive potassium (KATP) channels. Recent reports indicate that such mutations can increase channel activity by multiple molecular mechanisms. In this study, we determine the mechanism by which KATP function is altered by several mutations in distinct structural domains of SUR2: D207E in the intracellular L0-linker and Y985S, G989E, M1060I, and R1154Q/W in TMD2. Mutations were engineered at their equivalent position in rat SUR2A (D207E, Y981S, G985E, M1056I and R1150Q/W) and functional effects were investigated using macroscopic rubidium (86Rb+) efflux assays and patch clamp electrophysiology. The results show that D207E increases KATP activity by increasing intrinsic stability of the open state, whilst the cluster of Y981S/G985E/M1056I mutations, as well as R1150Q/W, augment Mg-nucleotide activation. The response of mutant channels to inhibition by the sulfonylurea drug glibenclamide, a potential pharmacotherapy for CS, was also tested. There was no major effect on glibenclamide sensitivity for the D207E, Y981S, G985E or M1056I mutations, but glutamine and tryptophan substitution at R1150 resulted in significant decreases in potency.