bluuebunny/crossref_metadata_2025_split huggingface dataset:
Conservative Substitutions of Buried Amino Acid Residues Distant from the Active Site of Horse Liver Alcohol Dehydrogenase Have Small Effects on Structure, Catalysis and Dynamics
Authors: Plapp Bryce V. , Shanmuganatham Karthik , Wallace Rachel S. , Lee Ann Ting‐I. 
Published: November 2023

Global protein dynamics are proposed to participate in enzyme catalysis. Conformational changes and proton transfers, on a relatively slow (~ms) time scale, can be important to establish the scaffold for preorganizing the enzyme‐substrate complex. The present study tests the hypothesis that global dynamics, on the fast time (~ps) scale, can affect protein promoting vibrations and the reorganization involved in the hydride transfer step of alcohol dehydrogenase. The rationale is that distal substitutions of buried, conserved amino acid residues should alter the stability of the protein and affect the fast global dynamics if they are coupled to the chemistry of catalysis. The fast dynamics can be reflected in the X‐ray crystallographic temperature factors. A prerequisite for the present test is that the global structure of the substituted enzymes should not be altered, because allosteric effects could change the structure of the active site. Five different amino acid residues that are buried in hydrophobic clusters and the hinge region around which the global conformation changes when coenzyme binds were substituted as a result of partially random site‐directed mutagenesis. The proteins were purified and the structures were determined by X‐ray crystallography at high resolution. The G173A; V197I; I220V, L or F; V222I; and F322L enzymes have almost identical three‐dimensional structures, except for local perturbations at the site of substitution, as compared to wild‐type enzyme. These enzymes have very similar kinetic constants for oxidation of benzyl alcohol and reduction of benzaldehyde. The random mutagenesis also led to other single substitutions of these amino acid residues (G173V, E, K, or R; V197G, S, or T; I220G, S, T, or N; and V222G, S, or T), but expression of these proteins was too poor to allow biochemical studies. The F322I or F322V enzymes were poorly expressed. It appears that the five residues are in critical locations because the non‐conservative substitutions can affect protein stability. We conclude that the enzyme can tolerate conservative, distal substitutions that do not affect the scaffold of the protein. Furthermore, it appears that fast, global dynamics do not participate significantly in catalysis. In contrast, similar studies of enzymes with substitutions of amino acid residues in the active site can have significant effects on catalysis, either by altering reorganization or local dynamics.Support or Funding InformationSupported by NIH grant GM078446
Transition Metal Cation Inhibition of Mycobacterium tuberculosis Esterase Rv0045c
Authors: Lancaster Benjamin , Hoops Geoffrey , Johnson R. Jeremy 
Published: November 2023

The survival of Mycobacterium tuberculosis within its human host is highly dependent upon the lipid metabolism performed by the numerous serine hydrolase enzymes at the bacterium's disposal. Among these serine hydrolases, Rv0045c is a mycobacterial‐specific serine hydrolase that has been characterized for global substrate specificity by scanning the hydrolase against a library of fluorogenic hydrolase substrates. Rv0045c expresses strong selectivity for short, straight‐chained alkyl esters, but the relative enzyme kinetic rate is slow in comparison to a majority of bacterial hydrolases. Herein, we determined the transition metal ion dependence of Rv0045c on hydrolysis of the four‐carbon methoxyacetic fluorogenic substrate. The catalytic activity of Rv0045c was significantly inhibited by low concentrations of Ni+2, Cu+2, and Zn+2 metal cations while other 3d metal cations displayed no effects on the activity of the hydrolase. The low inhibition constants for these three metals on Rv0045c suggest a likely physiological relevance for the metal dependent inhibition of Rv0045c.Support or Funding InformationFunding provided by a Butler University Holcomb research award and a Senior Research Grant from the Indiana Academy of Sciences.
The role of Aspartate 141 in the regulation of the Agrobacterium tumefaciens ADP‐Glc Pyrophosphorylase
Authors: Patel Hiralben , Gonzalez‐Martin Laura , Renta Vincent , Ballicora Miguel 
Published: November 2023

The main regulatory step of the glycogen and starch metabolic pathways takes place at the level of the synthesis of ADP‐glucose (ADP‐Glc), which is the first committed step and it is catalyzed by ADP‐Glc Pyrophosphorylase (ADP‐Glc PPase). This is an allosterically regulated enzyme, which catalyzes the conversion of ATP and glucose‐1‐phosphate to ADP‐Glc and inorganic pyrophosphate in the presence of Mg2. In previous studies, it was observed that an inter‐subunit conformational change in the potato tuber ADP‐Glc PPase may play a role in the allosteric regulation of the enzyme. Removal of a disulfide bond involving Cys12 in the small subunit, either by mutation or by reduction resulted in a more active enzyme (Ballicora et al, 2000, J. Biol. Chem. 275:1315–1320), indicating that the inter‐subunit interaction between α subunits is an important part of the allosteric mechanism (Jin, X. et al, 2005, EMBO J. 24:694–704). In the structure of the Agrobacterium tumefaciens ADP‐Glc PPase, we found that Asp141 is a candidate to interact with Arg11 of the opposing subunit (Cupp‐Vickery, et al. 2008, Biochemistry 47:12795–801). The extreme N‐terminal Arg5 and Arg11 are quite clearly involved in activation by pyruvate (Pyr) as it has been demonstrated before (Gomez‐Casati et al. Biochem, 40(34),2001, pp. 10169–10178). To evaluate the hypothesis that of Asp 141 is involved in regulation, we mutated it to Ala. D141A was successfully expressed and purified to near homogeneity. This mutant enzyme exhibited reduced Vmax values in the absence of activators (Pyr and Fru6P), and lower apparent affinity for ATP and Fru6P. In the presence of Fru6P, D141A enzyme exhibited only ~ 4.3% of the wild‐type activity, which indicates that it virtually insensitive to Fru6P. This mutant enzyme also displayed 2.5‐fold reduced apparent affinity to the activator Fru6P when compared to the wild‐type enzyme. Our results suggest a unique functional role of D141 in regulation, possibly by interacting with R11 in the opposing subunit.Support or Funding InformationNational Science Foundation (NSF)
Characterization of a Novel Flavin‐dependent Monooxygenase in Bacillus niacini
Authors: Dasovich Morgan A , Snider Mark J , Peterson Michael T 
Published: November 2023

Nicotinate metabolism is a model system for the degradation of N‐heterocyclic molecules, a common class of environmental contaminants. A putative monooxygenase in gram‐positive Bacillus niacini is hypothesized to carry out a decarboxylation‐hydroxylation reaction in a degradation pathway for nicotinate that is relatively uncharacterized. B. niacini is of particular interest because it exhibits a novel method of nicotinic acid degradation divergent from previously characterized pathways in Pseudomonas and Bordetella. Whole‐genome sequencing of B. niacini has identified a putative nicotinate‐degrading (nic) cluster and the proteins in this cluster have been expressed and purified, including the protein of interest. The putative monooxygenase contains a Rossman fold and is flavin‐dependent, utilizing NAD(P)H as a cofactor. It shows sequence homology with the 6‐hydroxynicotinate‐3‐monooxygenase (NicC) enzyme of P. fluorescens and B. bronchiseptica. NicC catalyzes a similar decarboxylation and hydroxylation reaction with 6‐hydroxynicotinate, thus it is hypothesized that the B. niacini homologue functions by catalyzing the decarboxylation‐hydroxylation of 2,6‐dihydroxynicotinic acid to 2,3,6‐trihydroxypyridine. To investigate this theory, 2,6‐dihydroxynicotinic acid and 2,3,6‐trihydroxypyridine were synthesized from commercially available 2,6‐dimethoxypyridine‐3‐carboxaldehyde. Compound purity has been assessed with NMR and mass spectrometry. UV spectrophotometry was used to determine the rate constants of the enzymatic reaction with NAD(P)H, and 2,6‐dihydroxynicotinic acid.Support or Funding InformationSupport and funding from the Department of Chemistry at the College of Wooster made this study possible.
Characterization and Identification of Metabolites in the Oxidation of Nicotinic Acid by Bacillus Niacini
Authors: Lesner Nicholas P. , Peterson Michael T. , Snider Mark J. 
Published: November 2023

N‐Heterocylic compounds are of environmental concern due to their ubiquitous contamination in groundwater and soil. Nicotinic acid is a simple N‐heterocylic compound that is known to be degraded by a select group of gram‐positive bacteria and can serve as a useful model compound for understanding biological degradation. Nicotinic acid's biological degradation has been well characterized in Pseudomonas, but a similar yet distinct pathway known in Bacillus niacini has yet to be fully characterized. Utilizing the recently sequenced genome of B. niacini, in which a gene cluster was hypothesized to code for the enzymes that catalyze the degradation of nicotinic acid, a enzyme hypothesized to catalyze the oxidative ring‐opening step in the pathway was characterized. Protein sequence analysis revealed identity with NicX in Pseudomonas, but does not reveal mechanistic insight concerning ring opening. Here we describe the synthesis of the hypothesized substrate 2,3,6‐trihydroxypyridine, confirm the enzyme catalyzed product, and report results of a kinetic assay developed to examine mechanistic proposals. The results provide insight to the chemistries involved in the degradation of N‐heterocylic compounds by aerobic bacteria found in the environment.Support or Funding InformationThe College of Wooster Department of Chemistry
Decoupling the Allosteric Activation of Ubiquitin Specific Protease 7 by Its Ubiquitin‐like Domains for the Development of Small Molecule Inhibitors
Authors: Hjortland Nicole M , Pepe Antonella , Molland Katrina , Mesecar Andrew 
Published: November 2023

Ubiquitin specific protease 7, USP7/HAUSP, regulates cellular levels of E3 ligase‐MDM2 and tumor suppressor p53 by the deubiquitination and subsequent stabilization of MDM2. The up‐regulation of USP7 in many types of cancer make it an ideal target for the development of anti‐cancer therapeutics. It is well known that the catalytic domain of USP7 has 100‐fold less activity than that of the full length enzyme. Recent work by Fasen et. al. (2011) reported that the catalytic function of USP7 is activated through the C‐terminal HAUSP ubiquitin‐like (HUBL) domain interaction with the switching loop of the catalytic domain. Our studies, utilizing a fluorescence based kinetic assay, define the kinetic parameters that surround the coupling of the activation of the catalytic domain by the HUBL domains. A small molecule inhibitor only capable of inhibiting the full length enzyme was identified by high‐throughput screening of a 20,000 compound library at Purdue University. Analogues of this compound were synthesized and significant structure activity relationships were defined. By kinetic studies, we probed the mode of inhibition of the best analogues and identified the need of specific HUBL domains for the inhibition of the enzyme by these compounds. These compounds can therefore serve as molecular probes in decoupling the allosteric activation of the HUBL domains for further studies of the intra‐molecular interactions of USP7.Support or Funding InformationThis work is funded by a grant from the Walther Cancer Foundation
The Agrobacterium tumefaciens ADP‐Glucose Pyrophosphorylase G329D Mutant Exists in a Pre‐Activated State
Authors: hill benjamin luke , Mascarenhas Romila , Koenig Amanda M , Huerta Fidel , Khan Samia , Iglesias Alberto A. , Liu Dali , Ballicora Miguel 
Published: November 2023

Allosteric regulation of ADP‐glucose (ADP‐Glc PPase, EC 2.7.7.27) is critical for determining the amount of glycogen and starch that gets synthesized in bacteria and plants, respectively [1]. This enzyme combines ATP and glucose‐1‐phosphate to form ADP‐glucose, which serves as the glucose moiety donor for polyglucan synthesis. ADP‐Glc PPases are regulated by intermediates of the major carbon assimilatory pathway that exists within a given species. To date, all identified regulators have a negative charge. The ADP‐Glc PPase from Agrobacterium tumefaciens is strongly activated by pyruvate and fructose‐6‐phosphate (Fru‐6P). Since ADP‐Glc PPase serves as a major control point for the production of glycogen and starch, which are renewable and biodegradable carbon sources, these enzymes have been attractive targets for protein engineering [1,2,3].A growing body of evidence suggests that the interface between the enzyme's distinct C‐ter and N‐ter domains is important for allosteric regulation [2,4,5]. Here, we report on the aspartate mutant of Gly329 in the A. tumefaciens enzyme, which, based on the crystal structure [2], is a residue that residues near this cleft. When this enzyme was characterized, it was found to be in a pre‐activated state, meaning that it did not need allosteric activators present in order to achieve levels of activity similar to the activated wild‐type enzyme. We suspect that the positioning of an aspartate group in this region mimics the action of negatively charged activators like pyruvate. Furthermore, these findings could help pinpoint the exact location of the allosteric binding site. This work could provide important information into the allosteric regulation of ADP‐Glc PPases.Support or Funding InformationNational Science Foundation Grant MCB‐1024945(MAB)
Determining the Structure of NRPS‐Independent Siderophore (NIS) Synthetase DesD Using X‐ray Crystallography
Authors: Karimi Kimya L , Hoffmann Katherine M 
Published: November 2023

A lack of iron leads to secretion of siderophores (small molecule metal chelators) by bacteria. Siderophores have high affinity for iron outcompeting host chelators, and making them virulence factors in infection. Some siderophores (of particularly virulent pathogens) are assembled via an NRPS‐independent siderophore (NIS) pathway. Understanding the structure of NIS siderophore biosynthesis enzymes may lead to the design of specific antibiotics to fight pathogenic bacteria.The Hoffmann lab is using x‐ray crystallography to determine the protein structure of NIS synthetase DesD, from Streptomyces coelicolor. DesD makes the siderophore desferrioxamine B, requiring three iterative bonds and a macrocyclization event. DesD is one of the best biochemically characterized NIS synthetases so far, and we are solving this structure as a model for the rest. The goal of this project is to solve the x‐ray structure of DesD bound to substrate and ATP, as well as apo (empty of substrate) structures, in order to characterize the conformational changes that may occur with binding. DesD crystalizes in 20% PED 8,000, 15% glycerol, and 15% ethylene glycol conditions, and crystals diffract to 2.48 Å with good statistics. We are solving the structure using molecular replacement and are pursuing substrate‐bound crystals. This structural information may allow us to design an inhibitor that will prevent the binding of substrate in future work.
Human alpha3 beta1 isoform of Na+/K+‐ATPase: expression in yeast, mutations and their relation to neurological disorders
Authors: Geletičová Jaroslava , Kubala Martin , Sorensen David , Pedersen Per Amstrup , Nissen Poul 
Published: November 2023

The Na+/K+‐ATPase (NKA) is a crucial membrane protein responsible for maintaining the membrane potential by actively transporting sodium and potassium ions. The proper function of this pump is essential for all cells (e. g. in homeostasis, membrane potential). The human body contains four isoforms of this enzyme, which are spread over whole organism. One of the most important localizations of NKA is the brain. Medical research of Heinzen et al. (2014) suggests that there is a possible connection between NKA alpha3 isoform mutations (mainly D801N, E815K, D923N, D923Y and G947R) and neurological disorders. Detailed studies of patient's genome revealed that all these mutations are closely linked to Rapid‐Onset Dystonia Parkinsonism and Alternating Hemiplegia of Childhood.Modern biotechnological methods, especially the production of a heterologous protein in expression systems, enable deeper study of a wide range of non‐abundant proteins. The preparation of alpha3 NKA proteins (wild type and mutants) was performed according to Pedersen et al. (2006) with some modifications. In general, changes in the level of protein expression and determination of the relative enzyme activity could be the first step towards better understanding of the relation between NKA function and neurological disorders.Support or Funding InformationThis project was supported by grant No. L01204 (Sustainable development of research in the Centre of the Region Haná) from the National Program of Sustainability I, MEYS and by “Palacky University Foundation 2015”. The project was awarded by EMBO Short‐Term fellowship ASTF 243‐2015.
Molecular Cloning and Expression of an Oleate Hydratase From Nocardia Cholesterolicum NRRL 5767
Authors: Vanderway David R , Alhmadi Hekmat B , Diaz Joshua D , Wen Lisa , Huang Jenq‐Kuen 
Published: November 2023

The production of household cooking oils in an industrial setting yields soapstock as a byproduct during the refining process. Soapstock contains a significant amount of oleic acid and due to soapstocks very low price, it serves as a cheap source of oleic acid. Oleate hydratase (Ohase) converts oleic acid to the industrially useful product 10(R)‐hydroxystearic acid (10‐HSA), in a regioselective manner. Classically, 10‐HSA is utilized to thicken grease, but it has potential to be used in the production of plastics, lubricants and other various industrially relevant materials. Barring the use of biocatalytic microorganisms containing Ohase, this reaction would take place under unfavorable conditions involving the use of high temperatures/pressures, strong acids or metal catalysts, and all of these methods lead to little regioselectivity in the final product. Therefore, the use of a biocatalyst that catalyzes the conversion of oleate hydratase to 10‐HSA is extremely desirable for industrial applications. The bacterium Nocardia Cholesterolicum species NRRL 5767 is able to convert 95% of oleic acid added into the bacterial culture to 10‐HSA. Ohase has never been isolated or characterized at either the protein level or DNA level from NRRL 5767. This work is the first report of molecular cloning of the Ohase gene from NRRL 5767 genomic DNA into the pET28a expression vector. Functional recombinant Ohase enzyme has been expressed and purified. Enzyme activity of Ohase was determined by the conversion of oleic acid to 10‐HSA or by spectrophotometrically through a coupling reaction.Support or Funding InformationWestern Illinois University Foundation and the Department of Chemistry.
Molecular Cloning and Expression of a Secondary Alcohol Dehydrogenase From Nocardia Cholesterolicum NRRL 5767
Authors: Diaz Joshua D , Vanderway David R , Flores Micah , Wen Lisa , Huang Jenq‐Kuen 
Published: November 2023

Norcardia cholesterolicum NRRL 5767 (N. cholesterolicum NRRL 5767) is a well‐known bacterium capable of transforming 95% of added oleic acid to a more valuable product, 10‐hydroxystearic acid (10‐HSA) (Appl Microbiol Biotechnol 32, 299–304, 1989). This reaction is catalyzed by oleate hydratase. However, a small amount of the 10‐HSA is subsequently converted to 10‐ketostearic acid (10‐KSA) by secondary alcohol dehydrogenase (2º‐ADH). Since 10‐HSA is more valuable than 10‐KSA in industry; 10‐KSA is considered a side product which complicates downstream separation and purification of 10‐HSA. It is attractive to hinder the production of 10‐KSA through bacterium strain improvement by genetic engineering to block the production of 2º‐ADH. The objective of this research is to clone, overexpress, and characterize functional recombinant 2º‐ADH. An annotated 2º‐ADH gene has been successfully amplified by PCR from genomic DNA of N. cholesterolicum NRRL 5767and sub‐cloned into pET28a expression vector. The 2º‐ADH protein has been overexpressed by IPTG induction and purified to homogeneity by Ni‐NTA affinity column. Enzyme activity of the 2º‐ADH was determined by monitoring the formation of NADH spectrophotometrically.Support or Funding InformationWestern Illinois University Foundation and the Department of Chemistry.
Antioxidant activity of the hexanic extract from Eryngium carlinae “in vitro” and in Saccharomyces cerevisiae
Authors: Peña‐Montes Donovan , Mejía‐Barajas Jorge , Clemente‐Guerrero Mónica , Salgado‐Garciglia Rafael , Cortés‐Rojo Christian , Montoya‐Pérez Rocío , Manzo‐Avalos Salvador , Saavedra‐Molina Alfredo 
Published: November 2023

Oxidative stress has been associated with the onset of chronic degenerative diseases such as cancer, cardiovascular and neurodegenerative disorders. Epidemiological studies have shown the use of plants containing antioxidant is beneficial to health. Eryngium carlinae commonly known in México as “Hierba Del Sapo” in traditional herbal medicine, attributed with diuretic and healing properties. Currently, ingested as daily infusion is use to regulate blood pressure and as a hypolipidemic agent; however. Different assays (“in vitro” and “in vivo”) have been developed to evaluate the antioxidant activity of plant extracts. The identification and quantification of constituents, through gas chromatography–mass spectrometry (GC/MS) analysis. The antioxidant activity was evaluated using “in vitro” assays (total antioxidant capacity, DPPH radical scavenging activity, scavenging capacity towards hydrogen peroxide (H2O2)), and, analyzing the antioxidant effect on biomarkers of oxidative stress and its influence on the viability in cells of Saccharomyces cerevisiae treated with the hexanic extract, and exposed to H2O2. In chromatographic analysis, the majority constituents found in the hexanic extract of E. carlinae were terpenes and sesquiterpenes, (Z)β‐Farnesene (38.79 %) β‐Pinene (17.53 %), Calamene (13.30 %), α‐Farnesene (10.38 %). “In vitro” tests, showed a similar reducing capacity of H2O2, and an approximate effect of anti‐lipid peroxidation obtained with ascorbic acid was observed in our results. In S. cerevisiae, the results of viability are correlated in the reduction of levels of cellular lipid peroxidation, protein carbonylation and glutathione ratio in accordance with control. In conclusion, the hexanic extract of E. carlinae has antioxidant activity “in vitro” and in S. cerevisiae, reducing the levels of lipid peroxidation, protein carbonylation, and increasing viability, attributable to farnesene and pinene, and the synergism between the constituents of the extract.Support or Funding InformationCONACYT 169093 to ASM; CIC‐UMSNH 2.16 to ASM.