scholarly journals Structural Determinants for Substrate Selectivity in Guanine Deaminase Enzymes of the Amidohydrolase Superfamily

Biochemistry ◽  
2019 ◽  
Vol 58 (30) ◽  
pp. 3280-3292
Author(s):  
Roger Shek ◽  
Tylene Hilaire ◽  
Jasper Sim ◽  
Jarrod B. French
Keyword(s):  
Substrate Selectivity ◽  
Structural Determinants ◽  
Guanine Deaminase ◽  
Amidohydrolase Superfamily

scholarly journals Novel Aldo-Keto Reductases for the Biocatalytic Conversion of 3-Hydroxybutanal to 1,3-Butanediol: Structural and Biochemical Studies

2017 ◽  
Vol 83 (7) ◽  
Author(s):  
Taeho Kim ◽  
Robert Flick ◽  
Joseph Brunzelle ◽  
Alex Singer ◽  
Elena Evdokimova ◽  
...  
Keyword(s):  
Rational Design ◽  
Molecular Mechanisms ◽  
Substrate Binding ◽  
Aromatic Aldehydes ◽  
Site Directed Mutagenesis ◽  
Substrate Selectivity ◽  
Significant Activity ◽  
Structural Determinants ◽  
One Pot ◽  
Content Type

ABSTRACT The nonnatural alcohol 1,3-butanediol (1,3-BDO) is a valuable building block for the synthesis of various polymers. One of the potential pathways for the biosynthesis of 1,3-BDO includes the biotransformation of acetaldehyde to 1,3-BDO via 3-hydroxybutanal (3-HB) using aldolases and aldo-keto reductases (AKRs). This pathway requires an AKR selective for 3-HB, but inactive toward acetaldehyde, so it can be used for one-pot synthesis. In this work, we screened more than 20 purified uncharacterized AKRs for 3-HB reduction and identified 10 enzymes with significant activity and nine proteins with detectable activity. PA1127 from Pseudomonas aeruginosa showed the highest activity and was selected for comparative studies with STM2406 from Salmonella enterica serovar Typhimurium, for which we have determined the crystal structure. Both AKRs used NADPH as a cofactor, reduced a broad range of aldehydes, and showed low activities toward acetaldehyde. The crystal structures of STM2406 in complex with cacodylate or NADPH revealed the active site with bound molecules of a substrate mimic or cofactor. Site-directed mutagenesis of STM2406 and PA1127 identified the key residues important for the activity against 3-HB and aromatic aldehydes, which include the residues of the substrate-binding pocket and C-terminal loop. Our results revealed that the replacement of the STM2406 Asn65 by Met enhanced the activity and the affinity of this protein toward 3-HB, resulting in a 7-fold increase in k cat/Km . Our work provides further insights into the molecular mechanisms of the substrate selectivity of AKRs and for the rational design of these enzymes toward new substrates. IMPORTANCE In this study, we identified several aldo-keto reductases with significant activity in reducing 3-hydroxybutanal to 1,3-butanediol (1,3-BDO), an important commodity chemical. Biochemical and structural studies of these enzymes revealed the key catalytic and substrate-binding residues, including the two structural determinants necessary for high activity in the biosynthesis of 1,3-BDO. This work expands our understanding of the molecular mechanisms of the substrate selectivity of aldo-keto reductases and demonstrates the potential for protein engineering of these enzymes for applications in the biocatalytic production of 1,3-BDO and other valuable chemicals.

scholarly journals Bacterial Ammeline Metabolism via Guanine Deaminase

2009 ◽  
Vol 192 (4) ◽  
pp. 1106-1112 ◽  
Author(s):  
Jennifer L. Seffernick ◽  
Anthony G. Dodge ◽  
Michael J. Sadowsky ◽  
John A. Bumpus ◽  
Lawrence P. Wackett
Keyword(s):  
Active Sites ◽  
Cyanuric Acid ◽  
Homo Sapiens ◽  
Wild Type ◽  
E Coli ◽  
Guanine Deaminase ◽  
Amidohydrolase Superfamily ◽  
Deamination Reaction

ABSTRACT Melamine toxicity in mammals has been attributed to the blockage of kidney tubules by insoluble complexes of melamine with cyanuric acid or uric acid. Bacteria metabolize melamine via three consecutive deamination reactions to generate cyanuric acid. The second deamination reaction, in which ammeline is the substrate, is common to many bacteria, but the genes and enzymes responsible have not been previously identified. Here, we combined bioinformatics and experimental data to identify guanine deaminase as the enzyme responsible for this biotransformation. The ammeline degradation phenotype was demonstrated in wild-type Escherichia coli and Pseudomonas strains, including E. coli K12 and Pseudomonas putida KT2440. Bioinformatics analysis of these and other genomes led to the hypothesis that the ammeline deaminating enzyme was guanine deaminase. An E. coli guanine deaminase deletion mutant was deficient in ammeline deaminase activity, supporting the role of guanine deaminase in this reaction. Two guanine deaminases from disparate sources (Bradyrhizobium japonicum USDA 110 and Homo sapiens) that had available X-ray structures were purified to homogeneity and shown to catalyze ammeline deamination at rates sufficient to support bacterial growth on ammeline as a sole nitrogen source. In silico models of guanine deaminase active sites showed that ammeline could bind to guanine deaminase in a similar orientation to guanine, with a favorable docking score. Other members of the amidohydrolase superfamily that are not guanine deaminases were assayed in vitro, and none had substantial ammeline deaminase activity. The present study indicated that widespread guanine deaminases have a promiscuous activity allowing them to catalyze a key reaction in the bacterial transformation of melamine to cyanuric acid and potentially contribute to the toxicity of melamine.

Structural domains involved in substrate selectivity in two neutral amino acid transporters

2004 ◽  
Vol 287 (3) ◽  
pp. C754-C761 ◽  
Author(s):  
Andrea Soragna ◽  
Stefania Anna Mari ◽  
Rossana Pisani ◽  
Antonio Peres ◽  
Michela Castagna ◽  
...  
Keyword(s):  
Amino Acid ◽  
Amino Acid Uptake ◽  
Transmembrane Domains ◽  
Neutral Amino Acid ◽  
Xenopus Laevis Oocytes ◽  
Amino Acid Transporters ◽  
Acid Uptake ◽  
Substrate Selectivity ◽  
Organic Substrates ◽  
Structural Determinants

The ability of the two highly homologous Na+/Cl−-dependent neutral amino acid transporters KAAT1 and CAATCH1, cloned from the midgut epithelium of the larva Manduca sexta, to transport different amino acids depends on the cotransported ion, on pH, and on the membrane voltage. Different organic substrates give rise to transport-associated currents with their own characteristics, which are notably distinct between the two proteins. Differences in amplitude, kinetics, and voltage dependence of the transport-associated currents have been observed, as well as different substrate selectivity patterns measured by radioactive amino acid uptake assays. These diversities represent useful tools to investigate the structural determinants involved in the substrate selectivity. To identify these regions, we built four chimeric proteins between the two transporters. These proteins, heterologously expressed in Xenopus laevis oocytes, were analyzed by two-electrode voltage clamp and uptake measurements. Initially, we exchanged the first three domains, obtaining the chimeras C3K9 and K3C9 (where numbers indicate the transmembrane domains and letters represent the original proteins), which showed electrophysiological and [3H]amino acid uptake characteristics resembling those of KAAT1 and CAATCH1, respectively. Subsequent substitution of the last four domains in C3K9 and K3C9 gave the proteins C3K5C4 and K3C5K4, which showed the same behavior as KAAT1 and CAATCH1 in electrophysiological and transport determinations. These results suggest that in KAAT1 and CAATCH1, only the central transmembrane domains (from 4 to 8) of the protein are responsible for substrate selectivity.

Induction of Endothelial Tissue Factor by Endotoxin and Its Precursors

1993 ◽  
Vol 70 (04) ◽  
pp. 702-706 ◽  
Author(s):  
Charles F Moldow ◽  
Ronald R Bach ◽  
Katherine Staskus ◽  
Paul D Rick
Keyword(s):  
Tissue Factor ◽  
Lipid A ◽  
Umbilical Vein ◽  
Structural Determinants ◽  
Monophosphoryl Lipid A ◽  
Maximal Induction ◽  
Umbilical Vein Endothelial Cells ◽  
Acyloxyacyl Hydrolase ◽  
Endothelial Tissue ◽  
Specific Mrna

SummaryThe structural determinants of lipopolysaccharide required for the induction of tissue factor in human umbilical vein endothelial cells were studied. Intact lipid A was essential for the induction of tissue factor whereas the incomplete lipid A precursors lipid IVA and lipid X, as well as monophosphoryl lipid A and acyloxyacyl hydrolase-treated lipopolysaccharide, were unable to induce tissue factor and tissue factor specific mRNA. However, the lipid A precursor, lipid IVA, was able to inhibit LPS-mediated induction of tissue factor; structural determinants distal to lipid A were found to be required for maximal induction of tissue factor activity and tissue factor mRNA. The presence of serum in the assay was found to amplify but was not obligate for tissue factor induction by LPS.

Structural Determinants of Homelessness in the United States

1991 ◽  
Vol 38 (1) ◽  
pp. 113-131 ◽  
Author(s):  
Marta Elliott ◽  
Lauren J. Krivo
Keyword(s):  
United States ◽  
The United States ◽  
Structural Determinants

Social Justice and Dietetic Education: Are We Preparing Practitioners to Lead?

2020 ◽  
Vol 81 (3) ◽  
pp. 120-126
Author(s):  
Jennifer Brady
Keyword(s):  
Social Justice ◽  
Education And Training ◽  
Determinants Of Health ◽  
Structural Determinants ◽  
Social Justice Advocacy ◽  
Dietetic Education ◽  
Qualitative Survey ◽  
The Social ◽  
Reflexive Practice ◽  
And Training

Purpose: To explore dietetic practitioners’ perceptions of their education and training in the knowledge, skills, and confidence to understand social justice issues and to engage in socially just dietetic practice and social justice advocacy. Methods: An online semi-qualitative survey sent to Canadian dietitians. Results: Most respondents (n = 264; 81.5%) felt that knowledge- and skill-based learning about social justice and social justice advocacy should be a part of dietetic education and training. Reasons given by respondents for the importance of social justice learning include: client-centred care and reflexive practice, effecting change to the social and structural determinants of health, preventing dietitian burnout, and relevance of the profession. Yet, over half of respondents either strongly disagreed or disagreed that they were adequately prepared with the knowledge (n = 186; 57.4%), skills (n = 195; 60.2%), or confidence (n = 196; 60.5%) to engage in advocacy related to social justice concerns. Some questioned the practicality of adding social justice learning via additional courses to already full programs, while others proposed infusing a social justice lens across dietetic education and practice areas. Conclusions: Dietetic education and training must do more to prepare dietitians to answer calls for dietitians to engage in social justice issues through practice and advocacy.

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