Structure, dynamics and function of the evolutionarily changing biliverdin reductase B family

Abstract Biliverdin reductase B (BLVRB) family members are general flavin reductases critical in maintaining cellular redox with recent findings revealing that BLVRB alone can dictate cellular fate. However, as opposed to most enzymes, the BLVRB family remains enigmatic with an evolutionarily changing active site and unknown structural and functional consequences. Here, we applied a multi-faceted approach that combines X-ray crystallography, NMR and kinetics methods to elucidate the structural and functional basis of the evolutionarily changing BLVRB active site. Using a panel of three BLVRB isoforms (human, lemur and hyrax) and multiple human BLVRB mutants, our studies reveal a novel evolutionary mechanism where coenzyme ‘clamps’ formed by arginine side chains at two co-evolving positions within the active site serve to slow coenzyme release (Positions 14 and 78). We find that coenzyme release is further slowed by the weaker binding substrate, resulting in relatively slow turnover numbers. However, different BLVRB active sites imposed by either evolution or mutagenesis exhibit a surprising inverse relationship between coenzyme release and substrate turnover that is independent of the faster chemical step of hydride transfer also measured here. Collectively, our studies have elucidated the role of the evolutionarily changing BLVRB active site that serves to modulate coenzyme release and has revealed that coenzyme release is coupled to substrate turnover.

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Role of Residues W228 and Y233 in the Structure and Activity of Metallo-β-Lactamase GIM-1

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.02017-15 ◽

2015 ◽

Vol 60 (2) ◽

pp. 990-1002 ◽

Cited By ~ 7

Author(s):

Susann Skagseth ◽

Trine Josefine Carlsen ◽

Gro Elin Kjæreng Bjerga ◽

James Spencer ◽

Ørjan Samuelsen ◽

...

Keyword(s):

Active Site ◽

Active Sites ◽

Point Mutations ◽

Resistant Bacteria ◽

Antibiotic Resistant ◽

X Ray Crystallography ◽

Steady State Kinetics ◽

Hydrophilic Residues

ABSTRACTMetallo-β-lactamases (MBLs) hydrolyze virtually all β-lactam antibiotics, including penicillins, cephalosporins, and carbapenems. The worldwide emergence of antibiotic-resistant bacteria harboring MBLs poses an increasing clinical threat. The MBL German imipenemase-1 (GIM-1) possesses an active site that is narrower and more hydrophobic than the active sites of other MBLs. The GIM-1 active-site groove is shaped by the presence of the aromatic side chains of tryptophan at residue 228 and tyrosine at residue 233, positions where other MBLs harbor hydrophilic residues. To investigate the importance of these two residues, eight site-directed mutants of GIM-1, W228R/A/Y/S and Y233N/A/I/S, were generated and characterized using enzyme kinetics, thermostability assays, and determination of the MICs of representative β-lactams. The structures of selected mutants were obtained by X-ray crystallography, and their interactions with β-lactam substrates were modeledin silico. Steady-state kinetics revealed that both positions are important to GIM-1 activity but that the effects of individual mutations vary depending on the β-lactam substrate. Activity against type 1 substrates bearing electron-donating C-3/C-4 substituents (cefoxitin, meropenem) could be enhanced by mutations at position 228, whereas hydrolysis of type 2 substrates (benzylpenicillin, ampicillin, ceftazidime, imipenem) with methyl or positively charged substituents was favored by mutations at position 233. The crystal structures showed that mutations at position 228 or the Y233A variant alters the conformation of GIM-1 loop L1 rather than that of loop L3, on which the mutations are located. Taken together, these data show that point mutations at both positions 228 and 233 can influence the catalytic properties and the structure of GIM-1.

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Allosteric effects in catalytic impaired variants of the enzyme cyclophilin A may be explained by changes in nano-microsecond time scale motions

10.1101/224329 ◽

2017 ◽

Cited By ~ 1

Author(s):

Pattama Wapeesittipan ◽

Antonia S. J. S. Mey ◽

Malcolm D. Walkinshaw ◽

Julien Michel

Keyword(s):

Active Site ◽

Time Scale ◽

Cyclophilin A ◽

Conformational Exchange ◽

Wild Type ◽

X Ray Crystallography ◽

Simulation Techniques ◽

Dynamics And Function ◽

A Minor ◽

And Function

AbstractThere is much debate about the mechanisms by which molecular motions influence catalysis in enzymes. This work investigates the connection between stochastic protein dynamics and function for the enzyme cyclophilin A (CypA) in wild-type (WT) form, and three variants that features several mutations that are distal from the active site. Previous biophysical studies have suggested that conformational exchange between a ‘major’ active and a ‘minor’ inactive state on millisecond time scales plays a key role in catalysis for CypA. Here this hypothesis was addressed by a variety of molecular dynamic (MD) simulation techniques. The simulations reproduce X-ray crystallography derived evidence for a shift in populations of major and minor active site conformations between the wild-type and mutant forms. Strikingly, exchange between these active site conformations occurs at a rate that is 5 to 6 orders of magnitude faster than previously proposed. Further analyses indicate that the minor active site conformation is catalytically impaired, and that decreased catalytic activity of the mutants may be explained by changes in Phe113 motions on a ns-μs time scale. Therefore previously described millisecond time scale motions may not be necessary to explain allosteric effects in CypA mutants.

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Prediction of Functional Consequences of Missense Mutations in ANO4 Gene

International Journal of Molecular Sciences ◽

10.3390/ijms22052732 ◽

2021 ◽

Vol 22 (5) ◽

pp. 2732

Author(s):

Nadine Reichhart ◽

Vladimir M. Milenkovic ◽

Christian H. Wetzel ◽

Olaf Strauß

Keyword(s):

Transmembrane Protein ◽

Functional Characterization ◽

Missense Mutations ◽

Mutant Proteins ◽

Functional Consequences ◽

New Perspective ◽

The Stability ◽

Dynamics Simulations ◽

And Function

The anoctamin (TMEM16) family of transmembrane protein consists of ten members in vertebrates, which act as Ca2+-dependent ion channels and/or Ca2+-dependent scramblases. ANO4 which is primarily expressed in the CNS and certain endocrine glands, has been associated with various neuronal disorders. Therefore, we focused our study on prioritizing missense mutations that are assumed to alter the structure and stability of ANO4 protein. We employed a wide array of evolution and structure based in silico prediction methods to identify potentially deleterious missense mutations in the ANO4 gene. Identified pathogenic mutations were then mapped to the modeled human ANO4 structure and the effects of missense mutations were studied on the atomic level using molecular dynamics simulations. Our data show that the G80A and A500T mutations significantly alter the stability of the mutant proteins, thus providing new perspective on the role of missense mutations in ANO4 gene. Results obtained in this study may help to identify disease associated mutations which affect ANO4 protein structure and function and might facilitate future functional characterization of ANO4.

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Characterization of anEscherichia coliSulfite Oxidase Homologue Reveals the Role of a Conserved Active Site Cysteine in Assembly and Function†

Biochemistry ◽

10.1021/bi050621a ◽

2005 ◽

Vol 44 (30) ◽

pp. 10339-10348 ◽

Cited By ~ 51

Author(s):

Stephen J. Brokx ◽

Richard A. Rothery ◽

Guijin Zhang ◽

Derek P. Ng ◽

Joel H. Weiner

Keyword(s):

Active Site ◽

Active Site Cysteine ◽

And Function

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Role of the Heme Active Site and Protein Environment in Structure, Spectra, and Function of the Cytochrome P450s

Chemical Reviews ◽

10.1021/cr980389x ◽

2000 ◽

Vol 100 (2) ◽

pp. 407-420 ◽

Cited By ~ 164

Author(s):

Gilda H. Loew ◽

Danni L. Harris

Keyword(s):

Active Site ◽

Cytochrome P450s ◽

And Function ◽

Protein Environment

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On the role of lysine in the active site Ser-X-X-Lys region of penicillin-recognizing enzymes

Canadian Journal of Chemistry ◽

10.1139/v09-148 ◽

2010 ◽

Vol 88 (1) ◽

pp. 1-4

Author(s):

Saul Wolfe ◽

Kiyull Yang

Keyword(s):

Active Site ◽

Active Sites ◽

Density Functional ◽

Basis Set ◽

Penicillin G ◽

Hydroxyl Group ◽

Functional Theory ◽

One Step ◽

Optimized Geometries

Using Autodock, docking of penicillin G to the crystal structures of penicillin-recognizing enzymes leads to an alignment in the active site Ser-X-X-Lys region consisting of the serine hydroxyl group, the terminal amino group of lysine, a second hydroxyl group, and the N–C=O of the β-lactam. This alignment is consistent with the notion that acylation of the serine hydroxyl group proceeds by a one-step cooperative mechanism in which C–O bond formation and proton transfer to the β-lactam nitrogen take place through a heteroatom bridge. For the cooperative ring opening of penam by two molecules of methanol and one molecule of methylamine or one molecule of water, density functional theory with the B3LYP DFT gradient-corrected functional and the 6–31G(d) basis set reproduces the alignment seen in the docked structures. Methylamine lowers the barrier calculated at MP2/6–31G(d) from the DFT-optimized geometries by 3 kcal/mol; water increases the barrier by 4 kcal/mol. The function of the conserved lysine in the active sites of penicillin-recognizing enzymes is therefore to catalyze the formation of an acyl enzyme by a cooperative mechanism.

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ChemInform Abstract: Role of the Heme Active Site and Protein Environment in Structure, Spectra, and Function of the Cytochrome P450s

ChemInform ◽

10.1002/chin.200016274 ◽

2010 ◽

Vol 31 (16) ◽

pp. no-no

Author(s):

Gilda H. Loew ◽

Danni L. Harris

Keyword(s):

Active Site ◽

Cytochrome P450s ◽

And Function ◽

Protein Environment

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Modulating Enzyme Function via Dynamic Allostery within Biliverdin Reductase B

Frontiers in Molecular Biosciences ◽

10.3389/fmolb.2021.691208 ◽

2021 ◽

Vol 8 ◽

Author(s):

Jasmina S. Redzic ◽

Michael R. Duff ◽

Ashley Blue ◽

Todd M. Pitts ◽

Pratul Agarwal ◽

...

Keyword(s):

Active Site ◽

Redox Regulation ◽

Nmr Relaxation ◽

Functional Coupling ◽

Global Dynamics ◽

Biliverdin Reductase ◽

Coenzyme Binding ◽

Catalytic Function ◽

Order Of Magnitude ◽

And Function

The biliverdin reductase B (BLVRB) class of enzymes catalyze the NADPH-dependent reduction of multiple flavin substrates and are emerging as critical players in cellular redox regulation. However, the role of dynamics and allostery have not been addressed, prompting studies here that have revealed a position 15 Å away from the active site within human BLVRB (T164) that is inherently dynamic and can be mutated to control global micro-millisecond motions and function. By comparing the inherent dynamics through nuclear magnetic resonance (NMR) relaxation approaches of evolutionarily distinct BLVRB homologues and by applying our previously developed Relaxation And Single Site Multiple Mutations (RASSMM) approach that monitors both the functional and dynamic effects of multiple mutations to the single T164 site, we have discovered that the most dramatic mutagenic effects coincide with evolutionary changes and these modulate coenzyme binding. Thus, evolutionarily changing sites distal to the active site serve as dynamic “dials” to globally modulate motions and function. Despite the distal dynamic and functional coupling modulated by this site, micro-millisecond motions span an order of magnitude in their apparent kinetic rates of motions. Thus, global dynamics within BLVRB are a collection of partially coupled motions tied to catalytic function.

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The role of endothelium in factor Xa regulation: the effect of plasma proteinase inhibitors and hirudin

Blood ◽

10.1182/blood.v71.5.1321.1321 ◽

1988 ◽

Vol 71 (5) ◽

pp. 1321-1328 ◽

Cited By ~ 13

Author(s):

RC Friedberg ◽

PO Hagen ◽

SV Pizzo

Keyword(s):

Active Site ◽

Proteinase Inhibitor ◽

Human Factor ◽

Antithrombin Iii ◽

Proteinase Inhibitors ◽

Factor Xa ◽

Proteolytic Degradation ◽

Chromogenic Substrates ◽

And Function

Abstract The role of endothelium in the inhibition of human factor Xa was studied in a plasma environment. Human factor Xa can bind to and function on bovine aortic endothelium in a manner similar to that of bovine factor Xa. Approximately 70% of the bound factor Xa is subject to inhibition by plasma proteinase inhibitors, and the remaining 30% is irreversibly bound as part of a 125 Kd membrane-associated complex not subject to proteolytic degradation. The proportion reversibly bound and its rate of release do not alter with changes in calcium, citrate, heparin, or active proteinase inhibitor concentrations. The principal plasma proteinase inhibitor of human factor Xa was antithrombin III, which accounted for 60% to 65% of factor Xa released from endothelium, with alpha 1-proteinase inhibitor inactivating 20% to 25% and alpha 2- macroglobulin approximately 15%. All of the reversibly bound factor Xa was identified in complex with one of these three proteinase inhibitors. The thrombin active-site inhibitor hirudin was found to markedly accelerate the displacement of reversibly bound factor Xa from the endothelium and to associate specifically with factor Xa without a loss of activity toward chromogenic substrates, perhaps accounting for a novel mechanism of anticoagulation.

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