scholarly journals Structural basis for substrate binding and specificity of a sodium/alanine symporter AgcS

2018 ◽  
Author(s):  
Jinming Ma ◽  
Hsiang-Ting Lei ◽  
Francis E. Reyes ◽  
Silvia Sanchez-Martinez ◽  
Maen Sarhan ◽  
...  
Keyword(s):  
Organic Molecules ◽  
Substrate Binding ◽  
Structural Features ◽  
Site Directed Mutagenesis ◽  
Structural Basis ◽  
Substrate Selectivity ◽  
Methanococcus Maripaludis ◽  
Functional Studies ◽  
Stereo Selectivity ◽  
Key Residues

AbstractThe amino acid, polyamine, and organocation (APC) superfamily is the second largest superfamily of membrane proteins forming secondary transporters that move a range of organic molecules across the cell membrane. Each transporter in APC superfamily is specific for a unique sub-set of substrates, even if they possess a similar structural fold. The mechanism of substrate selectivity remains, by and large, elusive. Here we report two crystal structures of an APC member from Methanococcus maripaludis, the alanine or glycine:cation symporter (AgcS), with L- or D-alanine bound. Structural analysis combined with site-directed mutagenesis and functional studies inform on substrate binding, specificity, and modulation of the AgcS family and reveal key structural features that allow this transporter to accommodate glycine and alanine while excluding all other amino acids. Mutation of key residues in the substrate binding site expand the selectivity to include valine and leucine. Moreover, as a transporter that binds both enantiomers of alanine, the present structures provide an unprecedented opportunity to gain insights into the mechanism of stereo-selectivity in APC transporters.

scholarly journals Structural basis for substrate binding and specificity of a sodium–alanine symporter AgcS

2019 ◽  
Vol 116 (6) ◽  
pp. 2086-2090 ◽  
Author(s):  
Jinming Ma ◽  
Hsiang-Ting Lei ◽  
Francis E. Reyes ◽  
Silvia Sanchez-Martinez ◽  
Maen F. Sarhan ◽  
...  
Keyword(s):  
Organic Molecules ◽  
Substrate Binding ◽  
Structural Features ◽  
Site Directed Mutagenesis ◽  
Structural Basis ◽  
Directed Mutagenesis ◽  
Substrate Selectivity ◽  
Methanococcus Maripaludis ◽  
Functional Studies ◽  
Key Residues

The amino acid, polyamine, and organocation (APC) superfamily is the second largest superfamily of membrane proteins forming secondary transporters that move a range of organic molecules across the cell membrane. Each transporter in the APC superfamily is specific for a unique subset of substrates, even if they possess a similar structural fold. The mechanism of substrate selectivity remains, by and large, elusive. Here, we report two crystal structures of an APC member fromMethanococcus maripaludis, the alanine or glycine:cation symporter (AgcS), withl- ord-alanine bound. Structural analysis combined with site-directed mutagenesis and functional studies inform on substrate binding, specificity, and modulation of the AgcS family and reveal key structural features that allow this transporter to accommodate glycine and alanine while excluding all other amino acids. Mutation of key residues in the substrate binding site expand the selectivity to include valine and leucine. These studies provide initial insights into substrate selectivity in AgcS symporters.

scholarly journals Structural basis for divergent and convergent evolution of catalytic machineries in plant aromatic amino acid decarboxylase proteins

2020 ◽  
Vol 117 (20) ◽  
pp. 10806-10817 ◽  
Author(s):  
Michael P. Torrens-Spence ◽  
Ying-Chih Chiang ◽  
Tyler Smith ◽  
Maria A. Vicent ◽  
Yi Wang ◽  
...  
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Convergent Evolution ◽  
Structural Features ◽  
Divergent Evolution ◽  
Structural Basis ◽  
Acid Decarboxylase ◽  
Substrate Selectivity ◽  
Amino Acid Decarboxylase ◽  
Catalytic Mechanisms

Radiation of the plant pyridoxal 5′-phosphate (PLP)-dependent aromatic l-amino acid decarboxylase (AAAD) family has yielded an array of paralogous enzymes exhibiting divergent substrate preferences and catalytic mechanisms. Plant AAADs catalyze either the decarboxylation or decarboxylation-dependent oxidative deamination of aromatic l-amino acids to produce aromatic monoamines or aromatic acetaldehydes, respectively. These compounds serve as key precursors for the biosynthesis of several important classes of plant natural products, including indole alkaloids, benzylisoquinoline alkaloids, hydroxycinnamic acid amides, phenylacetaldehyde-derived floral volatiles, and tyrosol derivatives. Here, we present the crystal structures of four functionally distinct plant AAAD paralogs. Through structural and functional analyses, we identify variable structural features of the substrate-binding pocket that underlie the divergent evolution of substrate selectivity toward indole, phenyl, or hydroxyphenyl amino acids in plant AAADs. Moreover, we describe two mechanistic classes of independently arising mutations in AAAD paralogs leading to the convergent evolution of the derived aldehyde synthase activity. Applying knowledge learned from this study, we successfully engineered a shortened benzylisoquinoline alkaloid pathway to produce (S)-norcoclaurine in yeast. This work highlights the pliability of the AAAD fold that allows change of substrate selectivity and access to alternative catalytic mechanisms with only a few mutations.

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.

Crystal structures and functional studies clarify substrate selectivity and catalytic residues for the unique orphan enzyme N-acetyl-D-mannosamine dehydrogenase

2014 ◽  
Vol 462 (3) ◽  
pp. 499-511 ◽  
Author(s):  
Agustín Sola-Carvajal ◽  
Fernando Gil-Ortiz ◽  
Francisco García-Carmona ◽  
Vicente Rubio ◽  
Álvaro Sánchez-Ferrer
Keyword(s):  
Substrate Specificity ◽  
Crystal Structures ◽  
Functional Site ◽  
Site Directed Mutagenesis ◽  
Short Chain ◽  
Directed Mutagenesis ◽  
Substrate Selectivity ◽  
Functional Studies ◽  
Short Chain Dehydrogenase ◽  
Orphan Enzyme

Functional site-directed mutagenesis and crystallographic studies with N-acetyl-D-mannosamine dehydrogenase reveal a homotetramer, a short-chain dehydrogenase/reductase subunit fold and a highly developed C-terminal tail interlinking subunit. The structures of the complexes explain substrate specificity. The four residues forming the catalytic tetrad are identified.

Probing the structural basis of oxygen binding in a cofactor-independent dioxygenase

2017 ◽  
Vol 73 (7) ◽  
pp. 573-580 ◽  
Author(s):  
Kunhua Li ◽  
Elisha N. Fielding ◽  
Heather L. Condurso ◽  
Steven D. Bruner
Keyword(s):  
Catalytic Mechanism ◽  
Binding Pocket ◽  
Binding Mode ◽  
Site Directed Mutagenesis ◽  
Structural Basis ◽  
General Mechanism ◽  
Oxygen Binding ◽  
Migration Pathway ◽  
And Migration ◽  
Key Residues

The enzyme DpgC is included in the small family of cofactor-independent dioxygenases. The chemistry of DpgC is uncommon as the protein binds and utilizes dioxygen without the aid of a metal or organic cofactor. Previous structural and biochemical studies identified the substrate-binding mode and the components of the active site that are important in the catalytic mechanism. In addition, the results delineated a putative binding pocket and migration pathway for the co-substrate dioxygen. Here, structural biology is utilized, along with site-directed mutagenesis, to probe the assigned dioxygen-binding pocket. The key residues implicated in dioxygen trafficking were studied to probe the process of binding, activation and chemistry. The results support the proposed chemistry and provide insight into the general mechanism of dioxygen binding and activation.

scholarly journals Structural basis for diamide modulation of ryanodine receptor

2021 ◽  
Vol 154 (9) ◽  
Author(s):  
Ruifang Ma ◽  
Omid Haji-Ghassemi ◽  
Dan Ma ◽  
Heng Jiang ◽  
Lianyun Lin ◽  
...  
Keyword(s):  
Mutant Cell ◽  
Diamondback Moth ◽  
Ryanodine Receptors ◽  
Dynamic Structure ◽  
Structural Features ◽  
Structural Basis ◽  
Loss Of Function ◽  
Resistance Mutations ◽  
A Site ◽  
Key Residues

Diamide insecticides target insect ryanodine receptors (RYRs) and cause dysregulation of calcium signaling in insect muscles and neurons, generating worldwide sales over 2 billion US dollars annually. Several resistance mutations have been reported to reduce the efficacy of the diamides, but the exact binding sites and mechanism of resistance mutations were not clear. Recently, we solved the cryo-electron microscopy (cryo-EM) structure of RYR in complex with the anthranilic diamide chlorantraniliprole (CHL). CHL binds to the pseudo–voltage-sensor domain (pVSD) of RYR, a site in proximity to the previously identified resistance mutations. Mutagenesis studies in silico, in mutant cell lines, and in transgenic Drosophila strains revealed the key residues involved in diamide coordination and the molecular mechanism under species-selectivity and resistance mutations. We also proposed that CHL may alleviate the loss-of-function effects of some central core disease (CCD) mutations by increasing the opening probability (Po) of RYR1. In addition, we solved the crystal structures of several RYR domains from the diamondback moth and the bee, revealing insect-specific structural features which could be potentially targeted by novel insecticides. Interestingly, we found that the phosphorylation of insect RYR is temperature dependent, facilitated by the low thermal stability and dynamic structure of the insect RYR. Our structures provide a foundation for developing novel pesticides to overcome the resistance crisis.

Direct neural induction and selective inhibition of mesoderm and epidermis inducers by Xnr3

Development ◽  
1997 ◽  
Vol 124 (2) ◽  
pp. 483-492 ◽  
Author(s):  
C.S. Hansen ◽  
C.D. Marion ◽  
K. Steele ◽  
S. George ◽  
W.C. Smith
Keyword(s):  
Family Members ◽  
Neural Induction ◽  
Structural Features ◽  
Site Directed Mutagenesis ◽  
Structural Basis ◽  
Mesoderm Induction ◽  
Related Factors ◽  
Spemann's Organizer ◽  
Secreted Factors ◽  
C Terminus

During gastrulation in amphibians, secreted factors from Spemann's organizer act on dorsal ectoderm to induce the central nervous system. A number of secreted factors produced by Spemann's organizer have recently been identified. The TGFbeta family member Xnr3 is similar in amino acid sequence to the mouse factor nodal and is expressed in a restricted group of cells in the superficial layer of Spemann's organizer. Xnr3, unlike the related factors nodal, Xnr1 and Xnr2, lacks mesoderm-inducing activity. We report here that Xnr3 can directly induce neural tissue in Xenopus ectoderm explants (animal caps). Injection of animal caps with either Xnr3 RNA or plasmids induces the expression of the pan-neural genes NCAM and nrp1, as well as the anterior neural marker Cpl1. A growing body of evidence suggests that neural induction in Xenopus proceeds as the default in the absence of epidermis inducers. The best candidates for the endogenous epidermis inducers are BMP-4 and BMP-7. The neural inducing activity of Xnr3 can be inhibited by overexpression of BMP-4, as has been observed with the neural inducers noggin, chordin and follistatin. Furthermore, Xnr3 can block mesoderm induction by BMP-4 and activin, but not by Xnr2. The structural basis underlying the divergent activities of Xnr2 and Xnr3 was analyzed using site-directed mutagenesis. Mutations introduced to the conserved cysteine residues characteristic of the TGFbeta family were found to inactivate Xnr2, but not Xnr3. The most unique feature of Xnr3 is the absence of a conserved cysteine at the C terminus of the protein. This feature distinguishes Xnr3 from other TGFbeta family members, including Xnr2. However, we observed that changing the C terminus of Xnr3 to more closely resemble other TGFbeta family members did not significantly alter its activity, suggesting that other structural features of Xnr3 distinguish its biological activity from Xnr2.

scholarly journals Structural basis of glycerophosphodiester recognition by the Mycobacterium tuberculosis substrate-binding protein UgpB

2019 ◽  
Author(s):  
Jonathan Fenn ◽  
Ridvan Nepravishta ◽  
Collette S Guy ◽  
James Harrison ◽  
Jesus Angulo ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Potential Role ◽  
Substrate Binding ◽  
Site Directed Mutagenesis ◽  
Structural Basis ◽  
X Ray Crystallography ◽  
Biochemical Analyses ◽  
Substrate Binding Domain ◽  
Substrate Binding Protein

AbstractMycobacterium tuberculosis (Mtb) is the causative agent of tuberculosis (TB) and has evolved an incredible ability to survive latently within the human host for decades. The Mtb pathogen encodes for a low number of ATP-binding cassette (ABC) importers for the acquisition of carbohydrates that may reflect the nutrient poor environment within the host macrophages. Mtb UgpB (Rv2833) is the substrate binding domain of the UgpABCE transporter that recognises glycerophosphocholine (GPC) indicating a potential role in glycerophospholipid recycling. By using a combination of saturation transfer difference (STD) NMR and X-ray crystallography we report the structural analysis of Mtb UgpB complexed with GPC and have identified that Mtb UgpB is promiscuous for other glycerophosphodiesters. Complementary biochemical analyses and site-directed mutagenesis define the molecular basis and specificity of glycerophosphodiester recognition. Our results provide critical insights into the structural and functional role of the Mtb UgpB transporter and reveal that the specificity of this ABC-transporter is not limited to GPC therefore optimising the ability of Mtb to scavenge scarce nutrients and essential glycerophospholipid metabolites during intracellular infection.

scholarly journals Structure and function of stator units of the bacterial flagellar motor

2020 ◽  
Author(s):  
Mònica Santiveri ◽  
Aritz Roa-Eguiara ◽  
Caroline Kühne ◽  
Navish Wadhwa ◽  
Howard C. Berg ◽  
...  
Keyword(s):  
Conformational Changes ◽  
Mechanistic Model ◽  
Structural Data ◽  
Structural Basis ◽  
Flagellar Motor ◽  
Functional Studies ◽  
Torque Generation ◽  
Bacterial Flagellar Motor ◽  
And Function ◽  
Key Residues

AbstractMany bacteria use the flagellum for locomotion and chemotaxis. Its bi-directional rotation is driven by the membrane-embedded motor, which uses energy from the transmembrane ion gradient to generate torque at the interface between stator units and rotor. The structural organization of the stator unit (MotAB), its conformational changes upon ion transport and how these changes power rotation of the flagellum, remain unknown. Here we present ~3 Å-resolution cryo-electron microscopy reconstructions of the stator unit in different functional states. We show that the stator unit consists of a dimer of MotB surrounded by a pentamer of MotA. Combining structural data with mutagenesis and functional studies, we identify key residues involved in torque generation and present a mechanistic model for motor function and switching of rotational direction.One Sentence SummaryStructural basis of torque generation in the bidirectional bacterial flagellar motor

scholarly journals Structural basis of the stereoselective formation of the spirooxindole ring in the biosynthesis of citrinadins

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Zhiwen Liu ◽  
Fanglong Zhao ◽  
Boyang Zhao ◽  
Jie Yang ◽  
Joseph Ferrara ◽  
...  
Keyword(s):  
High Resolution ◽  
Organic Synthesis ◽  
Indole Alkaloids ◽  
Site Directed Mutagenesis ◽  
Structural Basis ◽  
Directed Mutagenesis ◽  
Computational Studies ◽  
X Ray ◽  
Evolutionary Branch ◽  
Catalytic Mechanisms

AbstractPrenylated indole alkaloids featuring spirooxindole rings possess a 3R or 3S carbon stereocenter, which determines the bioactivities of these compounds. Despite the stereoselective advantages of spirooxindole biosynthesis compared with those of organic synthesis, the biocatalytic mechanism for controlling the 3R or 3S-spirooxindole formation has been elusive. Here, we report an oxygenase/semipinacolase CtdE that specifies the 3S-spirooxindole construction in the biosynthesis of 21R-citrinadin A. High-resolution X-ray crystal structures of CtdE with the substrate and cofactor, together with site-directed mutagenesis and computational studies, illustrate the catalytic mechanisms for the possible β-face epoxidation followed by a regioselective collapse of the epoxide intermediate, which triggers semipinacol rearrangement to form the 3S-spirooxindole. Comparing CtdE with PhqK, which catalyzes the formation of the 3R-spirooxindole, we reveal an evolutionary branch of CtdE in specific 3S spirocyclization. Our study provides deeper insights into the stereoselective catalytic machinery, which is important for the biocatalysis design to synthesize spirooxindole pharmaceuticals.

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