Structural and catalytic roles of amino acid residues located at substrate-binding pocket in Fibrobacter succinogenes 1,3-1,4-β-D -glucanase

2010 ◽  
Vol 78 (13) ◽  
pp. 2820-2830 ◽  
Author(s):  
Je-Hsin Chen ◽  
Li-Chu Tsai ◽  
Hsiao-Chuan Huang ◽  
Lie-Fen Shyur
Keyword(s):  
Amino Acid ◽  
Substrate Binding ◽  
Binding Pocket ◽  
Fibrobacter Succinogenes ◽  
Amino Acid Residues ◽  
Substrate Binding Pocket

scholarly journals New members of the brachyurins family in lobster include a trypsin-like enzyme with amino acid substitutions in the substrate-binding pocket

FEBS Journal ◽  
2010 ◽  
Vol 277 (17) ◽  
pp. 3489-3501 ◽  
Author(s):  
Erick Perera ◽  
Tirso Pons ◽  
Damir Hernandez ◽  
Francisco J. Moyano ◽  
Gonzalo Martínez-Rodríguez ◽  
...  
Keyword(s):  
Amino Acid ◽  
Substrate Binding ◽  
Binding Pocket ◽  
Amino Acid Substitutions ◽  
Substrate Binding Pocket ◽  
New Members

Structural basis for substrate specificity of heteromeric transporters of neutral amino acids

2021 ◽  
Vol 118 (49) ◽  
pp. e2113573118
Author(s):  
Carlos F. Rodriguez ◽  
Paloma Escudero-Bravo ◽  
Lucía Díaz ◽  
Paola Bartoccioni ◽  
Carmen García-Martín ◽  
...  
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Substrate Specificity ◽  
Molecular Mechanisms ◽  
Substrate Binding ◽  
Binding Pocket ◽  
Neutral Amino Acid ◽  
Substrate Preference ◽  
Structural Basis ◽  
Substrate Binding Pocket

Despite having similar structures, each member of the heteromeric amino acid transporter (HAT) family shows exquisite preference for the exchange of certain amino acids. Substrate specificity determines the physiological function of each HAT and their role in human diseases. However, HAT transport preference for some amino acids over others is not yet fully understood. Using cryo–electron microscopy of apo human LAT2/CD98hc and a multidisciplinary approach, we elucidate key molecular determinants governing neutral amino acid specificity in HATs. A few residues in the substrate-binding pocket determine substrate preference. Here, we describe mutations that interconvert the substrate profiles of LAT2/CD98hc, LAT1/CD98hc, and Asc1/CD98hc. In addition, a region far from the substrate-binding pocket critically influences the conformation of the substrate-binding site and substrate preference. This region accumulates mutations that alter substrate specificity and cause hearing loss and cataracts. Here, we uncover molecular mechanisms governing substrate specificity within the HAT family of neutral amino acid transporters and provide the structural bases for mutations in LAT2/CD98hc that alter substrate specificity and that are associated with several pathologies.

scholarly journals Amino acid residue 247 in canine sulphotransferase SULT1D1: a new determinant of substrate selectivity

2004 ◽  
Vol 378 (2) ◽  
pp. 687-692 ◽  
Author(s):  
Carrie TSOI ◽  
Mikael WIDERSTEN ◽  
Ralf MORGENSTERN ◽  
Stellan SWEDMARK
Keyword(s):  
Amino Acid ◽  
Critical Role ◽  
Fold Increase ◽  
Binding Pocket ◽  
Amino Acid Residues ◽  
Wild Type ◽  
Substrate Binding Pocket ◽  
Site Selectivity ◽  
Km Value ◽  
Exogenous Compounds

The SULT (sulphotransferase) family plays a critical role in the detoxification and activation of endogenous and exogenous compounds as well as in the regulation of steroid hormone actions and neurotransmitter functions. The structure–activity relationships of the human SULTs have been investigated with focus on the amino acid 146 in hSULT1A3 and its impact on dopamine/PNP (p-nitrophenol) specificity. In the present study, we have generated canine SULT1D1 (cSULT1D1) variants with mutations at amino acid residues in the substrate-binding pocket [A146E (Ala-146→Glu), A146D, A146Q, I86D or D247L]. These mutation sites were chosen with regard to their possible contribution to the marked dopamine/PNP preference of cSULT1D1. After characterization, we found that the overall sulphation efficiencies for the cSULT1D1 A146 and the I86 mutants were strongly decreased for both substrates compared with wild-type cSULT1D1 but the substrate preference was unchanged. In contrast, the D247L mutant was found to be more than 21-fold better at sulphating PNP (120-fold decrease in Km value) but 54-fold less efficient in sulphating dopamine (8-fold increase in Km value) and the preference was switched from dopamine to PNP, indicating the importance of this amino acid in the dopamine/PNP preference in cSULT1D1. Our results show that Asp-247 has a pronounced effect on the substrate specificity of cSULT1D1 and thus we have identified a previously unrecognized contributor to active-site selectivity.

scholarly journals Aromatic Amino Acid Mutagenesis at the Substrate Binding Pocket ofYarrowia lipolyticaLipase Lip2 Affects Its Activity and Thermostability

2014 ◽  
Vol 2014 ◽  
pp. 1-8 ◽  
Author(s):  
Guilong Wang ◽  
Zimin Liu ◽  
Li Xu ◽  
Yunjun Yan
Keyword(s):  
Amino Acid ◽  
Aromatic Amino Acid ◽  
Substrate Binding ◽  
Binding Pocket ◽  
Enzymatic Properties ◽  
Site Directed Mutagenesis ◽  
Wild Type ◽  
Substrate Binding Pocket ◽  
Significant Difference ◽  
Amino Acid Mutations

The lipase2 fromYarrowia lipolytica(YLLip2) is a yeast lipase exhibiting high homologous to filamentous fungal lipase family. Though its crystal structure has been resolved, its structure-function relationship has rarely been reported. By contrast, there are two amino acid residues (V94 and I100) with significant difference in the substrate binding pocket of YLLip2; they were subjected to site-directed mutagenesis (SDM) to introduce aromatic amino acid mutations. Two mutants (V94W and I100F) were created. The enzymatic properties of the mutant lipases were detected and compared with the wild-type. The activities of mutant enzymes dropped to some extent towardsp-nitrophenyl palmitate (pNPC16) and their optimum temperature was 35°C, which was 5°C lower than that of the wild-type. However, the thermostability of I100F increased 22.44% after incubation for 1 h at 40°C and its optimum substrate shifted fromp-nitrophenyl laurate (pNPC12) top-nitrophenyl caprate (pNPC10). The above results demonstrated that the two substituted amino acid residuals have close relationship with such enzymatic properties as thermostability and substrate selectivity.

Efficient enzymatic synthesis of α-keto acids by redesigned substrate-binding pocket of the l-amino acid deaminase (PmiLAAD)

2020 ◽  
Vol 132 ◽  
pp. 109393 ◽  
Author(s):  
Licheng Wu ◽  
Xiaolei Guo ◽  
Gaobing Wu ◽  
Pengfu Liu ◽  
Ziduo Liu
Keyword(s):  
Amino Acid ◽  
Enzymatic Synthesis ◽  
Substrate Binding ◽  
Binding Pocket ◽  
Substrate Binding Pocket ◽  
Keto Acids

scholarly journals Role of a Conserved Membrane Glycine Residue in a Dicarboxylate Transporter from Sinorhizobium meliloti

2006 ◽  
Vol 189 (5) ◽  
pp. 2160-2163 ◽  
Author(s):  
Maria A. Trainer ◽  
Svetlana N. Yurgel ◽  
Michael L. Kahn
Keyword(s):  
Amino Acid ◽  
Sinorhizobium Meliloti ◽  
Substrate Binding ◽  
Binding Pocket ◽  
Significant Activity ◽  
Nitrogen Fixing ◽  
Substrate Binding Pocket ◽  
Glycine Residue

ABSTRACT Nitrogen-fixing rhizobial bacteroids import dicarboxylates by using the DctA transporter. G114 of DctA is highly conserved. A G114D mutant is inactive, but DctA with a small amino acid (G114A) or a helix disrupter (G114P) retains significant activity. G114 probably interacts with other membrane helices in stabilizing a substrate-binding pocket.

Chitosanase from Streptomyces sp. strain N174: a comparative review of its structure and function

1997 ◽  
Vol 75 (6) ◽  
pp. 687-696 ◽  
Author(s):  
Tamo Fukamizo ◽  
Ryszard Brzezinski
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Substrate Binding ◽  
Structure And Function ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Amino Acid Residues ◽  
Streptomyces Sp ◽  
Binding Cleft ◽  
And Function

Novel information on the structure and function of chitosanase, which hydrolyzes the beta -1,4-glycosidic linkage of chitosan, has accumulated in recent years. The cloning of the chitosanase gene from Streptomyces sp. strain N174 and the establishment of an efficient expression system using Streptomyces lividans TK24 have contributed to these advances. Amino acid sequence comparisons of the chitosanases that have been sequenced to date revealed a significant homology in the N-terminal module. From energy minimization based on the X-ray crystal structure of Streptomyces sp. strain N174 chitosanase, the substrate binding cleft of this enzyme was estimated to be composed of six monosaccharide binding subsites. The hydrolytic reaction takes place at the center of the binding cleft with an inverting mechanism. Site-directed mutagenesis of the carboxylic amino acid residues that are conserved revealed that Glu-22 and Asp-40 are the catalytic residues. The tryptophan residues in the chitosanase do not participate directly in the substrate binding but stabilize the protein structure by interacting with hydrophobic and carboxylic side chains of the other amino acid residues. Structural and functional similarities were found between chitosanase, barley chitinase, bacteriophage T4 lysozyme, and goose egg white lysozyme, even though these proteins share no sequence similarities. This information can be helpful for the design of new chitinolytic enzymes that can be applied to carbohydrate engineering, biological control of phytopathogens, and other fields including chitinous polysaccharide degradation. Key words: chitosanase, amino acid sequence, overexpression system, reaction mechanism, site-directed mutagenesis.

scholarly journals Crystal structure of steroid reductase SRD5A reveals conserved steroid reduction mechanism

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Yufei Han ◽  
Qian Zhuang ◽  
Bo Sun ◽  
Wenping Lv ◽  
Sheng Wang ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Biochemical Characterization ◽  
Substrate Binding ◽  
Binding Pocket ◽  
Substrate Binding Pocket ◽  
Transmembrane Segments ◽  
Therapeutic Molecules ◽  
Binding Cavity ◽  
Immune System Regulation ◽  
Mediated Reduction

AbstractSteroid hormones are essential in stress response, immune system regulation, and reproduction in mammals. Steroids with 3-oxo-Δ4 structure, such as testosterone or progesterone, are catalyzed by steroid 5α-reductases (SRD5As) to generate their corresponding 3-oxo-5α steroids, which are essential for multiple physiological and pathological processes. SRD5A2 is already a target of clinically relevant drugs. However, the detailed mechanism of SRD5A-mediated reduction remains elusive. Here we report the crystal structure of PbSRD5A from Proteobacteria bacterium, a homolog of both SRD5A1 and SRD5A2, in complex with the cofactor NADPH at 2.0 Å resolution. PbSRD5A exists as a monomer comprised of seven transmembrane segments (TMs). The TM1-4 enclose a hydrophobic substrate binding cavity, whereas TM5-7 coordinate cofactor NADPH through extensive hydrogen bonds network. Homology-based structural models of HsSRD5A1 and -2, together with biochemical characterization, define the substrate binding pocket of SRD5As, explain the properties of disease-related mutants and provide an important framework for further understanding of the mechanism of NADPH mediated steroids 3-oxo-Δ4 reduction. Based on these analyses, the design of therapeutic molecules targeting SRD5As with improved specificity and therapeutic efficacy would be possible.

INFLUENCE OF G187W/K188F/D189Y MUTATION IN THE SUBSTRATE BINDING POCKET OF TRYPSIN ON ?-CASEIN PROCESSING

1998 ◽  
Vol 22 (6) ◽  
pp. 529-545 ◽  
Author(s):  
JEAN-MARC CHOBERT ◽  
LOIC BRIAND ◽  
THOMAS HAERTLE
Keyword(s):  
Substrate Binding ◽  
Binding Pocket ◽  
Substrate Binding Pocket

Characterization of the Acyl Substrate Binding Pocket of Acetyl-CoA Synthetase†

Biochemistry ◽  
2006 ◽  
Vol 45 (38) ◽  
pp. 11482-11490 ◽  
Author(s):  
Cheryl Ingram-Smith ◽  
Barrett I. Woods ◽  
Kerry S. Smith
Keyword(s):  
Substrate Binding ◽  
Binding Pocket ◽  
Substrate Binding Pocket ◽  
Acetyl Coa
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