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 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

Expanding Substrate Specificity of ω-Transaminase by Rational Remodeling of a Large Substrate-Binding Pocket

2015 ◽  
Vol 357 (12) ◽  
pp. 2712-2720 ◽  
Author(s):  
Sang-Woo Han ◽  
Eul-Soo Park ◽  
Joo-Young Dong ◽  
Jong-Shik Shin
Keyword(s):  
Substrate Specificity ◽  
Substrate Binding ◽  
Binding Pocket ◽  
Substrate Binding Pocket

scholarly journals Towards a better understanding of the substrate specificity of the UDP-N-acetylglucosamine C4 epimerase WbpP

2005 ◽  
Vol 389 (1) ◽  
pp. 173-180 ◽  
Author(s):  
Melinda DEMENDI ◽  
Noboru ISHIYAMA ◽  
Joseph S. LAM ◽  
Albert M. BERGHUIS ◽  
Carole CREUZENET
Keyword(s):  
Enzyme Activity ◽  
Substrate Specificity ◽  
Molecular Basis ◽  
Substrate Binding ◽  
Structural Data ◽  
Binding Pocket ◽  
Substrate Binding Pocket ◽  
Expected Effect ◽  
Biochemical Assays ◽  
Golden Opportunity

WbpP is the only genuine UDP-GlcNAc (UDP-N-acetylglucosamine) C4 epimerase for which both biochemical and structural data are available. This represents a golden opportunity to elucidate the molecular basis for its specificity for N-acetylated substrates. Based on the comparison of the substrate binding site of WbpP with that of other C4 epimerases that convert preferentially non-acetylated substrates, or that are able to convert both acetylated and non-acetylated substrates equally well, specific residues of WbpP were mutated, and the substrate specificity of the mutants was determined by direct biochemical assays and kinetic analyses. Most of the mutations tested were anticipated to trigger a significant switch in substrate specificity, mostly towards a preference for non-acetylated substrates. However, only one of the mutations (A209H) had the expected effect, and most others resulted in enhanced specificity of WbpP for N-acetylated substrates (Q201E, G102K, Q201E/G102K, A209N and S143A). One mutation (S144K) totally abolished enzyme activity. These data indicate that, although all residues targeted in the present study turned out to be important for catalysis, determinants of substrate specificity are not confined to the substrate-binding pocket and that longer range interactions are essential in allowing proper positioning of various ligands in the binding pocket. Hence prediction or engineering of substrate specificity solely based on sequence analysis, or even on modelling of the binding pocket, might lead to incorrect functional assignments.

scholarly journals Molecular mechanisms of the anti-cancer drug, LY2874455, in overcoming the FGFR4 mutation-based resistance

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Fariba Dehghanian ◽  
Shahryar Alavi
Keyword(s):  
Molecular Mechanisms ◽  
Kinase Inhibitors ◽  
Substrate Binding ◽  
Growth Factor Receptor ◽  
Binding Pocket ◽  
Cancer Drug ◽  
Inhibition Mechanism ◽  
Wild Type ◽  
Substrate Binding Pocket ◽  
Anti Cancer

AbstractIn recent years, many strategies have been used to overcome the fibroblast growth factor receptor (FGFR) tyrosine kinase inhibitors (TKIs) resistance caused by different mutations. LY2874455 (or 6LF) is a pan-FGFR inhibitor which is identified as the most efficient TKI for all resistant mutations in FGFRs. Here, we perform a comparative dynamics study of wild type (WT) and the FGFR4 V550L mutant for better understanding of the 6LF inhibition mechanism. Our results confirm that the pan-FGFR inhibitor 6LF can bind efficiently to both WT and V550L FGFR4. Moreover, the communication network analysis indicates that in apo-WT FGFR4, αD–αE loop behaves like a switch between open and close states of the substrate-binding pocket in searching of its ligand. In contrast, V550L mutation induces the active conformation of the FGFR4 substrate-binding pocket through disruption of αD–αE loop and αG helix anti-correlation. Interestingly, 6LF binding causes the rigidity of hinge and αD helix regions, which results in overcoming V550L induced resistance. Collectively, the results of this study would be informative for designing more efficient TKIs for more effective targeting of the FGFR signaling pathway.

scholarly journals Mutagenesis of the l-Amino Acid Ligase RizA Increased the Production of Bioactive Dipeptides

Catalysts ◽  
2021 ◽  
Vol 11 (11) ◽  
pp. 1385
Author(s):  
Sven Bordewick ◽  
Ralf G. Berger ◽  
Franziska Ersoy
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Aspartic Acid ◽  
Product Yield ◽  
Binding Pocket ◽  
Site Directed Mutagenesis ◽  
Product Concentration ◽  
Substrate Binding Pocket ◽  
E Coli ◽  
Set Up

The l-amino acid ligase RizA from B. subtilis selectively synthesizes dipeptides containing an N-terminal arginine. Many arginyl dipeptides have salt-taste enhancing properties while Arg-Phe has been found to have an antihypertensive effect. A total of 21 RizA variants were created by site-directed mutagenesis of eight amino acids in the substrate binding pocket. The variants were recombinantly produced in E. coli and purified by affinity chromatography. Biocatalytic reactions were set up with arginine and four amino acids differing in size and polarity (aspartic acid, serine, alanine, and phenylalanine) and were analyzed by RP-HPLC with fluorescence detection. Variant T81F significantly improved the yield in comparison to wild type RizA for aspartic acid (7 to 17%), serine (33 to 47%) and alanine (12 to 17%). S84F increased product yield similarly for aspartic acid (7 to 17%) and serine (33 to 42%). D376E increased the yield with alanine (12 to 19%) and phenylalanine (11 to 26%). The largest change was observed for S156A, which showed a yield for Arg-Phe of 40% corresponding to a 270% increase in product concentration. This study expands the knowledge about positions governing the substrate specificity of RizA and may help to inform future protein engineering endeavors.

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.

scholarly journals Structural Insights for Core Scaffold and Substrate Specificity of B1, B2, and B3 Metallo-β-Lactamases

2022 ◽  
Vol 12 ◽  
Author(s):  
Yeongjin Yun ◽  
Sangjun Han ◽  
Yoon Sik Park ◽  
Hyunjae Park ◽  
Dogyeong Kim ◽  
...  
Keyword(s):  
Substrate Specificity ◽  
Active Site ◽  
Chemical Structure ◽  
Substrate Binding ◽  
Binding Pocket ◽  
Structural Features ◽  
Amino Acid Sequences ◽  
Zinc Ions ◽  
Substrate Binding Pocket ◽  
Inhibitory Spectrum

Metallo-β-lactamases (MBLs) hydrolyze almost all β-lactam antibiotics, including penicillins, cephalosporins, and carbapenems; however, no effective inhibitors are currently clinically available. MBLs are classified into three subclasses: B1, B2, and B3. Although the amino acid sequences of MBLs are varied, their overall scaffold is well conserved. In this study, we systematically studied the primary sequences and crystal structures of all subclasses of MBLs, especially the core scaffold, the zinc-coordinating residues in the active site, and the substrate-binding pocket. We presented the conserved structural features of MBLs in the same subclass and the characteristics of MBLs of each subclass. The catalytic zinc ions are bound with four loops from the two central β-sheets in the conserved αβ/βα sandwich fold of MBLs. The three external loops cover the zinc site(s) from the outside and simultaneously form a substrate-binding pocket. In the overall structure, B1 and B2 MBLs are more closely related to each other than they are to B3 MBLs. However, B1 and B3 MBLs have two zinc ions in the active site, while B2 MBLs have one. The substrate-binding pocket is different among all three subclasses, which is especially important for substrate specificity and drug resistance. Thus far, various classes of β-lactam antibiotics have been developed to have modified ring structures and substituted R groups. Currently available structures of β-lactam-bound MBLs show that the binding of β-lactams is well conserved according to the overall chemical structure in the substrate-binding pocket. Besides β-lactam substrates, B1 and cross-class MBL inhibitors also have distinguished differences in the chemical structure, which fit well to the substrate-binding pocket of MBLs within their inhibitory spectrum. The systematic structural comparison among B1, B2, and B3 MBLs provides in-depth insight into their substrate specificity, which will be useful for developing a clinical inhibitor targeting MBLs.

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