scholarly journals Rational Engineering of the Substrate Specificity of a Thermostable D-Hydantoinase (Dihydropyrimidinase)

2020 ◽  
Vol 9 (1) ◽  
pp. 5
Author(s):  
Hovsep Aganyants ◽  
Pierre Weigel ◽  
Yeranuhi Hovhannisyan ◽  
Michèle Lecocq ◽  
Haykanush Koloyan ◽  
...  
Keyword(s):  
Substrate Specificity ◽  
Substrate Binding ◽  
Kinetic Constant ◽  
3D Structure ◽  
Binding Pocket ◽  
Site Directed Mutagenesis ◽  
Rational Engineering ◽  
Moderate Thermophiles ◽  
Hydantoin Derivatives ◽  
Optically Pure

D-hydantoinases catalyze an enantioselective opening of 5- and 6-membered cyclic structures and therefore can be used for the production of optically pure precursors for biomedical applications. The thermostable D-hydantoinase from Geobacillus stearothermophilus ATCC 31783 is a manganese-dependent enzyme and exhibits low activity towards bulky hydantoin derivatives. Homology modeling with a known 3D structure (PDB code: 1K1D) allowed us to identify the amino acids to be mutated at the substrate binding site and in its immediate vicinity to modulate the substrate specificity. Both single and double substituted mutants were generated by site-directed mutagenesis at appropriate sites located inside and outside of the stereochemistry gate loops (SGL) involved in the substrate binding. Substrate specificity and kinetic constant data demonstrate that the replacement of Phe159 and Trp287 with alanine leads to an increase in the enzyme activity towards D,L-5-benzyl and D,L-5-indolylmethyl hydantoins. The length of the side chain and the hydrophobicity of substrates are essential parameters to consider when designing the substrate binding pocket for bulky hydantoins. Our data highlight that D-hydantoinase is the authentic dihydropyrimidinase involved in the pyrimidine reductive catabolic pathway in moderate thermophiles.

scholarly journals Crystal structure and mechanism of human carboxypeptidase O: Insights into its specific activity for acidic residues

2018 ◽  
Vol 115 (17) ◽  
pp. E3932-E3939 ◽  
Author(s):  
Maria C. Garcia-Guerrero ◽  
Javier Garcia-Pardo ◽  
Esther Berenguer ◽  
Roberto Fernandez-Alvarez ◽  
Gifty B. Barfi ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Specific Activity ◽  
Dominant Role ◽  
Substrate Binding ◽  
Digestive Enzyme ◽  
Binding Pocket ◽  
Site Directed Mutagenesis ◽  
Enzyme Specificity ◽  
Substrate Binding Pocket ◽  
Acidic Residues

Human metallocarboxypeptidase O (hCPO) is a recently discovered digestive enzyme localized to the apical membrane of intestinal epithelial cells. Unlike pancreatic metallocarboxypeptidases, hCPO is glycosylated and produced as an active enzyme with distinctive substrate specificity toward C-terminal (C-t) acidic residues. Here we present the crystal structure of hCPO at 1.85-Å resolution, both alone and in complex with a carboxypeptidase inhibitor (NvCI) from the marine snail Nerita versicolor. The structure provides detailed information regarding determinants of enzyme specificity, in particular Arg275, placed at the bottom of the substrate-binding pocket. This residue, located at “canonical” position 255, where it is Ile in human pancreatic carboxypeptidases A1 (hCPA1) and A2 (hCPA2) and Asp in B (hCPB), plays a dominant role in determining the preference of hCPO for acidic C-t residues. Site-directed mutagenesis to Asp and Ala changes the specificity to C-t basic and hydrophobic residues, respectively. The single-site mutants thus faithfully mimic the enzymatic properties of CPB and CPA, respectively. hCPO also shows a preference for Glu over Asp, probably as a consequence of a tighter fitting of the Glu side chain in its S1′ substrate-binding pocket. This unique preference of hCPO, together with hCPA1, hCPA2, and hCPB, completes the array of C-t cleavages enabling the digestion of the dietary proteins within the intestine. Finally, in addition to activity toward small synthetic substrates and peptides, hCPO can also trim C-t extensions of proteins, such as epidermal growth factor, suggesting a role in the maturation and degradation of growth factors and bioactive peptides.

scholarly journals Main Structural Targets for Engineering Lipase Substrate Specificity

Catalysts ◽  
2020 ◽  
Vol 10 (7) ◽  
pp. 747
Author(s):  
Samah Hashim Albayati ◽  
Malihe Masomian ◽  
Siti Nor Hasmah Ishak ◽  
Mohd Shukuri bin Mohamad Ali ◽  
Adam Leow Thean ◽  
...  
Keyword(s):  
Substrate Specificity ◽  
Rational Design ◽  
Molecular Mechanisms ◽  
Experimental Studies ◽  
Random Mutagenesis ◽  
Binding Pocket ◽  
Amino Acid Sequences ◽  
Site Directed Mutagenesis ◽  
Saturation Mutagenesis ◽  
Process Conditions

Microbial lipases represent one of the most important groups of biotechnological biocatalysts. However, the high-level production of lipases requires an understanding of the molecular mechanisms of gene expression, folding, and secretion processes. Stable, selective, and productive lipase is essential for modern chemical industries, as most lipases cannot work in different process conditions. However, the screening and isolation of a new lipase with desired and specific properties would be time consuming, and costly, so researchers typically modify an available lipase with a certain potential for minimizing cost. Improving enzyme properties is associated with altering the enzymatic structure by changing one or several amino acids in the protein sequence. This review detailed the main sources, classification, structural properties, and mutagenic approaches, such as rational design (site direct mutagenesis, iterative saturation mutagenesis) and direct evolution (error prone PCR, DNA shuffling), for achieving modification goals. Here, both techniques were reviewed, with different results for lipase engineering, with a particular focus on improving or changing lipase specificity. Changing the amino acid sequences of the binding pocket or lid region of the lipase led to remarkable enzyme substrate specificity and enantioselectivity improvement. Site-directed mutagenesis is one of the appropriate methods to alter the enzyme sequence, as compared to random mutagenesis, such as error-prone PCR. This contribution has summarized and evaluated several experimental studies on modifying the substrate specificity of lipases.

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.

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 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.

Molecular engineering of cycloisomaltooligosaccharide glucanotransferase from Bacillus circulans T-3040: structural determinants for the reaction product size and reactivity

2015 ◽  
Vol 467 (2) ◽  
pp. 259-270 ◽  
Author(s):  
Ryuichiro Suzuki ◽  
Nobuhiro Suzuki ◽  
Zui Fujimoto ◽  
Mitsuru Momma ◽  
Keitarou Kimura ◽  
...  
Keyword(s):  
Binding Sites ◽  
Catalytic Domain ◽  
Substrate Binding ◽  
3D Structure ◽  
Site Directed Mutagenesis ◽  
Bacillus Circulans ◽  
Glycoside Hydrolase Family ◽  
Product Size ◽  
Carbohydrate Binding Modules ◽  
Substrate Binding Sites

Cycloisomaltooligosaccharide glucanotransferase (CITase) is a member of glycoside hydrolase family 66 and it produces cycloisomaltooligosaccharides (CIs). Small CIs (CI-7–9) and large CIs (CI-≥10) are designated as oligosaccharide-type CIs (oligo-CIs) and megalosaccharide-type CIs (megalo-CIs) respectively. CITase from Bacillus circulans T-3040 (BcCITase) produces mainly CI-8 with little megalo-CIs. It has two family 35 carbohydrate-binding modules (BcCBM35-1 and BcCBM35-2). BcCBM35-1 is inserted in a catalytic domain of BcCITase and BcCBM35-2 is located at the C-terminal region. Our previous studies suggested that BcCBM35-1 has two substrate-binding sites (B-1 and B-2) [Suzuki et al. (2014) J. Biol. Chem. 289, 12040–12051]. We implemented site-directed mutagenesis of BcCITase to explore the preference for product size on the basis of the 3D structure of BcCITase. Mutational studies provided evidence that B-1 and B-2 contribute to recruiting substrate and maintaining product size respectively. A mutant (mutant-R) with four mutations (F268V, D469Y, A513V and Y515S) produced three times as much megalo-CIs (CI-10–12) and 1.5 times as much total CIs (CI-7–12) as compared with the wild-type (WT) BcCITase. The 3D structure of the substrate–enzyme complex of mutant-R suggested that the modified product size specificity was attributable to the construction of novel substrate-binding sites in the B-2 site of BcCBM35-1 and reactivity was improved by mutation on subsite −3 on the catalytic domain.

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.

scholarly journals Semirational design and engineering of grapevine glucosyltransferases for enhanced activity and modified product selectivity

Glycobiology ◽  
2019 ◽  
Vol 29 (11) ◽  
pp. 765-775 ◽  
Author(s):  
Rakesh Joshi ◽  
Johanna Trinkl ◽  
Annika Haugeneder ◽  
Katja Härtl ◽  
Katrin Franz-Oberdorf ◽  
...  
Keyword(s):  
Substrate Specificity ◽  
Structural Changes ◽  
Binding Pocket ◽  
Site Directed Mutagenesis ◽  
Uridine Diphosphate ◽  
Amino Acid Residues ◽  
Product Selectivity ◽  
Active Site Residues ◽  
Product Specificity ◽  
Increased Activity

Abstract Uridine diphosphate-dependent glycosyltransferases (UGTs) catalyze the transfer of a diversity of sugars to several acceptor molecules and often exhibit distinct substrate specificity. Modulation of glycosyltransferases for increased catalytic activity and altered substrate or product specificity are the key manipulations for the biotechnological use of glycosyltransferases in various biosynthetic processes. Here, we have engineered the binding pocket of three previously characterized Vitis vinifera glycosyltransferases, UGT88F12, UGT72B27 and UGT92G6, by structure-guided in silico mutagenesis to facilitate the interactions of active site residues with flavonol glucosides and thus modify substrate specificity and activity. Site-directed mutagenesis at selected sites, followed with liquid chromatography–mass spectrometry based activity assays, exhibited that mutant UGTs were altered in product selectivity and activity as compared to the wild-type enzymes. Mutant UGTs produced larger amounts of flavonol di-monosaccharide glucosides, which imply that the mutations led to structural changes that increased the volume of the binding pocket to accommodate a larger substrate and to release larger products at ease. Mutants showed increased activity and modified product specificity. Thus, structure-based systematic mutations of the amino acid residues in the binding pocket can be explored for the generation of engineered UGTs for diverse biotechnological applications.

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