Single-component and two-component para -nitrophenol monooxygenases: structural basis for their catalytic difference

2021 ◽  
Author(s):  
Yuan Guo ◽  
De-Feng Li ◽  
Jianting Zheng ◽  
Ying Xu ◽  
Ning-Yi Zhou
Keyword(s):  
Crystal Structure ◽  
Rational Design ◽  
Single Component ◽  
Site Directed Mutagenesis ◽  
Structural Basis ◽  
Side Chain ◽  
Amino Acid Residues ◽  
Gram Positive ◽  
Two Component ◽  
Group D

para -Nitrophenol (PNP) is a hydrolytic product of organophosphate insecticides, such as parathion and methylparathion, in soil. Aerobic microbial degradation of PNP has been classically shown to proceed via ‘Hydroquinone (HQ) pathway’ in Gram-negative degraders, whereas via ‘Benzenetriol (BT) pathway’ in Gram-positive ones. ‘HQ pathway’ is initiated by a single-component PNP 4-monooxygenase and ‘BT pathway’ by a two-component PNP 2-monooxygenase. Their rigio-selectivity intrigues us to investigate their catalytic difference through structural study. PnpA1 is the oxygenase component of the two-component PNP 2-monooxygenase from Gram-positive Rhodococcus imtechensis RKJ300. It also catalyzes the hydroxylation of 4-nitrocatechol (4NC) and 2-chloro-4-nitrophenol (2C4NP). However, the mechanisms are unknown. Here, PnpA1 was structurally determined to be a member of group D flavin-dependent monooxygenases with an acyl-CoA dehydrogenase fold. The crystal structure and site-directed mutagenesis underlined the direct involvement of Arg100 and His293 in catalysis. The bulky side chain of Val292 was proposed to push the substrate towards FAD, hence positioning the substrate properly. A variant N450A was found with improved activity for 4NC and 2C4NP, probably because of the reduced steric hindrance. PnpA1 shows obvious difference in substrate selectivity with its close homologues TcpA and TftD, which may be determined by Thr296 and loop 449–454. Above all, our study allows the structural comparison between the two types of PNP monooxygenases. An explanation that accounts for their regio-selectivity was proposed: the different PNP binding manner determines their choice of ortho - or para -hydroxylation on PNP. IMPORTANCE Single-component PNP monoxygenases hydroxylate PNP at 4-position while two-component ones at 2-position. However, their catalytic and structural differences remain elusive. The structure of single-component PNP 4-monooxygenase has previously been determined. In this study, to illustrate their catalytic difference, we resolved the crystal structure of, PnpA1, a typical two-component PNP 2-monooxygenase. The roles of several key amino acid residues in substrate binding and catalysis were revealed and a variant with improved activities towards 4NC and 2C4NP was obtained. Moreover, through comparing the two types of PNP monooxygenases, a hypothesis was proposed to account for their catalytic difference, which gives us a better understanding of these two similar reactions at molecular level. And these results will also be of further aid in enzyme rational design in bioremediation and biosynthesis.

scholarly journals Severity of the Streptomycin Resistance and Streptomycin Dependence Phenotypes of Ribosomal Protein S12 of Thermus thermophilus Depends on the Identity of Highly Conserved Amino Acid Residues

2005 ◽  
Vol 187 (10) ◽  
pp. 3548-3550 ◽  
Author(s):  
Jennifer F. Carr ◽  
Steven T. Gregory ◽  
Albert E. Dahlberg
Keyword(s):  
Ribosomal Protein ◽  
Thermus Thermophilus ◽  
Gene Replacement ◽  
Streptomycin Resistance ◽  
Site Directed Mutagenesis ◽  
Structural Basis ◽  
Side Chain ◽  
Amino Acid Residues ◽  
Drug Dependent ◽  
Ribosomal Protein S12

ABSTRACT The structural basis for the streptomycin dependence phenotype of ribosomal protein S12 mutants is poorly understood. Here we describe the application of site-directed mutagenesis and gene replacement of Thermus thermophilus rpsL to assess the importance of side chain identity and tertiary interactions as phenotypic determinants of drug-dependent mutants.

scholarly journals Identification of Highly Conserved Residues Involved in Inhibition of HIV-1 RNase H Function by Diketo Acid Derivatives

2014 ◽  
Vol 58 (10) ◽  
pp. 6101-6110 ◽  
Author(s):  
Angela Corona ◽  
Francesco Saverio Di Leva ◽  
Sylvain Thierry ◽  
Luca Pescatori ◽  
Giuliana Cuzzucoli Crucitti ◽  
...  
Keyword(s):  
Rational Design ◽  
Acid Ethyl Ester ◽  
Rnase H ◽  
Site Directed Mutagenesis ◽  
Amino Acid Residues ◽  
Conserved Residues ◽  
Essential Function ◽  
Catalytic Motif ◽  
Hiv 1 ◽  
Micromolar Range

ABSTRACTHIV-1 reverse transcriptase (RT)-associated RNase H activity is an essential function in viral genome retrotranscription. RNase H is a promising drug target for which no inhibitor is available for therapy. Diketo acid (DKA) derivatives are active site Mg2+-binding inhibitors of both HIV-1 RNase H and integrase (IN) activities. To investigate the DKA binding site of RNase H and the mechanism of action, six couples of ester and acid DKAs, derived from 6-[1-(4-fluorophenyl)methyl-1H-pyrrol-2-yl)]-2,4-dioxo-5-hexenoic acid ethyl ester (RDS1643), were synthesized and tested on both RNase H and IN functions. Most of the ester derivatives showed selectivity for HIV-1 RNase H versus IN, while acids inhibited both functions. Molecular modeling and site-directed mutagenesis studies on the RNase H domain demonstrated different binding poses for ester and acid DKAs and proved that DKAs interact with residues (R448, N474, Q475, Y501, and R557) involved not in the catalytic motif but in highly conserved portions of the RNase H primer grip motif. The ester derivative RDS1759 selectively inhibited RNase H activity and viral replication in the low micromolar range, making contacts with residues Q475, N474, and Y501. Quantitative PCR studies and fluorescence-activated cell sorting (FACS) analyses showed that RDS1759 selectively inhibited reverse transcription in cell-based assays. Overall, we provide the first demonstration that RNase H inhibition by DKAs is due not only to their chelating properties but also to specific interactions with highly conserved amino acid residues in the RNase H domain, leading to effective targeting of HIV retrotranscription in cells and hence offering important insights for the rational design of RNase H inhibitors.

scholarly journals Protein engineering of the 2-haloacid halidohydrolase IVa from Pseudomonas cepacia MBA4

1993 ◽  
Vol 292 (1) ◽  
pp. 69-74 ◽  
Author(s):  
W Asmara ◽  
U Murdiyatmo ◽  
A J Baines ◽  
A T Bull ◽  
D J Hardman
Keyword(s):  
Amino Acid ◽  
Rational Design ◽  
Catalytic Mechanism ◽  
Protonated Form ◽  
Amino Acid Sequences ◽  
Site Directed Mutagenesis ◽  
Pseudomonas Cepacia ◽  
Amino Acid Residues ◽  
Substrate Complex ◽  
Key Residues

The chemical modification of L-2-haloacid halidohydrolase IVa (Hdl IVa), originally identified in Pseudomonas cepacia MBA4, produced as a recombinant protein in Escherichia coli DH5 alpha, led to the identification of histidine and arginine as amino acid residues likely to play a part in the catalytic mechanism of the enzyme. These results, together with DNA sequence and analyses [Murdiyatmo, Asmara, Baines, Bull and Hardman (1992) Biochem. J. 284, 87-93] provided the basis for the rational design of a series of random- and site-directed-mutagenesis experiments of the Hdl IVa structural gene (hdl IVa). Subsequent apparent kinetic analyses of purified mutant enzymes identified His-20 and Arg-42 as the key residues in the activity of this halidohydrolase. It is also proposed that Asp-18 is implicated in the functioning of the enzyme, possibly by positioning the correct tautomer of His-20 for catalysis in the enzyme-substrate complex and stabilizing the protonated form of His-20 in the transition-state complex. Comparison of conserved amino acid sequences between the Hdl IVa and other halidohydrolases suggests that L-2-haloacid halidohydrolases contain conserved amino acid sequences that are not found in halidohydrolases active towards both D- and L-2-monochloropropionate.

scholarly journals Family D DNA polymerase interacts with GINS to promote CMG-helicase in the archaeal replisome

2021 ◽  
Author(s):  
Keisuke Oki ◽  
Mariko Nagata ◽  
Takeshi Yamagami ◽  
Tomoyuki Numata ◽  
Sonoko Ishino ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Dna Polymerase ◽  
Direct Interaction ◽  
Crystal Structure Analysis ◽  
Site Directed Mutagenesis ◽  
Structural Basis ◽  
Functional Connection ◽  
Terminal Domain ◽  
The Family ◽  
Lagging Strand

Abstract Genomic DNA replication requires replisome assembly. We show here the molecular mechanism by which CMG (GAN–MCM–GINS)-like helicase cooperates with the family D DNA polymerase (PolD) in Thermococcus kodakarensis. The archaeal GINS contains two Gins51 subunits, the C-terminal domain of which (Gins51C) interacts with GAN. We discovered that Gins51C also interacts with the N-terminal domain of PolD’s DP1 subunit (DP1N) to connect two PolDs in GINS. The two replicases in the replisome should be responsible for leading- and lagging-strand synthesis, respectively. Crystal structure analysis of the DP1N–Gins51C–GAN ternary complex was provided to understand the structural basis of the connection between the helicase and DNA polymerase. Site-directed mutagenesis analysis supported the interaction mode obtained from the crystal structure. Furthermore, the assembly of helicase and replicase identified in this study is also conserved in Eukarya. PolD enhances the parental strand unwinding via stimulation of ATPase activity of the CMG-complex. This is the first evidence of the functional connection between replicase and helicase in Archaea. These results suggest that the direct interaction of PolD with CMG-helicase is critical for synchronizing strand unwinding and nascent strand synthesis and possibly provide a functional machinery for the effective progression of the replication fork.

scholarly journals Crystal structure of Arabidopsis thaliana HPPK/DHPS, a bifunctional enzyme and target of the herbicide asulam

2021 ◽  
Author(s):  
Grishma Vadlamani ◽  
Kirill V Sukhoverkov ◽  
Joel Haywood ◽  
Karen J Breese ◽  
Mark F Fisher ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Arabidopsis Thaliana ◽  
Mode Of Action ◽  
Rational Design ◽  
Binding Pocket ◽  
Structural Basis ◽  
Metabolic Resistance ◽  
Folate Biosynthesis ◽  
Limited Opportunity ◽  
Regulatory Scrutiny

Herbicides are vital for modern agriculture, but their utility is threatened by genetic or metabolic resistance in weeds as well as heightened regulatory scrutiny. Of the known herbicide modes of action, 6-hydroxymethyl-7,8-dihydropterin synthase (DHPS) which is involved in folate biosynthesis, is targeted by just one commercial herbicide, asulam. A mimic of the substrate para-aminobenzoic acid, asulam is chemically similar to sulfonamide antibiotics - and while still in widespread use, asulam has faced regulatory scrutiny. With an entire mode of action represented by just one commercial agrochemical, we sought to improve the understanding of its plant target. Here we solve a 2.6 Å resolution crystal structure for Arabidopsis thaliana DHPS that is conjoined to 6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase (HPPK) and reveal a strong structural conservation with bacterial counterparts at the sulfonamide-binding pocket of DHPS. We demonstrate asulam and the antibiotics sulfacetamide and sulfamethoxazole have herbicidal as well as antibacterial activity and explore the structural basis of their potency by modelling these compounds in mitochondrial HPPK/DHPS. Our findings suggest limited opportunity for the rational design of plant selectivity from asulam and that pharmacokinetic or delivery differences between plants and microbes might be the best approaches to safeguard this mode of action.

scholarly journals High-Resolution Crystal Structure of the Subclass B3 Metallo-β-Lactamase BJP-1: Rational Basis for Substrate Specificity and Interaction with Sulfonamides

2010 ◽  
Vol 54 (10) ◽  
pp. 4343-4351 ◽  
Author(s):  
Jean-Denis Docquier ◽  
Manuela Benvenuti ◽  
Vito Calderone ◽  
Magdalena Stoczko ◽  
Nicola Menciassi ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Active Site ◽  
Bradyrhizobium Japonicum ◽  
Active Sites ◽  
Structural Diversity ◽  
Binding Pocket ◽  
Structural Basis ◽  
Side Chain ◽  
Functional Heterogeneity ◽  
Hydrophobic Binding

ABSTRACT Metallo-β-lactamases (MBLs) are important enzymatic factors in resistance to β-lactam antibiotics that show important structural and functional heterogeneity. BJP-1 is a subclass B3 MBL determinant produced by Bradyrhizobium japonicum that exhibits interesting properties. BJP-1, like CAU-1 of Caulobacter vibrioides, overall poorly recognizes β-lactam substrates and shows an unusual substrate profile compared to other MBLs. In order to understand the structural basis of these properties, the crystal structure of BJP-1 was obtained at 1.4-Å resolution. This revealed significant differences in the conformation and locations of the active-site loops, determining a rather narrow active site and the presence of a unique N-terminal helix bearing Phe-31, whose side chain binds in the active site and represents an obstacle for β-lactam substrate binding. In order to probe the potential of sulfonamides (known to inhibit various zinc-dependent enzymes) to bind in the active sites of MBLs, the structure of BJP-1 in complex with 4-nitrobenzenesulfonamide was also obtained (at 1.33-Å resolution), thereby revealing the mode of interaction of these molecules in MBLs. Interestingly, sulfonamide binding resulted in the displacement of the side chain of Phe-31 from its hydrophobic binding pocket, where the benzene ring of the molecule is now found. These data further highlight the structural diversity shown by MBLs but also provide interesting insights in the structure-function relationships of these enzymes. More importantly, we provided the first structural observation of MBL interaction with sulfonamides, which might represent an interesting scaffold for the design of MBL inhibitors.

scholarly journals Tuning of pKa values activates substrates in flavin-dependent aromatic hydroxylases

2020 ◽  
Vol 295 (12) ◽  
pp. 3965-3981 ◽  
Author(s):  
Warintra Pitsawong ◽  
Pirom Chenprakhon ◽  
Taweesak Dhammaraj ◽  
Dheeradhach Medhanavyn ◽  
Jeerus Sucharitakul ◽  
...  
Keyword(s):  
Active Sites ◽  
Single Component ◽  
Electrophilic Aromatic Substitution ◽  
Aromatic Substitution ◽  
Substrate Analogs ◽  
Two Component ◽  
Group A ◽  
Catalytic Mechanisms ◽  
Protein Variants ◽  
Group D

Hydroxylation of substituted phenols by flavin-dependent monooxygenases is the first step of their biotransformation in various microorganisms. The reaction is thought to proceed via electrophilic aromatic substitution, catalyzed by enzymatic deprotonation of substrate, in single-component hydroxylases that use flavin as a cofactor (group A). However, two-component hydroxylases (group D), which use reduced flavin as a co-substrate, are less amenable to spectroscopic investigation. Herein, we employed 19F NMR in conjunction with fluorinated substrate analogs to directly measure pKa values and to monitor protein events in hydroxylase active sites. We found that the single-component monooxygenase 3-hydroxybenzoate 6-hydroxylase (3HB6H) depresses the pKa of the bound substrate analog 4-fluoro-3-hydroxybenzoate (4F3HB) by 1.6 pH units, consistent with previously proposed mechanisms. 19F NMR was applied anaerobically to the two-component monooxygenase 4-hydroxyphenylacetate 3-hydroxylase (HPAH), revealing depression of the pKa of 3-fluoro-4-hydroxyphenylacetate by 2.5 pH units upon binding to the C2 component of HPAH. 19F NMR also revealed a pKa of 8.7 ± 0.05 that we attributed to an active-site residue involved in deprotonating bound substrate, and assigned to His-120 based on studies of protein variants. Thus, in both types of hydroxylases, we confirmed that binding favors the phenolate form of substrate. The 9 and 14 kJ/mol magnitudes of the effects for 3HB6H and HPAH-C2, respectively, are consistent with pKa tuning by one or more H-bonding interactions. Our implementation of 19F NMR in anaerobic samples is applicable to other two-component flavin-dependent hydroxylases and promises to expand our understanding of their catalytic mechanisms.

scholarly journals Crystal structure of fibrinogen-Aα peptide 1–23 (F8Y) bound to bovine thrombin explains why the mutation of Phe-8 to tyrosine strongly inhibits normal cleavage at Arg-16

1997 ◽  
Vol 326 (3) ◽  
pp. 815-822 ◽  
Author(s):  
Michael G. MALKOWSKI ◽  
Philip D. MARTIN ◽  
Susan T. LORD ◽  
Brian F. P. EDWARDS
Keyword(s):  
Crystal Structure ◽  
Structural Basis ◽  
Side Chain ◽  
Asymmetric Unit ◽  
Bovine Thrombin ◽  
R Factor ◽  
Native Peptide ◽  
Cell Dimensions ◽  
Scissile Bond ◽  
Unit Cell Dimensions

A peptide containing residues 1–50 of the Aα-chain of fibrinogen, expressed as a fusion peptide with β-galactosidase, is rapidly cleaved by thrombin at Arg-16, similarly to whole fibrinogen. When Phe-8, which is highly conserved, is replaced with tyrosine (F8Y), the cleavage is slowed drastically [Lord, Byrd, Hede, Wei and Colby (1990) J. Biol. Chem. 265, 838–843]. To examine the structural basis for this result, we have determined the crystal structure of bovine thrombin complexed with a synthetic peptide containing residues 1–23 of fibrinogen Aα and the F8Y mutation. The crystals are in space group P43212, with unit-cell dimensions of a = 88.3 Å (1 Å = 0.1 nm), c = 195.5 Å and two complexes in the asymmetric unit. The final R factor is 0.183 for 2σ data from 7.0 to 2.5 Å resolution. There is continuous density for the five residues in the P3, P2, P1, P1′ and P2′ positions of the peptide (Gly-14f to Pro-18f) at the active site of thrombin, and isolated but well-defined density for Tyr-8f at position P9 in the hydrophobic pocket of thrombin. The tyrosine residue is shifted relative to phenylalanine in the native peptide because the phenol side chain is larger and makes a novel, intrapeptide hydrogen bond with Gly-14f. Adjacent peptide residues cannot form the hydrogen bonds that stabilize the secondary structure of the native peptide. Consequently, the ‘reaction’ geometry at the scissile bond, eight residues from the mutation, is perturbed and the peptide is mostly uncleaved in the crystal structure.

scholarly journals Molecular Dynamics Simulations of a Cytochrome P450 from Tepidiphilus thermophilus (P450-TT) Reveal How Its Substrate-Binding Channel Opens

Molecules ◽  
2021 ◽  
Vol 26 (12) ◽  
pp. 3614
Author(s):  
Abayomi S. Faponle ◽  
Anupom Roy ◽  
Ayodeji A. Adelegan ◽  
James W. Gauld
Keyword(s):  
Crystal Structure ◽  
Molecular Dynamics ◽  
Cytochrome P450 ◽  
Amino Acid ◽  
Molecular Dynamics Simulations ◽  
Organic Molecules ◽  
Side Chain ◽  
Relative Solvent Accessibility ◽  
Amino Acid Residues ◽  
Dynamics Simulations

Cytochrome P450s (P450) are important enzymes in biology with useful biochemical reactions in, for instance, drug and xenobiotics metabolisms, biotechnology, and health. Recently, the crystal structure of a new member of the CYP116B family has been resolved. This enzyme is a cytochrome P450 (CYP116B46) from Tepidiphilus thermophilus (P450-TT) and has potential for the oxy-functionalization of organic molecules such as fatty acids, terpenes, steroids, and statins. However, it was thought that the opening to its hitherto identified substrate channel was too small to allow organic molecules to enter. To investigate this, we performed molecular dynamics simulations on the enzyme. The results suggest that the crystal structure is not relaxed, possibly due to crystal packing effects, and that its tunnel structure is constrained. In addition, the simulations revealed two key amino acid residues at the mouth of the channel; a glutamyl and an arginyl. The glutamyl’s side chain tightens and relaxes the opening to the channel in conjunction with the arginyl’s, though the latter’s side chain is less dramatically changed after the initial relaxation of its conformations. Additionally, it was observed that the effect of increased temperature did not considerably affect the dynamics of the enzyme fold, including the relative solvent accessibility of the amino acid residues that make up the substrate channel wall even as compared to the changes that occurred at room temperature. Interestingly, the substrate channel became distinguishable as a prominent tunnel that is likely to accommodate small- to medium-sized organic molecules for bioconversions. That is, P450-TT has the ability to pass appropriate organic substrates to its active site through its elaborate substrate channel, and notably, is able to control or gate any molecules at the opening to this channel.

scholarly journals Ligand-induced activation of human TRPM2 requires the terminal ribose of ADPR and involves Arg1433 and Tyr1349

2017 ◽  
Vol 474 (13) ◽  
pp. 2159-2175 ◽  
Author(s):  
Ralf Fliegert ◽  
Joanna M. Watt ◽  
Anja Schöbel ◽  
Monika D. Rozewitz ◽  
Christelle Moreau ◽  
...  
Keyword(s):  
Hydrogen Bonding ◽  
Rational Design ◽  
Transient Receptor Potential ◽  
Therapeutic Potential ◽  
Receptor Potential ◽  
Transient Receptor Potential Channel ◽  
Site Directed Mutagenesis ◽  
Structural Basis ◽  
Pharmacological Intervention ◽  
Hydroxyl Groups

TRPM2 (transient receptor potential channel, subfamily melastatin, member 2) is a Ca2+-permeable non-selective cation channel activated by the binding of adenosine 5′-diphosphoribose (ADPR) to its cytoplasmic NUDT9H domain (NUDT9 homology domain). Activation of TRPM2 by ADPR downstream of oxidative stress has been implicated in the pathogenesis of many human diseases, rendering TRPM2 an attractive novel target for pharmacological intervention. However, the structural basis underlying this activation is largely unknown. Since ADP (adenosine 5′-diphosphate) alone did not activate or antagonize the channel, we used a chemical biology approach employing synthetic analogues to focus on the role of the ADPR terminal ribose. All novel ADPR derivatives modified in the terminal ribose, including that with the seemingly minor change of methylating the anomeric-OH, abolished agonist activity at TRPM2. Antagonist activity improved as the terminal substituent increasingly resembled the natural ribose, indicating that gating by ADPR might require specific interactions between hydroxyl groups of the terminal ribose and the NUDT9H domain. By mutating amino acid residues of the NUDT9H domain, predicted by modelling and docking to interact with the terminal ribose, we demonstrate that abrogating hydrogen bonding of the amino acids Arg1433 and Tyr1349 interferes with activation of the channel by ADPR. Taken together, using the complementary experimental approaches of chemical modification of the ligand and site-directed mutagenesis of TRPM2, we demonstrate that channel activation critically depends on hydrogen bonding of Arg1433 and Tyr1349 with the terminal ribose. Our findings allow for a more rational design of novel TRPM2 antagonists that may ultimately lead to compounds of therapeutic potential.

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