scholarly journals Improved System for Protein Engineering of the Hydroxylase Component of Soluble Methane Monooxygenase

2002 ◽  
Vol 68 (11) ◽  
pp. 5265-5273 ◽  
Author(s):  
Thomas J. Smith ◽  
Susan E. Slade ◽  
Nicolas P. Burton ◽  
J. Colin Murrell ◽  
Howard Dalton
Keyword(s):  
Protein Engineering ◽  
Active Site ◽  
Methane Monooxygenase ◽  
Expression System ◽  
Active Form ◽  
Wild Type ◽  
Active Site Residues ◽  
Α Subunit ◽  
Soluble Methane Monooxygenase ◽  
Type Activity

ABSTRACT Soluble methane monooxygenase (sMMO) of Methylosinus trichosporium OB3b is a three-component oxygenase that catalyses the O2- and NAD(P)H-dependent oxygenation of methane and numerous other substrates. Despite substantial interest in the use of genetic techniques to study the mechanism of sMMO and manipulate its substrate specificity, directed mutagenesis of active-site residues was previously impossible because no suitable heterologous expression system had been found for expression in a highly active form of the hydroxylase component, which is an (αβγ)2 complex containing the binuclear iron active site. A homologous expression system that enabled the expression of recombinant wild-type sMMO in a derivative of M. trichosporium OB3b from which the chromosomal copy of the sMMO-encoding operon had been partially deleted was previously reported. Here we report substantial development of this method to produce a system for the facile construction and expression of mutants of the hydroxylase component of sMMO. This new system has been used to investigate the functions of Cys 151 and Thr 213 of the α subunit, which are the only nonligating protonated side chains in the hydrophobic active site. Both residues were found to be critical for the stability and/or activity of sMMO, but neither was essential for oxygenation reactions. The T213S mutant was purified to >98% homogeneity. It had the same iron content as the wild type and had 72% wild-type activity toward toluene but only 17% wild-type activity toward propene; thus, its substrate profile was significantly altered. With these results, we have demonstrated proof of the principle for protein engineering of this uniquely versatile enzyme.

scholarly journals Mutagenesis of the “Leucine Gate” To Explore the Basis of Catalytic Versatility in Soluble Methane Monooxygenase

2007 ◽  
Vol 73 (20) ◽  
pp. 6460-6467 ◽  
Author(s):  
Elena Borodina ◽  
Tim Nichol ◽  
Marc G. Dumont ◽  
Thomas J. Smith ◽  
J. Colin Murrell
Keyword(s):  
Active Site ◽  
Methane Monooxygenase ◽  
New Products ◽  
Wild Type ◽  
Α Subunit ◽  
Soluble Methane Monooxygenase ◽  
Homologous Expression ◽  
Aromatic Substrates ◽  
Methane Oxidizing Bacteria ◽  
Wide Range

ABSTRACT Soluble methane monooxygenase (sMMO) from methane-oxidizing bacteria is a multicomponent nonheme oxygenase that naturally oxidizes methane to methanol and can also cooxidize a wide range of adventitious substrates, including mono- and diaromatic hydrocarbons. Leucine 110, at the mouth of the active site in the α subunit of the hydroxylase component of sMMO, has been suggested to act as a gate to control the access of substrates to the active site. Previous crystallography of the wild-type sMMO has indicated at least two conformations of the enzyme that have the “leucine gate” open to different extents, and mutagenesis of homologous enzymes has indicated a role for this residue in the control of substrate range and regioselectivity with aromatic substrates. By further refinement of the system for homologous expression of sMMO that we developed previously, we have been able to prepare a range of site-directed mutations at position 110 in the α subunit of sMMO. All the mutants (with Gly, Cys, Arg, and Tyr, respectively, at this position) showed relaxations of regioselectivity compared to the wild type with monoaromatic substrates and biphenyl, including the appearance of new products arising from hydroxylation at the 2- and 3- positions on the benzene ring. Mutants with the larger Arg and Trp residues at position 110 also showed shifts in regioselectivity during naphthalene hydroxylation from the 2- to the 1- position. No evidence that mutagenesis of Leu 110 could allow very large substrates to enter the active site was found, however, since the mutants (like the wild type) were inactive toward the triaromatic hydrocarbons anthracene and phenanthrene. Thus, our results indicate that the “leucine gate” in sMMO is more important in controlling the precision of regioselectivity than the sizes of substrates that can enter the active site.

scholarly journals Activation and selectivity of OTUB-1 and OTUB-2 deubiquitinylases

2020 ◽  
Vol 295 (20) ◽  
pp. 6972-6982
Author(s):  
Dakshinamurthy Sivakumar ◽  
Vikash Kumar ◽  
Michael Naumann ◽  
Matthias Stein
Keyword(s):  
Active Site ◽  
Electrostatic Interactions ◽  
Active Form ◽  
Cysteine Proteases ◽  
Binding Pocket ◽  
Catalytic Triad ◽  
Catalytic Site ◽  
Dynamics Simulation ◽  
Active Site Residues ◽  
Catalytically Active

The ovarian tumor domain (OTU) deubiquitinylating cysteine proteases OTUB1 and OTUB2 (OTU ubiquitin aldehyde binding 1 and 2) are representative members of the OTU subfamily of deubiquitinylases. Deubiquitinylation critically regulates a multitude of important cellular processes, such as apoptosis, cell signaling, and growth. Moreover, elevated OTUB expression has been observed in various cancers, including glioma, endometrial cancer, ovarian cancer, and breast cancer. Here, using molecular dynamics simulation approaches, we found that both OTUB1 and OTUB2 display a catalytic triad characteristic of proteases but differ in their configuration and protonation states. The OTUB1 protein had a prearranged catalytic site, with strong electrostatic interactions between the active-site residues His265 and Asp267. In OTUB2, however, the arrangement of the catalytic triad was different. In the absence of ubiquitin, the neutral states of the catalytic-site residues in OTUB2 were more stable, resulting in larger distances between these residues. Only upon ubiquitin binding did the catalytic triad in OTUB2 rearrange and bring the active site into a catalytically feasible state. An analysis of water access channels revealed only a few diffusion trajectories for the catalytically active form of OTUB1, whereas in OTUB2 the catalytic site was solvent-accessible, and a larger number of water molecules reached and left the binding pocket. Interestingly, in OTUB2, the catalytic residues His224 and Asn226 formed a stable hydrogen bond. We propose that the observed differences in activation kinetics, protonation states, water channels, and active-site accessibility between OTUB1 and OTUB2 may be relevant for the selective design of OTU inhibitors.

scholarly journals Roles of key active-site residues in flavocytochrome P450 BM3

1999 ◽  
Vol 339 (2) ◽  
pp. 371-379 ◽  
Author(s):  
Michael A. NOBLE ◽  
Caroline S. MILES ◽  
Stephen K. CHAPMAN ◽  
Dominikus A. LYSEK ◽  
Angela C. MACKAY ◽  
...  
Keyword(s):  
Electron Transfer ◽  
Fatty Acid ◽  
Fatty Acid Oxidation ◽  
Active Site ◽  
Side Chain ◽  
Acid Oxidation ◽  
Dodecanoic Acid ◽  
Wild Type ◽  
Active Site Residues ◽  
P450 Bm3

The effects of mutation of key active-site residues (Arg-47, Tyr-51, Phe-42 and Phe-87) in Bacillus megaterium flavocytochrome P450 BM3 were investigated. Kinetic studies on the oxidation of laurate and arachidonate showed that the side chain of Arg-47 contributes more significantly to stabilization of the fatty acid carboxylate than does that of Tyr-51 (kinetic parameters for oxidation of laurate: R47A mutant, Km 859 µM, kcat 3960 min-1; Y51F mutant, Km 432 µM, kcat 6140 min-1; wild-type, Km 288 µM, kcat 5140 min-1). A slightly increased kcat for the Y51F-catalysed oxidation of laurate is probably due to decreased activation energy (ΔG‡) resulting from a smaller ΔG of substrate binding. The side chain of Phe-42 acts as a phenyl ‘cap ’ over the mouth of the substrate-binding channel. With mutant F42A, Km is massively increased and kcat is decreased for oxidation of both laurate (Km 2.08 mM, kcat 2450 min-1) and arachidonate (Km 34.9 µM, kcat 14620 min-1; compared with values of 4.7 µM and 17100 min-1 respectively for wild-type). Amino acid Phe-87 is critical for efficient catalysis. Mutants F87G and F87Y not only exhibit increased Km and decreased kcat values for fatty acid oxidation, but also undergo an irreversible conversion process from a ‘fast ’ to a ‘slow ’ rate of substrate turnover [for F87G (F87Y)-catalysed laurate oxidation: kcat ‘fast ’, 760 (1620) min-1; kcat ‘slow ’, 48.0 (44.6) min-1; kconv (rate of conversion from fast to slow form), 4.9 (23.8) min-1]. All mutants showed less than 10% uncoupling of NADPH oxidation from fatty acid oxidation. The rate of FMN-to-haem electron transfer was shown to become rate-limiting in all mutants analysed. For wild-type P450 BM3, the rate of FMN-to-haem electron transfer (8340 min-1) is twice the steady-state rate of oxidation (4100 min-1), indicating that other steps contribute to rate limitation. Active-site structures of the mutants were probed with the inhibitors 12-(imidazolyl)dodecanoic acid and 1-phenylimidazole. Mutant F87G binds 1-phenylimidazole > 10-fold more tightly than does the wild-type, whereas mutant Y51F binds the haem-co-ordinating fatty acid analogue 12-(imidazolyl)dodecanoic acid > 30-fold more tightly than wild-type.

scholarly journals Site-directed mutagenesis of proposed active-site residues of penicillin-binding protein 5 from Escherichia coli

1994 ◽  
Vol 303 (2) ◽  
pp. 357-362 ◽  
Author(s):  
M P G van der Linden ◽  
L de Haan ◽  
O Dideberg ◽  
W Keck
Keyword(s):  
Active Site ◽  
Catalytic Mechanism ◽  
Binding Capacity ◽  
Binding Protein ◽  
Complete Loss ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Penicillin Binding Protein ◽  
Wild Type ◽  
Active Site Residues

Alignment of the amino acid sequence of penicillin-binding protein 5 (PBP5) with the sequences of other members of the family of active-site-serine penicillin-interacting enzymes predicted the residues playing a role in the catalytic mechanism of PBP5. Apart from the active-site (Ser44), Lys47, Ser110-Gly-Asn, Asp175 and Lys213-Thr-Gly were identified as the residues making up the conserved boxes of this protein family. To determine the role of these residues, they were replaced using site-directed mutagenesis. The mutant proteins were assayed for their penicillin-binding capacity and DD-carboxypeptidase activity. The Ser44Cys and the Ser44Gly mutants showed a complete loss of both penicillin-binding capacity and DD-carboxypeptidase activity. The Lys47Arg mutant also lost its DD-carboxypeptidase activity but was able to bind and hydrolyse penicillin, albeit at a considerably reduced rate. Mutants in the Ser110-Gly-Asn fingerprint were affected in both acylation and deacylation upon reaction with penicillin and lost their DD-carboxypeptidase activity with the exception of Asn112Ser and Asn112Thr. The Asp175Asn mutant showed wild-type penicillin-binding but a complete loss of DD-carboxypeptidase activity. Mutants of Lys213 lost both penicillin-binding and DD-carboxypeptidase activity except for Lys213His, which still bound penicillin with a k+2/K' of 0.2% of the wild-type value. Mutation of His216 and Thr217 also had a strong effect on DD-carboxypeptidase activity. Thr217Ser and Thr217Ala showed augmented hydrolysis rates for the penicillin acyl-enzyme. This study reveals the residues in the conserved fingerprints to be very important for both DD-carboxypeptidase activity and penicillin-binding, and confirms them to play crucial roles in catalysis.

scholarly journals Active Residues and Viral Substrate Cleavage Sites of the Protease of the Birnavirus Infectious Pancreatic Necrosis Virus

2000 ◽  
Vol 74 (5) ◽  
pp. 2057-2066 ◽  
Author(s):  
Stéphanie Petit ◽  
Nathalie Lejal ◽  
Jean-Claude Huet ◽  
Bernard Delmas
Keyword(s):  
Active Site ◽  
Pancreatic Necrosis ◽  
Infectious Pancreatic Necrosis Virus ◽  
Target Sequence ◽  
Cleavage Sites ◽  
Wild Type ◽  
Necrosis Virus ◽  
Type Activity ◽  
Infectious Pancreatic Necrosis ◽  
Pancreatic Necrosis Virus

ABSTRACT The polyprotein of infectious pancreatic necrosis virus (IPNV), a birnavirus, is processed by the viral protease VP4 (also named NS) to generate three polypeptides: pVP2, VP4, and VP3. Site-directed mutagenesis at 42 positions of the IPNV VP4 protein was performed to determine the active site and the important residues for the protease activity. Two residues (serine 633 and lysine 674) were critical for cleavage activity at both the pVP2-VP4 and the VP4-VP3 junctions. Wild-type activity at the pVP2-VP4 junction and a partial block (with an alteration of the cleavage specificity) at the VP4-VP3 junction were observed when replacement occurred at histidines 547 and 679. A similar observation was made when aspartic acid 693 was replaced by leucine, but wild-type activity and specificity were found when substituted by glutamine or asparagine. Sequence comparison between IPNV and two birnavirus (infectious bursal disease virus and DrosophilaX virus) VP4s revealed that serine 633 and lysine 674 are conserved in these viruses, in contrast to histidines 547 and 679. The importance of serine 633 and lysine 674 is reminiscent of the protease active site of bacterial leader peptidases and their mitochondrial homologs and of the bacterial LexA-like proteases. Self-cleavage sites of IPNV VP4 were determined at the pVP2-VP4 and VP4-VP3 junctions by N-terminal sequencing and mutagenesis. Two alternative cleavage sites were also identified in the carboxyl domain of pVP2 by cumulative mutagenesis. The results suggest that VP4 cleaves the (Ser/Thr)-X-Ala↓(Ser/Ala)-Gly motif, a target sequence with similarities to bacterial leader peptidases and herpesvirus protease cleavage sites.

scholarly journals Protein Engineering of Toluene-o-Xylene Monooxygenase from Pseudomonas stutzeri OX1 for Synthesizing 4-Methylresorcinol, Methylhydroquinone, and Pyrogallol

2004 ◽  
Vol 70 (6) ◽  
pp. 3253-3262 ◽  
Author(s):  
G�n�l Vardar ◽  
Thomas K. Wood
Keyword(s):  
Active Site ◽  
High Performance ◽  
Agar Plate ◽  
Pseudomonas Stutzeri ◽  
Saturation Mutagenesis ◽  
Dna Shuffling ◽  
Natural Substrate ◽  
Wild Type ◽  
Active Site Residues ◽  
Derivatives Of

ABSTRACT Toluene-o-xylene monooxygenase (ToMO) from Pseudomonas stutzeri OX1 oxidizes toluene to 3- and 4-methylcatechol and oxidizes benzene to form phenol; in this study ToMO was found to also form catechol and 1,2,3-trihydroxybenzene (1,2,3-THB) from phenol. To synthesize novel dihydroxy and trihydroxy derivatives of benzene and toluene, DNA shuffling of the alpha-hydroxylase fragment of ToMO (TouA) and saturation mutagenesis of the TouA active site residues I100, Q141, T201, and F205 were used to generate random mutants. The mutants were initially identified by screening with a rapid agar plate assay and then were examined further by high-performance liquid chromatography and gas chromatography. Several regiospecific mutants with high rates of activity were identified; for example, Escherichia coli TG1/pBS(Kan)ToMO expressing the F205G TouA saturation mutagenesis variant formed 4-methylresorcinol (0.78 nmol/min/mg of protein), 3-methylcatechol (0.25 nmol/min/mg of protein), and methylhydroquinone (0.088 nmol/min/mg of protein) from o-cresol, whereas wild-type ToMO formed only 3-methylcatechol (1.1 nmol/min/mg of protein). From o-cresol, the I100Q saturation mutagenesis mutant and the M180T/E284G DNA shuffling mutant formed methylhydroquinone (0.50 and 0.19 nmol/min/mg of protein, respectively) and 3-methylcatechol (0.49 and 1.5 nmol/min/mg of protein, respectively). The F205G mutant formed catechol (0.52 nmol/min/mg of protein), resorcinol (0.090 nmol/min/mg of protein), and hydroquinone (0.070 nmol/min/mg of protein) from phenol, whereas wild-type ToMO formed only catechol (1.5 nmol/min/mg of protein). Both the I100Q mutant and the M180T/E284G mutant formed hydroquinone (1.2 and 0.040 nmol/min/mg of protein, respectively) and catechol (0.28 and 2.0 nmol/min/mg of protein, respectively) from phenol. Dihydroxybenzenes were further oxidized to trihydroxybenzenes with different regiospecificities; for example, the I100Q mutant formed 1,2,4-THB from catechol, whereas wild-type ToMO formed 1,2,3-THB (pyrogallol). Regiospecific oxidation of the natural substrate toluene was also checked; for example, the I100Q mutant formed 22% o-cresol, 44% m-cresol, and 34% p-cresol, whereas wild-type ToMO formed 32% o-cresol, 21% m-cresol, and 47% p-cresol.

scholarly journals Asymmetric Subunit Organization of Heterodimeric Rous Sarcoma Virus Reverse Transcriptase αβ: Localization of the Polymerase and RNase H Active Sites in the α Subunit

2000 ◽  
Vol 74 (7) ◽  
pp. 3245-3252 ◽  
Author(s):  
Susanne Werner ◽  
Birgitta M. Wöhrl
Keyword(s):  
Reverse Transcriptase ◽  
Active Site ◽  
Rous Sarcoma Virus ◽  
Active Sites ◽  
Polymerase Activity ◽  
Rnase H ◽  
Wild Type ◽  
Α Subunit ◽  
Rous Sarcoma ◽  
Sarcoma Virus

ABSTRACT The genes encoding the α (63-kDa) and β (95-kDa) subunits of Rous sarcoma virus (RSV) reverse transcriptase (RT) or the entire Pol polypeptide (99 kDa) were mutated in the conserved aspartic acid residue Asp 181 of the polymerase active site (YMDD) or in the conserved Asp 505 residue of the RNase H active site. We have analyzed heterodimeric recombinant RSV αβ and αPol RTs within which one subunit was selectively mutated. When αβ heterodimers contained the Asp 181→Asn mutation in their β subunits, about 42% of the wild-type polymerase activity was detected, whereas when the heterodimers contained the same mutation in their α subunits, only 7.5% of the wild-type polymerase activity was detected. Similar results were obtained when the conserved Asp 505 residue of the RNase H active site was mutated to Asn. RNase H activity was clearly detectable in αβ heterodimers mutated in the β subunit but was lost when the mutation was present in the α subunit. In summary, our data imply that the polymerase and RNase H active sites are located in the α subunit of the heterodimeric RSV RT αβ.

Active-site mutations impairing the catalytic function of the catalytic subunit of human protein phosphatase 2A permit baculovirus-mediated overexpression in insect cells

2001 ◽  
Vol 357 (1) ◽  
pp. 225-232 ◽  
Author(s):  
Timothy MYLES ◽  
Karsten SCHMIDT ◽  
David R. H. EVANS ◽  
Peter CRON ◽  
Brian A. HEMMINGS
Keyword(s):  
Active Site ◽  
Protein Phosphatase ◽  
Insect Cells ◽  
Specific Activity ◽  
Catalytic Subunit ◽  
Regulatory Control ◽  
Wild Type ◽  
Active Site Residues ◽  
Catalytic Function

Members of the phosphoprotein phosphatase (PPP) family of protein serine/threonine phosphatases, including protein phosphatase (PP)1, PP2A and PP2B, share invariant active-site residues that are critical for catalytic function [Zhuo, Clemens, Stone and Dixon (1994) J. Biol. Chem. 269, 26234–26238]. Mutation of the active-site residues Asp88 or His118 within the human PP2A catalytic subunit (PP2Ac)α impaired catalytic activity in vitro; the D88N and H118N substitutions caused a 9- and 23-fold reduction in specific activity respectively, when compared with wild-type recombinant PP2Ac, indicating an important role for these residues in catalysis. Consistent with this, the D88N and H118N substituted forms failed to provide PP2A function in vivo, because, unlike wild-type human PP2Acα, neither substituted for the endogenous PP2Ac enzyme of budding yeast. Relative to wild-type PP2Ac, the active-site mutants were dramatically overexpressed in High Five® insect cells using the baculovirus system. Milligram quantities of PP2Ac were purified from 1×109 High Five cells and the kinetic constants for dephosphorylation of the peptide RRA(pT)VA (single-letter amino-acid notation) by PP2Ac (Km = 337.5μM; kcat = 170s−1) and D88N (Km = 58.4μM; kcat = 2s−1) were determined. The results show that the substitution impairs catalysis severely without a significant effect on substrate binding, consistent with the PPP catalytic mechanism. Combination of the baculovirus and yeast systems provides a strategy whereby the structure–function of PP2Ac may be fully explored, a goal which has previously proven difficult, owing to the stringent auto-regulatory control of PP2Ac protein levels in vivo.

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