Selectivity of Fungal Sesquiterpene Synthases: Role of the Active Site's H-1α Loop in Catalysis

ABSTRACT Sesquiterpene synthases are responsible for the cyclization of farnesyl pyrophosphate into a myriad of structurally diverse compounds with various biological activities. We examine here the role of the conserved active site H-α1 loop in catalysis in three previously characterized fungal sesquiterpene synthases. The H-α1 loops of Cop3, Cop4, and Cop6 from Coprinus cinereus were altered by site-directed mutagenesis and the resultant product profiles were analyzed by gas chromatography-mass spectrometry and compared to the wild-type enzymes. In addition, we examine the effect of swapping the H-α1 loop from the promiscuous enzyme Cop4 with the more selective Cop6 and the effect of acidic or basic conditions on loop mutations in Cop4. Directed mutations of the H-α1 loop had a marked effect on the product profile of Cop3 and Cop4, while little to no change was shown in Cop6. Swapping of the Cop4 and Cop6 loops with one another was again shown to influence the product profile of Cop4, while the product profile of Cop6 remained identical to the wild-type enzyme. The loop mutations in Cop4 also implicate specific residues responsible for the pH sensitivity of the enzyme. These results affirm the role of the H-α1 loop in catalysis and provide a potential target to increase the product diversity of terpene synthases.

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Role of Arg-401 of cytosolic serine hydroxymethyltransferase in subunit assembly and interaction with the substrate carboxy group

Biochemical Journal ◽

10.1042/bj3270877 ◽

1997 ◽

Vol 327 (3) ◽

pp. 877-882 ◽

Cited By ~ 9

Author(s):

Junutula Reddy JAGATH ◽

Naropantul APPAJI RAO ◽

Handanahal SubbaRao SAVITHRI

Keyword(s):

Carboxy Group ◽

Gel Filtration ◽

Serine Hydroxymethyltransferase ◽

Site Directed Mutagenesis ◽

Mutant Enzyme ◽

Wild Type ◽

Wild Type Enzyme ◽

Partial Dissociation ◽

Tetrameric Form

In an attempt to identify the arginine residue involved in binding of the carboxylate group of serine to mammalian serine hydroxymethyltransferase, a highly conserved Arg-401 was mutated to Ala by site-directed mutagenesis. The mutant enzyme had a characteristic visible absorbance at 425 nm indicative of the presence of bound pyridoxal 5ʹ-phosphate as an internal aldimine with a lysine residue. However, it had only 0.003% of the catalytic activity of the wild-type enzyme. It was also unable to perform reactions with glycine, β-phenylserine or D-alanine, suggesting that the binding of these substrates to the mutant enzyme was affected. This was also evident from the interaction of amino-oxyacetic acid, which was very slow (8.4×10-4 s-1 at 50 μM) for the R401A mutant enzyme compared with the wild-type enzyme (44.6 s-1 at 50 μM). In contrast, methoxyamine (which lacks the carboxy group) reacted with the mutant enzyme (1.72 s-1 at 250 μM) more rapidly than the wild-type enzyme (0.2 s-1 at 250 μM). Further, both wild-type and the mutant enzymes were capable of forming unique quinonoid intermediates absorbing at 440 and 464 nm on interaction with thiosemicarbazide, which also does not have a carboxy group. These results implicate Arg-401 in the binding of the substrate carboxy group. In addition, gel-filtration profiles of the apoenzyme and the reconstituted holoenzyme of R401A and the wild-type enzyme showed that the mutant enzyme remained in a tetrameric form even when the cofactor had been removed. However, the wild-type enzyme underwent partial dissociation to a dimer, suggesting that the oligomeric structure was rendered more stable by the mutation of Arg-401. The increased stability of the mutant enzyme was also reflected in the higher apparent melting temperature (Tm) (61 °C) than that of the wild-type enzyme (56 °C). The addition of serine or serinamide did not change the apparent Tm of R401A mutant enzyme. These results suggest that the mutant enzyme might be in a permanently ‘open’ form and the increased apparent Tm could be due to enhanced subunit interactions.

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Substitution of Asp-309 by Asn in the Arg-Asp-Pro (RDP) motif of Acetobacter diazotrophicus levansucrase affects sucrose hydrolysis, but not enzyme specificity

Biochemical Journal ◽

10.1042/bj3370503 ◽

1999 ◽

Vol 337 (3) ◽

pp. 503-506 ◽

Cited By ~ 23

Author(s):

Frank R. BATISTA ◽

Lázaro HERNÁNDEZ ◽

Julio R. FERNÁNDEZ ◽

Juan ARRIETA ◽

Carmen MENÉNDEZ ◽

...

Keyword(s):

Functional Role ◽

Site Directed Mutagenesis ◽

Enzyme Specificity ◽

Wild Type ◽

Wild Type Enzyme ◽

Sucrose Hydrolysis ◽

Acetobacter Diazotrophicus ◽

Optimal Activity ◽

Mechanism of action of dd-peptidases: role of asparagine-161 in the Streptomyces R61 dd-peptidase

Biochemical Journal ◽

10.1042/bj2930195 ◽

1993 ◽

Vol 293 (1) ◽

pp. 195-201 ◽

Cited By ~ 10

Author(s):

J M Wilkin ◽

M Jamin ◽

B Joris ◽

J M Frere

Keyword(s):

Site Directed Mutagenesis ◽

Beta Lactamase ◽

Wild Type ◽

Peptide Substrate ◽

Beta Lactam ◽

Beta Lactams ◽

Wild Type Enzyme ◽

Class A ◽

First Case

The role of residue Asn-161 in the interaction between the Streptomyces R61 DD-peptidase and various substrates or beta-lactam inactivators was probed by site-directed mutagenesis. The residue was successively replaced by serine and alanine. In the first case, acylation rates were mainly affected with the peptide and ester substrates but not with the thiol-ester substrates and beta-lactams. However, the deacylation rates were decreased 10-30-fold with the substrates yielding benzoylglycyl and benzoylalanyl adducts. The Asn161Ala mutant was more generally affected, although the acylation rates with cefuroxime and cefotaxime remained similar to those observed with the wild-type enzyme. Surprisingly, the deacylation rates of the benzoylglycyl and benzoylalanyl adducts were very close to those observed with the wild-type enzyme. The results also indicate that the interaction with the peptide substrate and the transpeptidation reaction were more sensitive to the mutations than the other reactions studied. The results are discussed and compared with those obtained with the Asn-132 mutants of a class A beta-lactamase.

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Asp-89: a critical residue in maintaining the oligomeric structure of sheep liver cytosolic serine hydroxymethyltransferase

Biochemical Journal ◽

10.1042/bj3430257 ◽

1999 ◽

Vol 343 (1) ◽

pp. 257-263 ◽

Cited By ~ 3

Author(s):

J. V. KRISHNA RAO ◽

Junutula R. JAGATH ◽

Balasubramanya SHARMA ◽

N. APPAJI RAO ◽

H. S. SAVITHRI

Keyword(s):

Specific Activity ◽

Serine Hydroxymethyltransferase ◽

Site Directed Mutagenesis ◽

Ionic Interactions ◽

Wild Type ◽

Wild Type Enzyme ◽

Sheep Liver ◽

Critical Residue ◽

Similar Kinetic

Aspartate residues function as proton acceptors in catalysis and are involved in ionic interactions stabilizing subunit assembly. In an attempt to unravel the role of a conserved aspartate (D89) in sheep-liver tetrameric serine hydroxymethyltransferase (SHMT), it was converted into aspargine by site-directed mutagenesis. The purified D89N mutant enzyme had a lower specific activity compared with the wild-type enzyme. It was a mixture of dimers and tetramers with the proportion of tetramers increasing with an increase in the pyridoxal-5′-phosphate (PLP) concentration used during purification. The D89N mutant tetramer was as active as the wild-type enzyme and had similar kinetic and spectral properties in the presence of 500 μM PLP. The quinonoid spectral intermediate commonly seen in the case of SHMT was also seen in the case of D89N mutant tetramer, although the amount of intermediate formed was lower. Although the purified dimer exhibited visible absorbance at 425 nm, it had a negligible visible CD spectrum at 425 nm and was only 5% active. The apo-D89N mutant tetramer was a dimer unlike the apo-form of the wild-type enzyme which was present predominantly as a tetramer. Furthermore the apo mutant dimer could not be reconstituted to the holo-form by the addition of excess PLP, suggesting that dimer-dimer interactions are weak in this mutant. The recently published crystal structure of human liver cytosolic recombinant SHMT indicates that this residue (D90 in the human enzyme) is located at the N-terminal end of the fourth helix of one subunit and packs against K39 from the second N-terminal helix of the other symmetry related subunit forming the tight dimer. D89 is at the interface of tight dimers where the PLP 5′-phosphate is also bound. Mutation of D89 could lead to weakened ionic interactions in the tight dimer interface, resulting in decreased affinity of the enzyme for the cofactor.

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The potent inhibition of human cytosolic sulfotransferase 1A1 by 17α-ethinylestradiol is due to interactions with isoleucine 89 on loop 1

Hormone Molecular Biology and Clinical Investigation ◽

10.1515/hmbci-2014-0028 ◽

2014 ◽

Vol 20 (3) ◽

Cited By ~ 1

Author(s):

Katie Jo Rohn-Glowacki ◽

Charles N. Falany

Keyword(s):

Docking Studies ◽

Site Directed Mutagenesis ◽

Wild Type ◽

Binding Assays ◽

Wild Type Enzyme ◽

Sulfotransferase 1A1 ◽

Mechanism Of Inhibition ◽

Potent Inhibition

AbstractDrug-drug interactions (DDI) with oral contraceptives containing 17α-ethinylestradiol (EE2) have been well characterized with regard to interactions with phase I drug metaolizing enzymes; however, DDI with EE2 and phase II enzymes have not been as thoroughly addressed. Our laboratory recently reported that in vitro EE2 potently inhibits human cytosolic sulfotransferase (SULT) 1A1 while EE2 was not sulfated until micromolar concentrations. Molecular docking studies demonstrated that Tyr169 and isoleucine 89 (Ile89) may play a role in the inhibitory and/or catalytic positioning of EE2 within the active site of SULT1A1. Therefore, the current study focused on determining the role of Ile89 in the inhibition of SULT1A1 utilizing site-directed mutagenesis. Ile89 was mutated to an alanine and the effect of the mutation was characterized using kinetic and binding assays. SULT1A1-Ile89Ala was found to have a Km for EE2 that was 11-fold greater than wild-type enzyme. A decreased affinity (Kd) of EE2 for SULT1A1-Ile89Ala was apparently responsible for the increase in Km, and also resulted in the loss of the potent inhibition. Molecular modeling was used in an attempt to determine the atomic level changes in binding of EE2 to SULT1A1-Ile89Ala. However, analysis of the effect of the single Ile89 mutation on both the open and closed homology models was not consistent with the docking and kinetic results. Overall, the mechanism of inhibition of EE2 for SULT1A1 is apparently the result of interactions of Ile89 with EE2 holding it in a potent inhibitory conformation, and mutation of the Ile89 significantly decreases the inhibition.

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Site-directed mutagenesis of β-lactamase I: role of Glu-166

Biochemical Journal ◽

10.1042/bj2990671 ◽

1994 ◽

Vol 299 (3) ◽

pp. 671-678 ◽

Cited By ~ 30

Author(s):

Y C Leung ◽

C V Robinson ◽

R T Aplin ◽

S G Waley

Keyword(s):

Active Site ◽

Site Directed Mutagenesis ◽

Side Chain ◽

Kinetic Properties ◽

Ionization Mass ◽

Beta Lactamase ◽

Wild Type ◽

Wild Type Enzyme ◽

Important Residue

Two Glu-166 mutants of beta-lactamase I from Bacillus cereus 569/H were constructed: one with a lengthened side chain (E166Cmc, the S-carboxymethylcysteine mutant) and the other with the side chain shortened and made non-polar (E166A). Their kinetic properties were studied and compared with those of the wild-type and the E166D mutant (with a shortened side chain) previously made by Gibson, Christensen and Waley (1990) (Biochem. J. 272, 613-619). Surprisingly, with good penicillin substrates, Km, kcat. and kcat./Km of the two conservative mutants (E166Cmc and E166D) are similar to those of the non-conservative mutant E166A. Their kcat. values are 3000-fold lower than that of the wild-type enzyme, showing that Glu-166 is a very important residue. The acylenzyme intermediate of E166A and a good substrate, penicillin V, was trapped by acid-quench and observed by electrospray ionization mass spectrometry, suggesting that Glu-166 is more important in catalysing the deacylation step than the acylation step. The beta-lactamase I E166A mutant is about 200-fold more active than the Bacillus licheniformis E166A mutant with nitrocefin or 6 beta-furylacryloyl-amidopenicillanic acid as substrate. This suggested that other groups in the active site of the beta-lactamase I mutant may activate the catalytic water molecule for deacylation.

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Functional control of polypeptide GalNAc-transferase 3 through an acetylation site in the C-terminal lectin domain

Biological Chemistry ◽

10.1515/hsz-2017-0130 ◽

2017 ◽

Vol 398 (11) ◽

pp. 1237-1246

Author(s):

Virginia Lorenz ◽

Romina B. Cejas ◽

Eric P. Bennett ◽

Gustavo A. Nores ◽

Fernando J. Irazoqui

Keyword(s):

Biological Properties ◽

Site Directed Mutagenesis ◽

Wild Type ◽

Wild Type Enzyme ◽

Lectin Domain ◽

Acetylation Site ◽

Functional Control ◽

High Level ◽

A Site

AbstractO-GalNAc glycans are important structures in cellular homeostasis. Their biosynthesis is initiated by members of the polypeptide GalNAc-transferase (ppGalNAc-T) enzyme family. Mutations in ppGalNAc-T3 isoform cause diseases (congenital disorders of glycosylation) in humans. The K626 residue located in the C-terminal β-trefoil fold of ppGalNAc-T3 was predicted to be a site with high likelihood of acetylation by CBP/p300 acetyltransferase. We used a site-directed mutagenesis approach to evaluate the role of this acetylation site in biological properties of the enzyme. Two K626 mutants of ppGalNAc-T3 (T3K626Qand T3K626A) had GalNAc-T activities lower than that of wild-type enzyme. Direct and competitive interaction assays revealed that GalNAc recognition by the lectin domain was altered in the mutants. The presence of GlcNAc glycosides affected the interaction of the three enzymes with mucin-derived peptides. In GalNAc-T activity assays, the presence of GlcNAc glycosides significantly inhibited activity of the mutant (T3K626Q) that mimicked acetylation. Our findings, taken together, reveal the crucial role of the K626 residue in the C-terminal β-trefoil fold in biological properties of human ppGalNAc-T3. We propose that acetylated residues on ppGalNAc-T3 function as control points for enzyme activity, and high level of GlcNAc glycosides promote a synergistic regulatory mechanism, leading to a metabolically disordered state.

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N-glycosylation of human acetylcholinesterase: effects on activity, stability and biosynthesis

Biochemical Journal ◽

10.1042/bj2960649 ◽

1993 ◽

Vol 296 (3) ◽

pp. 649-656 ◽

Cited By ~ 63

Author(s):

B Velan ◽

C Kronman ◽

A Ordentlich ◽

Y Flashner ◽

M Leitner ◽

...

Keyword(s):

Site Directed Mutagenesis ◽

Heat Inactivation ◽

Detectable Effect ◽

Wild Type ◽

Wild Type Enzyme ◽

Triple Mutant ◽

Glycosylation Sites ◽

Peripheral Site ◽

Increased Susceptibility

The role of N-glycosylation in the function of human acetylcholinesterase (HuAChE) was examined by site-directed mutagenesis (Asn to Gln substitution) of the three potential N-glycosylation sites Asn-265, Asn-350 and Asn-464. Analysis of HuAChE mutants, defective in a single or multiple N-glycosylation sites, by expression in transiently or stably transfected human embryonal 293 kidney cells suggests the following. (a) All three AChE glycosylation signals are utilized, but not all the secreted molecules are fully glycosylated. (b) Glycosylation at all sites is important for effective biosynthesis and secretion; extracellular AChE levels in mutants defective in one, two or all three sites amounted to 20-30%, 2-4% and about 0.5% of wild-type level respectively. (c) Some glycosylation mutants display impaired stability, as reflected by increased susceptibility to heat inactivation; substitution of Asn-464 has the most pronounced effect on thermostability. (d) Abrogation of N-glycosylation has no detectable effect on the enzyme activity of HuAChE; all glycosylation mutants, including the triple mutant, hydrolyse acetylthiocholine efficiently, displaying Km, kcat. and kcat./Km values similar to those of the wild-type enzyme. (e) In most mutants, inhibition profiles with edrophonium and bisquaternary ammonium ligands are identical with those of wild-type enzyme; the Asn-350 mutants, however, exhibit a slight decrease in their affinity towards these ligands. (f) Elimination of oligosaccharide side chains has no detectable effect on the surface-related ‘peripheral-site’ functions; like the wild-type enzyme, all mutants were inhibited by propidium and by increased concentrations of acetylthiocholine.

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Active-site serine mutants of the Streptomyces albus G β-lactamase

Biochemical Journal ◽

10.1042/bj2770647 ◽

1991 ◽

Vol 277 (3) ◽

pp. 647-652 ◽

Cited By ~ 10

Author(s):

F Jacob ◽

B Joris ◽

J M Frère

Keyword(s):

Active Site ◽

Specific Activity ◽

Site Directed Mutagenesis ◽

Beta Lactamase ◽

Wild Type ◽

Serine Residue ◽

Wild Type Enzyme ◽

Ph Values ◽

Streptomyces Albus ◽

Small Production

By using site-directed mutagenesis, the active-site serine residue of the Streptomyces albus G beta-lactamase was substituted by alanine and cysteine. Both mutant enzymes were produced in Streptomyces lividans and purified to homogeneity. The cysteine beta-lactamase exhibited a substrate-specificity profile distinct from that of the wild-type enzyme, and its kcat./Km values at pH 7 were never higher than 0.1% of that of the serine enzyme. Unlike the wild-type enzyme, the activity of the mutant increased at acidic pH values. Surprisingly, the alanine mutant exhibited a weak but specific activity for benzylpenicillin and ampicillin. In addition, a very small production of wild-type enzyme, probably due to mistranslation, was detected, but that activity could be selectively eliminated. Both mutant enzymes were nearly as thermostable as the wild-type.

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Key Role of Cysteine Residues in Catalysis and Subcellular Localization of Sulfur Oxygenase-Reductase of Acidianus tengchongensis

Applied and Environmental Microbiology ◽

10.1128/aem.71.2.621-628.2005 ◽

2005 ◽

Vol 71 (2) ◽

pp. 621-628 ◽

Cited By ~ 37

Author(s):

Zhi-Wei Chen ◽

Cheng-Ying Jiang ◽

Qunxin She ◽

Shuang-Jiang Liu ◽

Pei-Jin Zhou

Keyword(s):

Cytoplasmic Membrane ◽

Sulfur Oxidation ◽

Site Directed Mutagenesis ◽

Cysteine Residues ◽

Wild Type ◽

Immune Electron Microscopy ◽

Mutant Proteins ◽

Close Proximity ◽

Catalytic Sites

ABSTRACT Analysis of known sulfur oxygenase-reductases (SORs) and the SOR-like sequences identified from public databases indicated that they all possess three cysteine residues within two conserved motifs (V-G-P-K-V-C31 and C101-X-X-C104; numbering according to the Acidianus tengchongensis numbering system). The thio-modifying reagent N-ethylmaleimide and Zn2+ strongly inhibited the activities of the SORs of A. tengchongensis, suggesting that cysteine residues are important. Site-directed mutagenesis was used to construct four mutant SORs with cysteines replaced by serine or alanine. The purified mutant proteins were investigated in parallel with the wild-type SOR. Replacement of any cysteine reduced SOR activity by 98.4 to 100%, indicating that all the cysteine residues are crucial to SOR activities. Circular-dichroism and fluorescence spectrum analyses revealed that the wild-type and mutant SORs have similar structures and that none of them form any disulfide bond. Thus, it is proposed that three cysteine residues, C31 and C101-X-X-C104, in the conserved domains constitute the putative binding and catalytic sites of SOR. Furthermore, enzymatic activity assays of the subcellular fractions and immune electron microscopy indicated that SOR is not only present in the cytoplasm but also associated with the cytoplasmic membrane of A. tengchongensis. The membrane-associated SOR activity was colocalized with the activities of sulfite:acceptor oxidoreductase and thiosulfate:acceptor oxidoreductase. We tentatively propose that these enzymes are located in close proximity on the membrane to catalyze sulfur oxidation in A. tengchongensis.

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