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

1999 ◽  
Vol 343 (1) ◽  
pp. 257-263 ◽  
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.

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

1997 ◽  
Vol 327 (3) ◽  
pp. 877-882 ◽  
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.

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

1991 ◽  
Vol 277 (3) ◽  
pp. 647-652 ◽  
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.

scholarly journals Production of l-Ribose from l-Ribulose by a Triple-Site Variant of Mannose-6-Phosphate Isomerase from Geobacillus thermodenitrificans

2012 ◽  
Vol 78 (11) ◽  
pp. 3880-3884 ◽  
Author(s):  
Yu-Ri Lim ◽  
Soo-Jin Yeom ◽  
Deok-Kun Oh
Keyword(s):  
Specific Activity ◽  
Thermus Thermophilus ◽  
Catalytic Efficiency ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Wild Type ◽  
Geobacillus Thermodenitrificans ◽  
Wild Type Enzyme ◽  
Content Type ◽  
Mannose 6 Phosphate

ABSTRACTA triple-site variant (W17Q N90A L129F) of mannose-6-phosphate isomerase fromGeobacillus thermodenitrificanswas obtained by combining variants with residue substitutions at different positions after random and site-directed mutagenesis. The specific activity and catalytic efficiency (kcat/Km) forl-ribulose isomerization of this variant were 3.1- and 7.1-fold higher, respectively, than those of the wild-type enzyme at pH 7.0 and 70°C in the presence of 1 mM Co2+. The triple-site variant produced 213 g/literl-ribose from 300 g/literl-ribulose for 60 min, with a volumetric productivity of 213 g liter−1h−1, which was 4.5-fold higher than that of the wild-type enzyme. Thekcat/Kmand productivity of the triple-site variant were approximately 2-fold higher than those of theThermus thermophilusR142N variant of mannose-6-phosphate isomerase, which exhibited the highest values previously reported.

scholarly journals The role of cysteine residues 129 and 329 in Escherichia coli K1 CMP-NeuAc synthase

1993 ◽  
Vol 295 (2) ◽  
pp. 485-491 ◽  
Author(s):  
G Zapata ◽  
P P Roller ◽  
J Crowley ◽  
W F Vann
Keyword(s):  
Escherichia Coli ◽  
Specific Activity ◽  
Site Directed Mutagenesis ◽  
Cysteine Residues ◽  
Directed Mutagenesis ◽  
Wild Type ◽  
Acetylneuraminic Acid ◽  
Denatured States ◽  
Escherichia Coli K1

N-Acetylneuraminic acid cytidyltransferase (CMP-NeuAc synthase) of Escherichia coli K1 is sensitive to mercurials and has cysteine residues only at positions 129 and 329. The role of these residues in the catalytic activity and structure of the protein has been investigated by site-directed mutagenesis and chemical modification. The enzyme is inactivated by the thiol-specific reagent dithiodipyridine. Inactivation by this reagent is decreased in the presence of the nucleotide substrate CTP, suggesting that a thiol residue is at or near the active site. Site-directed mutagenesis of either residue Cys-129 to serine or Cys-329 to selected amino acids has minor effects on the specific activity of the enzyme, suggesting that cysteine is not essential for catalysis and that a disulphide bond is not an essential structural component. The limited reactivity of the enzyme to other thiol-blocking reagents suggests that its cysteine residues are partially exposed. The accessibility and role of the cysteine residues in enzyme structure were investigated by fluorescence, c.d. and denaturation studies of wild-type and mutant enzymes. The mutation of Cys-129 to serine makes the enzyme more sensitive to heat and chemical denaturation, but does not cause gross changes in the protein structure as judged by the c.d. spectrum. The mutant containing Ser-129 instead of Cys-129 had a complex denaturation pathway similar to that of wild-type E. coli K1 CMP-NeuAc synthase consisting of several partially denatured states. Cys-329 reacts more readily with N-[14C]ethylmaleimide when the enzyme is in a heat-induced relaxed state. Cys-129 is less reactive and is probably a buried residue.

scholarly journals Substitution of asparagine residues in Aspergillus awamori glucoamylase by site-directed mutagenesis to eliminate N-glycosylation and inactivation by deamidation

1994 ◽  
Vol 301 (1) ◽  
pp. 275-281 ◽  
Author(s):  
H M Chen ◽  
C Ford ◽  
P J Reilly
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cell Membrane ◽  
Specific Activity ◽  
Site Directed Mutagenesis ◽  
Aspergillus Awamori ◽  
Directed Mutagenesis ◽  
Wild Type ◽  
Wild Type Enzyme ◽  
Recognition Sites ◽  
Membrane Components

Aspergillus awamori glucoamylase is a secreted glycoprotein containing N-linked carbohydrate recognition sites at Asn-171, Asn-182 and Asn-395. Site-directed mutagenesis was performed at Asn-182 and Asn-395 to determine whether these residues were N-glycosylated by Saccharomyces cerevisiae, to investigate the function of any glycans linked to them, and to determine the effect of their deamidation on glucoamylase thermostability. Asn-171 and Asn-395, but not Asn-182, were N-glycosylated. Deletion of the glycan N-linked to Asn-395 did not affect specific activity, but greatly decreased enzyme secretion and thermostability. The mutant lacking the N-glycan linked to Asn-395 was synthesized very slowly, and was more associated with cell membrane components and susceptible to proteinase degradation than were wild-type or other mutant glucoamylases. Its secreted form was 30-fold less thermostable than wild-type enzyme at pH 4.5. Replacement of Asn-182 by Gln to eliminate deamidation at this site did not change glucoamylase specific activity or thermostability, while replacement by Asp decreased specific activity about 25%, but increased thermostability moderately at pH 4.5 below 70 degrees C. Both mutations of Asn-182 increased glucoamylase production.

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

2010 ◽  
Vol 76 (23) ◽  
pp. 7723-7733 ◽  
Author(s):  
Fernando L�pez-Gallego ◽  
GraysonT. Wawrzyn ◽  
Claudia Schmidt-Dannert
Keyword(s):  
Marked Effect ◽  
Biological Activities ◽  
Site Directed Mutagenesis ◽  
Gas Chromatography Mass Spectrometry ◽  
Farnesyl Pyrophosphate ◽  
Wild Type ◽  
Wild Type Enzyme ◽  
Terpene Synthases ◽  
Product Profile

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.

scholarly journals Identification by site-directed mutagenesis of three essential histidine residues in membrane dipeptidase, a novel mammalian zinc peptidase

1997 ◽  
Vol 326 (1) ◽  
pp. 47-51 ◽  
Author(s):  
Shoshana KEYNAN ◽  
Nigel M. HOOPER ◽  
Anthony J. TURNER
Keyword(s):  
Specific Activity ◽  
Mammalian Species ◽  
Competitive Inhibitor ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Wild Type ◽  
Wild Type Enzyme ◽  
Renal Metabolism ◽  
Leukotriene D4 ◽  
Histidine Residues

Membrane dipeptidase (EC 3.4.13.19) is a plasma membrane zinc peptidase that is involved in the renal metabolism of glutathione and its conjugates, such as leukotriene D4. The enzyme lacks the classical signatures of other zinc-dependent hydrolases and shows no homology with any other mammalian protein. We have used site-directed mutagenesis to explore the roles of five histidine residues in pig membrane dipeptidase that are conserved among mammalian species. When expressed in COS-1 cells, the mutants H49K and H128L exhibited a specific activity and Km for the substrate Gly-D-Phe comparable with those of the wild-type enzyme. However, the mutants H20L, H152L and H198K were inactive, but were expressed at the cell surface at equivalent levels to the wild-type, as assessed by immunoblotting and immunofluorescence. These three mutants were compared with regard to their ability to bind to the competitive inhibitor cilastatin, which binds with equal efficacy to native and EDTA-treated pig kidney membrane dipeptidase. Expressed wild-type enzyme and mutants H20L and H198K were efficiently bound by cilastatin–Sepharose, but H152L failed to bind. Thus His-152 appears to be involved in the binding of substrate or inhibitor, whereas His-20 and His-198 appear to be involved in catalysis. Membrane dipeptidase shares some similarity with a dipeptidase recently cloned from Acinetobacter calcoaceticus. In particular, His-20 and His-198 of membrane dipeptidase are conserved in the bacterial enzyme, as are Glu-125 and His-219, previously shown to be required for catalytic activity.

scholarly journals PGRP-LB: An Inside View into the Mechanism of the Amidase Reaction

2021 ◽  
Vol 22 (9) ◽  
pp. 4957
Author(s):  
Julien Orlans ◽  
Carole Vincent-Monegat ◽  
Isabelle Rahioui ◽  
Catherine Sivignon ◽  
Agata Butryn ◽  
...  
Keyword(s):  
Specific Activity ◽  
Insect Immunity ◽  
Site Directed Mutagenesis ◽  
Nucleophilic Attack ◽  
Wild Type ◽  
X Ray ◽  
Enzymatic Mechanism ◽  
Imd Pathway ◽  
Essential Function

Peptidoglycan recognition proteins (PGRPs) are ubiquitous among animals and play pivotal functions in insect immunity. Non-catalytic PGRPs are involved in the activation of immune pathways by binding to the peptidoglycan (PGN), whereas amidase PGRPs are capable of cleaving the PGN into non-immunogenic compounds. Drosophila PGRP-LB belongs to the amidase PGRPs and downregulates the immune deficiency (IMD) pathway by cleaving meso-2,6-diaminopimelic (meso-DAP or DAP)-type PGN. While the recognition process is well analyzed for the non-catalytic PGRPs, little is known about the enzymatic mechanism for the amidase PGRPs, despite their essential function in immune homeostasis. Here, we analyzed the specific activity of different isoforms of Drosophila PGRP-LB towards various PGN substrates to understand their specificity and role in Drosophila immunity. We show that these isoforms have similar activity towards the different compounds. To analyze the mechanism of the amidase activity, we performed site directed mutagenesis and solved the X-ray structures of wild-type Drosophila PGRP-LB and its mutants, with one of these structures presenting a protein complexed with the tracheal cytotoxin (TCT), a muropeptide derived from the PGN. Only the Y78F mutation abolished the PGN cleavage while other mutations reduced the activity solely. Together, our findings suggest the dynamic role of the residue Y78 in the amidase mechanism by nucleophilic attack through a water molecule to the carbonyl group of the amide function destabilized by Zn2+.

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

1999 ◽  
Vol 337 (3) ◽  
pp. 503-506 ◽  
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 ◽  
Similar Product

β-Fructofuranosidases share a conserved aspartic acid-containing motif (Arg-Asp-Pro; RDP) which is absent from α-glucopyranosidases. The role of Asp-309 located in the RDP motif of levansucrase (EC 2.4.1.10) from Acetobacter diazotrophicus SRT4 was studied by site-directed mutagenesis. Substitution of Asp-309 by Asn did not affect enzyme secretion. The kcat of the mutant levansucrase was reduced 75-fold, but its Km was similar to that of the wild-type enzyme, indicating that Asp-309 plays a major role in catalysis. The two levansucrases showed optimal activity at pH 5.0 and yielded similar product profiles. Thus the mutation D309N affected the efficiency of sucrose hydrolysis, but not the enzyme specificity. Since the RDP motif is present in a conserved position in fructosyltransferases, invertases, levanases, inulinases and sucrose-6-phosphate hydrolases, it is likely to have a common functional role in β-fructofuranosidases.

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

1993 ◽  
Vol 293 (1) ◽  
pp. 195-201 ◽  
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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