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

1994 ◽  
Vol 299 (3) ◽  
pp. 671-678 ◽  
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.

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 The mutation Lys234His yields a class A β-lactamase with a novel pH-dependence

1991 ◽  
Vol 278 (3) ◽  
pp. 673-678 ◽  
Author(s):  
J Brannigan ◽  
A Matagne ◽  
F Jacob ◽  
C Damblon ◽  
B Joris ◽  
...  
Keyword(s):  
Active Site ◽  
Positive Charge ◽  
Ph Dependence ◽  
Side Chain ◽  
Beta Lactamase ◽  
Wild Type ◽  
Wild Type Enzyme ◽  
Beta Lactamases ◽  
Class A ◽  
Transition State Stabilization

The lysine-234 residue is highly conserved in beta-lactamases and in nearly all active-site-serine penicillin-recognizing enzymes. Its replacement by a histidine residue in the Streptomyces albus G class A beta-lactamase yielded an enzyme the pH-dependence of which was characterized by the appearance of a novel pK, which could be attributed to the newly introduced residue. At low pH, the kcat, value for benzylpenicillin was as high as 50% of that of the wild-type enzyme, demonstrating that an efficient active site was maintained. Both kcat. and kcat/Km dramatically decreased above pH 6 but the decrease in kcat./Km could not be attributed to larger Km values. Thus a positive charge on the side chain of residue 234 appears to be more essential for transition-state stabilization than for initial recognition of the substrate ground state.

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.

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 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 The role of residues glutamate-50 and phenylalanine-496 in Zymomonas mobilis pyruvate decarboxylase

1996 ◽  
Vol 315 (3) ◽  
pp. 745-751 ◽  
Author(s):  
Judith M. CANDY ◽  
Jinichiro KOGA ◽  
Peter F. NIXON ◽  
Ronald G. DUGGLEBY
Keyword(s):  
Fluorescence Quenching ◽  
Zymomonas Mobilis ◽  
Specific Interaction ◽  
Pyruvate Decarboxylase ◽  
Site Directed Mutagenesis ◽  
Kinetic Properties ◽  
Subunit Structure ◽  
Wild Type ◽  
Cloned Gene

Several enzymes require thiamine diphosphate (ThDP) as an essential cofactor, and we have used one of these, pyruvate decarboxylase (PDC; EC 4.1.1.1) from Zymomonas mobilis, as a model for this group of enzymes. It is well suited for this purpose because of its stability, ease of purification, homotetrameric subunit structure and simple kinetic properties. Crystallographic analyses of three ThDP-dependent enzymes [Müller, Lindqvist, Furey, Schulz, Jordan and Schneider (1993) Structure 1, 95–103] have suggested that an invariant glutamate participates in catalysis. In order to evaluate the role of this residue, identified in PDC from Zymomonas mobilis as Glu-50, it has been altered to glutamine and aspartate by site-directed mutagenesis of the cloned gene. The mutant proteins were expressed in Escherichia coli. Here we demonstrate that substitution with aspartate yields an enzyme with 3% of the activity of the wild-type, but with normal kinetics for pyruvate. Replacement of Glu-50 with glutamine yields an enzyme with only 0.5% of the catalytic activity of the wild-type enzyme. Each of these mutant enzymes has a decreased affinity for both ThDP and Mg2+. It has been reported that the binding of cofactors to apoPDC quenches the intrinsic tryptophan fluorescence [Diefenbach and Duggleby (1991) Biochem. J. 276, 439–445] and we have identified the residue responsible as Trp-487 [Diefenbach, Candy, Mattick and Duggleby (1992) FEBS Lett. 296, 95–98]. Although this residue is some distance from the cofactor binding site, it lies in the dimer interface, and the proposal has been put forward [Dyda, Furey, Swaminathan, Sax, Farrenkopf and Jordan (1993) Biochemistry 32, 6165–6170] that alteration of ring stacking with Phe-496 of the adjacent subunit is the mechanism of fluorescence quenching when cofactors bind. The closely related enzyme indolepyruvate decarboxylase (from Enterobacter cloacae) has a leucine residue at the position corresponding to Phe-496 but shows fluorescence quenching properties that are similar to those of PDC. This suggests that the fluorescence quenching is due to some perturbation of the local environment of Trp-487 rather than to a specific interaction with Phe-496. This latter hypothesis is supported by our data: mutation of this phenylalanine to leucine, isoleucine or histidine in PDC does not eliminate the fluorescence quenching upon addition of cofactors.

scholarly journals Point mutations of two arginine residues in the Streptomyces R61 dd-peptidase

1992 ◽  
Vol 283 (1) ◽  
pp. 123-128 ◽  
Author(s):  
C Bourguignon-Bellefroid ◽  
B Joris ◽  
J Van Beeumen ◽  
J M Ghuysen ◽  
J M Frère
Keyword(s):  
Enzyme Activity ◽  
Enzyme Stability ◽  
Point Mutations ◽  
Site Directed Mutagenesis ◽  
Side Chain ◽  
Directed Mutagenesis ◽  
Serine Residue ◽  
Wild Type Enzyme ◽  
Arginine Residues

Incubation of the exocellular DD-carboxypeptidase/transpeptidase of Streptomyces R61 with phenylglyoxal resulted in a time-dependent decrease in the enzyme activity. This inactivation was demonstrated to be due to modification of the Arg-99 side chain. In consequence, the role of that residue was investigated by site-directed mutagenesis. Mutation of Arg-99 into leucine appeared to be highly detrimental to enzyme stability, reflecting a determining structural role for this residue. The conserved Arg-103 residue was also substituted by using site-directed mutagenesis. The modification to a serine residue yielded a stable enzyme, the catalytic properties of which were similar to those of the wild-type enzyme. Thus Arg-103, although strictly conserved or replaced by a lysine residue in most of the active-site penicillin-recognizing proteins, did not appear to fulfil any essential role in either the enzyme activity or structure.

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.

A mutant of Escherichia coli citrate synthase that affects the allosteric equilibrium

1991 ◽  
Vol 69 (4) ◽  
pp. 232-238 ◽  
Author(s):  
Deborah H. Anderson ◽  
Lynda J. Donald ◽  
Mary V. Jacob ◽  
Harry W. Duckworth
Keyword(s):  
Escherichia Coli ◽  
Model Building ◽  
Citrate Synthase ◽  
Salt Bridge ◽  
Difference Spectrum ◽  
Site Directed Mutagenesis ◽  
Kinetic Properties ◽  
Wild Type ◽  
Wild Type Enzyme ◽  
In The Wild

We describe a mutant of Escherichia coli citrate synthase, CS R319L, in which the arginine residue at position 319 of the sequence has been replaced by leucine. In this mutant, saturation by the substrate acetyl-CoA is changed from sigmoid (Hill parameter = 1.75 ± 0.2) to hyperbolic (Hill parameter = 1.0 ± 0.1) and dependence on the activator KCl is greatly reduced. Further mutations at this site and at position 343 (which model building predicts is close enough to allow a side-chain interaction with position 319) are also described. In the wild-type enzyme, the model suggests the possibility of a salt-bridge interaction between Arg-319 (located on the P helix in the small domain) and Glu-343 (in the Q helix in the same domain), but mutation of Glu-343 to Ala (CS E343A) produced a much smaller difference in the kinetic properties than the Arg-319 to Leu mutation did. Small changes in kinetic properties were also obtained with an Arg-319 → Glu (CS R319E) mutation. In CS R319L, oxaloacetate, the first substrate to bind, induces an ultraviolet difference spectrum which is obtained with wild-type enzyme only in the presence of KCl. To account for these observations we postulate that wild-type E. coli citrate synthase exists in two conformational states, T and R, which are equilibrium; T state binds NADH, the allosteric inhibitor, while R state binds substrates and can be converted to another substrate-binding state, R′, by KCl. In the CS R319L mutant, it is proposed that the T ↔ R equilibrium is shifted significantly towards R state, permitting an easier interaction with substrates in the absence of KCl. To account for the behaviour of enzymes mutated at amino acids 319 and 343, we propose that the allosteric transition between T and R states involves a subtle adjustment of the relative positions of the P and Q helices, which is affected by some of the mutations tested.Key words: citrate synthase, allostery, site-directed mutagenesis.

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