scholarly journals Re-design of Saccharomyces cerevisiae flavocytochrome b2: introduction of l-mandelate dehydrogenase activity

1998 ◽  
Vol 333 (1) ◽  
pp. 117-120 ◽  
Author(s):  
Rhona SINCLAIR ◽  
Graeme A. REID ◽  
Stephen K. CHAPMAN
Keyword(s):  
Lactate Dehydrogenase ◽  
Dehydrogenase Activity ◽  
Active Site ◽  
Mutant Enzyme ◽  
Activity Levels ◽  
Wild Type ◽  
Wild Type Enzyme ◽  
Activity Increase ◽  
Double Mutation ◽  
Flavocytochrome B2

Flavocytochrome b2 from Saccharomyces cerevisiaeis an l-lactate dehydrogenase which exhibits only barely detectable activity levels towards another 2-hydroxyacid, l-mandelate. Using protein engineering methods we have altered the active site of flavocytochrome b2 and successfully introduced substantial mandelate dehydrogenase activity into the enzyme. Changes to Ala-198 and Leu-230 have significant effects on the ability of the enzyme to utilize l-mandelate as a substrate. The double mutation of Ala-198 → Gly and Leu-230 → Ala results in an enzyme with a kcat value (25 °C) with l-mandelate of 8.5 s-1, which represents an increase of greater than 400-fold over the wild-type enzyme. Perhaps more significantly, the mutant enzyme has a catalytic efficiency (as judged by kcat/Km values) that is 6-fold higher with l-mandelate than it is with l-lactate. Closer examination of the X-ray structure of S. cerevisiae flavocytochrome b2 led us to conclude that one of the haem propionate groups might interfere with the binding of l-mandelate at the active site of the enzyme. To test this idea, the activity with l-mandelate of the independently expressed flavodehydrogenase domain (FDH), was examined and found to be higher than that seen with the wild-type enzyme. In addition, the double mutation of Ala-198 → Gly and Leu-230 → Ala introduced into FDH produced the greatest mandelate dehydrogenase activity increase, with a kcat value more than 700-fold greater than that seen with the wild-type holoenzyme. In addition, the enzyme efficiency (kcat/Km) of this mutant enzyme was more than 20-fold greater with l-mandelate than with l-lactate. We have therefore succeeded in constructing an enzyme which is now a better mandelate dehydrogenase than a lactate dehydrogenase.

scholarly journals Tyr-143 facilitates interdomain electron transfer in flavocytochrome b2

1992 ◽  
Vol 285 (1) ◽  
pp. 187-192 ◽  
Author(s):  
C S Miles ◽  
N Rouvière-Fourmy ◽  
F Lederer ◽  
F S Mathews ◽  
G A Reid ◽  
...  
Keyword(s):  
Electron Transfer ◽  
Steady State ◽  
Isotope Effects ◽  
Mutant Enzyme ◽  
Stopped Flow ◽  
Wild Type ◽  
Wild Type Enzyme ◽  
Flavocytochrome B2 ◽  
Rate Determining Step ◽  
Kinetic Isotope

The role of Tyr-143 in the catalytic cycle of flavocytochrome b2 (L-lactate:cytochrome c oxidoreductase) has been examined by replacement of this residue with phenylalanine. The electron-transfer steps in wild-type and mutant flavocytochromes b2 have been investigated by using steady-state and stopped-flow kinetic methods. The most significant effect of the Tyr-143----Phe mutation is a change in the rate-determining step in the reduction of the enzyme. For wild-type enzyme the main rate-determining step is proton abstraction at the C-2 position of lactate, as shown by the 2H kinetic-isotope effect. However, for the mutant enzyme it is clear that the slowest step is interdomain electron transfer between the FMN and haem prosthetic groups. In fact, the rate of haem reduction by lactate, as determined by the stopped-flow method, is decreased by more than 20-fold, from 445 +/- 50 s-1 (25 degrees C, pH 7.5) in the wild-type enzyme to 21 +/- 2 s-1 in the mutant enzyme. Decreases in kinetic-isotope effects seen with [2-2H]lactate for mutant enzyme compared with wild-type, both for flavin reduction (from 8.1 +/- 1.4 to 4.3 +/- 0.8) and for haem reduction (from 6.3 +/- 1.2 to 1.6 +/- 0.5) also provide support for a change in the nature of the rate-determining step. Other kinetic parameters determined by stopped-flow methods and with two external electron acceptors (cytochrome c and ferricyanide) under steady-state conditions are all consistent with this mutation having a dramatic effect on interdomain electron transfer. We conclude that Tyr-143, an active-site residue which lies between the flavodehydrogenase and cytochrome domains of flavocytochrome b2, plays a key role in facilitating electron transfer between FMN and haem groups.

scholarly journals Contribution of a buried aspartate residue towards the catalytic efficiency and structural stability of Bacillus stearothermophilus lactate dehydrogenase

1994 ◽  
Vol 300 (2) ◽  
pp. 491-499 ◽  
Author(s):  
T J Nobbs ◽  
A Cortés ◽  
J L Gelpi ◽  
J J Holbrook ◽  
T Atkinson ◽  
...  
Keyword(s):  
Hydrogen Bonding ◽  
Lactate Dehydrogenase ◽  
Active Site ◽  
Bacillus Stearothermophilus ◽  
Mutant Enzyme ◽  
Hydroxyl Group ◽  
Side Chain ◽  
General Effect ◽  
Carboxylate Group ◽  
Wild Type

The X-ray structure of lactate dehydrogenase (LDH) shows the side-chain carboxylate group of Asp-143 to be buried in the hydrophobic interior of the enzyme, where it makes hydrogen-bonding interactions with both the side-chain hydroxyl group of Ser-273 and the main-chain amide group of His-195. This is an unusual environment for a carboxylate side-chain as hydrogen bonding normally occurs with water molecules at the surface of the protein. A charged hydrogen-bonding interaction in the interior of a protein would be expected to be much stronger than a similar interaction on the solvent-exposed exterior. In this respect the side-chain carboxylate group of Asp-143 appears to be important for maintaining tertiary structure by providing a common linkage point between three discontinuous elements of the secondary structure, alpha 1F, beta K and the beta-turn joining beta G and beta H. The contribution of the Asp-143 side-chain to the structure and function of Bacillus stearothermophilus LDH was assessed by creating a mutant enzyme containing Asn-143. The decreased thermal stability of both unactivated and fructose-1,6-diphosphate (Fru-1,6-P2)-activated forms of the mutant enzyme support a structural role for Asp-143. Furthermore, the difference in stability of the wild-type and mutant enzymes in guanidinium chloride suggested that the carboxylate group of Asp-143 contributes at least 22 kJ/mol to the conformational stability of the wild-type enzyme. However, there was no alteration in the amount of accessible tryptophan fluorescence in the mutant enzyme, indicating that the mutation caused a structural weakness rather than a gross conformational change. Comparison of the wild-type and mutant enzyme steady-state parameters for various 2-keto acid substrates showed the mutation to have a general effect on catalysis, with an average difference in binding energy of 11 kJ/mol for the transition-state complexes. The different effects of pH and Fru-1,6-P2 on the wild-type and mutant enzymes also confirmed a perturbation of the catalytic centre in the mutant enzyme. As the side-chain of Asp-143 is not sufficiently close to the active site to be directly involved in catalysis or substrate binding it is proposed that the effects on catalysis shown by the mutant enzyme are induced either by a structural change or by charge imbalance at the active site.(ABSTRACT TRUNCATED AT 400 WORDS)

scholarly journals Modulation of flavocytochrome b2 intraprotein electron transfer via an interdomain hinge region

1996 ◽  
Vol 316 (2) ◽  
pp. 507-513 ◽  
Author(s):  
R. Eryl SHARP ◽  
Stephen K. CHAPMAN ◽  
Graeme A. REID
Keyword(s):  
Electron Transfer ◽  
Steady State ◽  
Lactate Dehydrogenase ◽  
Structural Integrity ◽  
Hinge Region ◽  
Wild Type ◽  
Wild Type Enzyme ◽  
Lactate Dehydrogenase Activity ◽  
Flavocytochrome B2

The two domains of flavocytochrome b2 are connected by a typical hinge peptide. To probe the role of the hinge in modulating the efficiency of intraprotein electron transfer between these two domains, a number of mutant enzymes with truncated hinge regions were previously constructed and characterized [Sharp, Chapman and Reid (1996) Biochemistry 35, 891–899]. In the present study two mutant enzymes with elongated hinge regions have been constructed (HI3 and HI6) to further our understanding of the controlling influence of hinge length and primary structure on intraprotein electron transfer. Modification of the hinge had little effect on the lactate dehydrogenase activity of the enzyme, as was evident from steady-state experiments using ferricyanide as electron acceptor and from pre-steady-state experiments monitoring flavin reduction. However, the hinge insertions lowered the enzyme's effectiveness as a cytochrome c reductase. This effect results from a defect at the first interdomain electron-transfer step (FMNH2 → haem electron transfer), where the rate constants for haem reduction in HI3 and HI6 were 50-and 100-fold lower than the corresponding value for the wild-type enzyme. Preservation of structural integrity within the hinge region is apparently essential for efficient intraprotein electron transfer.

scholarly journals Complementation and enzyme studies of revertants induced in an am mutant of N. crassa

1965 ◽  
Vol 6 (3) ◽  
pp. 419-432 ◽  
Author(s):  
J. A. Pateman ◽  
J. R. S. Fincham
Keyword(s):  
Glutamate Dehydrogenase ◽  
Dehydrogenase Activity ◽  
Ultra Violet ◽  
Mutant Enzyme ◽  
Ammonium Ion ◽  
Wild Type ◽  
Wild Type Enzyme ◽  
Ultra Violet Light ◽  
Violet Light ◽  
Glutamate Dehydrogenase Activity

A total of eighty-seven revertants were induced by ultra-violet light in an am3 strain. All of these revertants appear to be the result of mutation at sites in or close to the am locus. Fourteen of the eighty-seven revertants were partial revertants in that under some conditions of assay they possessed low glutamate dehydrogenase activity compared with the wild-type although their growth rate was similar to that of the wild-type. Enzyme extracts of thirteen of the partial revertants were assayed for glutamate dehydrogenase in various ways in order to establish qualitative distinctions between different kinds of mutant enzyme. On the basis of these tests six different groups were established, of which one contained six revertants, one three and the others one. All except one of the mutant enzyme types showed a marked activation when incubated with α-oxoglutarate plus NADPH2, and all of these had Michaelis constants for ammonium ion much higher than is found for the wild-type enzyme. The remaining group of three revertants gave, at first, no enzyme activity in any of the assay systems. Two of these (the third was not tested) were shown to produce an enzyme variety which becomes quite inactive in phosphate buffer at pH 8·0 but can be fully activated by the addition of ethylenediamine tetra-acetic acid. Forced heterocaryons between each of six partial revertants and eleven am mutants were made and the resultant sixty-six heterocaryons assayed for glutamate dehydrogenase activity. The partial revertants differed among themselves in their complementation characteristics. Some complemented with none of the am mutants, some with am1 only, and some with am1 or with am7. The complementation tests confirmed the differences established by the enzyme studies. The data presented here, together with previous work, demonstrate that ultra-violet light induced mutation in an am strain can result in at least eight types of revertant differing from each other in respect of the glutamate dehydrogenase variety which each can produce.

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 importance of the interdomain hinge in intramolecular electron transfer in flavocytochrome b2

1993 ◽  
Vol 291 (1) ◽  
pp. 89-94 ◽  
Author(s):  
P White ◽  
F D C Manson ◽  
C E Brunt ◽  
S K Chapman ◽  
G A Reid
Keyword(s):  
Electron Transfer ◽  
Steady State ◽  
Cytochrome C ◽  
Kinetic Properties ◽  
Stopped Flow ◽  
Wild Type ◽  
Wild Type Enzyme ◽  
Cytochrome C Reductase ◽  
Flavocytochrome B2 ◽  
Cytochrome C Reduction

The two distinct domains of flavocytochrome b2 (L-lactate:cytochrome c oxidoreductase) are connected by a typical hinge peptide. The amino acid sequence of this interdomain hinge is dramatically different in flavocytochromes b2 from Saccharomyces cerevisiae and Hansenula anomala. This difference in the hinge is believed to contribute to the difference in kinetic properties between the two enzymes. To probe the importance of the hinge, an interspecies hybrid enzyme has been constructed comprising the bulk of the S. cerevisiae enzyme but containing the H. anomala flavocytochrome b2 hinge. The kinetic properties of this ‘hinge-swap’ enzyme have been investigated by steady-state and stopped-flow methods. The hinge-swap enzyme remains a good lactate dehydrogenase as is evident from steady-state experiments with ferricyanide as acceptor (only 3-fold less active than wild-type enzyme) and stopped-flow experiments monitoring flavin reduction (2.5-fold slower than in wild-type enzyme). The major effect of the hinge-swap mutation is to lower dramatically the enzyme's effectiveness as a cytochrome c reductase; kcat. for cytochrome c reduction falls by more than 100-fold, from 207 +/- 10 s-1 (25 degrees C, pH 7.5) in the wild-type enzyme to 1.62 +/- 0.41 s-1 in the mutant enzyme. This fall in cytochrome c reductase activity results from poor interdomain electron transfer between the FMN and haem groups. This can be demonstrated by the fact that the kcat. for haem reduction in the hinge-swap enzyme (measured by the stopped-flow method) has a value of 1.61 +/- 0.42 s-1, identical with the value for cytochrome c reduction and some 300-fold lower than the value for the wild-type enzyme. From these and other kinetic parameters, including kinetic isotope effects with [2-2H]lactate, we conclude that the hinge plays a crucial role in allowing efficient electron transfer between the two domains of flavocytochrome b2.

scholarly journals Hydration change during the aging of phosphorylated human butyrylcholinesterase: importance of residues aspartate-70 and glutamate-197 in the water network as probed by hydrostatic and osmotic pressures

1999 ◽  
Vol 343 (2) ◽  
pp. 361-369 ◽  
Author(s):  
Patrick MASSON ◽  
Cécile CLÉRY ◽  
Patrice GUERRA ◽  
Arnaud REDSLOB ◽  
Christine ALBARET ◽  
...  
Keyword(s):  
Molecular Dynamics ◽  
Hydrostatic Pressure ◽  
Transition State ◽  
Osmotic Pressure ◽  
Active Site ◽  
Aging Process ◽  
Wild Type ◽  
Water Network ◽  
Wild Type Enzyme ◽  
Human Butyrylcholinesterase

Wild-type human butyrylcholinesterase (BuChE) and Glu-197 → Asp and Asp-70 → Gly mutants (E197D and D70G respectively) were inhibited by di-isopropyl phosphorofluoridate under standard conditions of pH, temperature and pressure. The effect of hydrostatic and osmotic pressures on the aging process (dealkylation of an isopropyl chain) of phosphorylated enzymes [di-isopropylated (DIP)-BuChE] was investigated. Hydrostatic pressure markedly increased the rate of aging of wild-type enzyme. The average activation volume (δV≠) for the dealkylation reaction was -170 ml/mol for DIP wild-type BuChE. On the other hand, hydrostatic pressure had little effect on the aging of the DIP mutants (δV≠ = -2.6 ml/mol for E197D and -2 ml/mol for D70G), suggesting that the transition state of the aging process was associated with an extended hydration and conformational change in wild-type BuChE, but not in the mutants. The rate of aging of wild-type and mutant enzymes decreased with osmotic pressure, allowing very large positive osmotic activation volumes (δV≠osm) to be estimated, thus probing the participation of water in the aging process. Molecular dynamics simulations performed on the active-site gorge of the wild-type DIP adduct showed that the isopropyl chain involved in aging was highly solvated, supporting the idea that water is important for stabilizing the transition state of the dealkylation reaction. Wild-type BuChE was inhibited by soman (pinacolyl methylphosphonofluoridate). Electrophoresis performed under high pressure [up to 2.5 kbar (1 bar = 105 Pa)] showed that the soman-aged enzyme did not pass through a pressure-induced, molten-globule transition, unlike the native wild-type enzyme. Likewise, this transition was not seen for the native E197D and D70G mutants, indicating that these mutants are resistant to the penetration of water into their structure. The stability energetics of native and soman-aged wild-type BuChE were determined by differential scanning calorimetry. The pH-dependence of the midpoint transition temperature of endotherms indicated that the high difference in stabilization energy between aged and native BuChE (δδG = 23.7 kJ/mol at pH 8.0) is mainly due to the salt bridge between protonated His-438 and PO-, with pKHis-438 = 8.3. A molecular dynamics simulation on the MIP adduct showed that there is no water molecule around the ion pair. The ‘hydrostatic versus osmotic pressure’ approach probed the importance of water in aging, and also revealed that Asp-70 and Glu-197 are the major residues controlling both the dynamics and the structural organization of the water/hydrogen-bond network in the active-site gorge of BuChE. In wild-type BuChE both residues function like valves, whereas in the mutant enzymes the water network is slack, and residues Gly-70 and Asp-197 function like check valves, i.e. forced penetration of water into the gorge is not easily achieved, thereby facilitating the release of water.

scholarly journals Inhibition of Inositol Phosphorylceramide Synthase by the Cyclic Peptide Aureobasidin A

2009 ◽  
Vol 53 (2) ◽  
pp. 496-504 ◽  
Author(s):  
Paul A. Aeed ◽  
Casey L. Young ◽  
Marek M. Nagiec ◽  
Åke P. Elhammer
Keyword(s):  
Cyclic Peptide ◽  
Time Dependent ◽  
Mutant Enzyme ◽  
Kinetic Properties ◽  
Dependent Manner ◽  
Wild Type ◽  
Wild Type Enzyme ◽  
Noncompetitive Inhibitor ◽  
Aureobasidin A ◽  
Comparison Of The Results

ABSTRACT By using a detergent-washed membrane preparation, the interaction of the fungal natural product inhibitor aureobasidin A (AbA) with inositol phosphorylceramide synthase (IPC synthase) was studied by kinetic analysis of wild-type and mutant enzyme-catalyzed reactions. AbA inhibited the wild-type enzyme from both Candida albicans and Saccharomyces cerevisiae in an irreversible, time-dependent manner, with apparent Ki values of 183 and 234 pM, respectively. Three synthetic chemistry-derived AbA derivatives, PHA-533179, PHA-556655, and PHA-556656, had affinities 4 to 5 orders of magnitude lower and were reversible inhibitors that competed with the donor substrate phosphatidylinositol (PI). AbA was a reversible, apparently noncompetitive inhibitor, with a Ki of 1.4 μM, of the IPC synthase from an AbA-resistant S. cerevisiae mutant. The Km values for both substrates (ceramide and PI) were similar when they interacted with the mutant and the wild-type enzymes. By contrast, the V max for the mutant enzyme was less than 10% of that for the wild-type enzyme. A comparison of the results obtained with AbA with those obtained with two other natural products inhibitors, rustmicin and khafrefungin, revealed that while rustmicin appeared to be a reversible, noncompetitive inhibitor of the wild-type enzyme, with a Ki of 16.0 nM, khafrefungin had the kinetic properties of a time-dependent inhibitor and an apparent Ki of 0.43 nM. An evaluation of the efficiencies of these compounds as inhibitors of the mutant enzyme revealed for both a drop in the apparent affinity for the enzyme of more than 2 orders of magnitude.

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