Identification of three essential lysine residues probably involved in substrate binding of the NAD+ - dependent glutamate dehydrogenase of Clostridium symbiosum

1988 ◽  
Vol 16 (5) ◽  
pp. 875-876 ◽  
Author(s):  
K. S. LILLEY ◽  
P. C. ENGEL
Keyword(s):  
Glutamate Dehydrogenase ◽  
Substrate Binding ◽  
Lysine Residues ◽  
Clostridium Symbiosum ◽  
Essential Lysine Residues

The active site of Rubisco

1986 ◽  
Vol 313 (1162) ◽  
pp. 379-395 ◽  
Keyword(s):  
Active Site ◽  
Rhodospirillum Rubrum ◽  
Substrate Binding ◽  
Cross Linking ◽  
Ribulose Bisphosphate ◽  
Sequence Analyses ◽  
Lysine Residues ◽  
Affinity Labelling ◽  
Ph Curve ◽  
Essential Lysine Residues

Previous affinity-labelling studies and comparative sequence analyses have identified two different lysine residues at the active site of ribulose bisphosphate carboxylaseoxygenase and have suggested that they are essential to function. The essential lysine residues occupy positions 166 and 329 in the Rhodospirillum rubrum enzyme and positions 175 and 334 in the spinach enzyme. Based on the pH-dependences of inactivations of the two enzymes by trinitrobenzene sulphonate, Lys 166 ( R. rubrum enzyme) exhibits a p K a of 7.9 and Lys 334 (spinach enzyme) exhibits a p K a of 9.0. These low p K a values, as well as the enhanced nucleophilicities of the lysine residues, argue that both are important to catalysis rather than to substrate binding. Lys 166 may correspond to the essential base that initiates catalysis and that displays a p K a of 7.5 in the pH-curve for V max / K m . Cross-linking experiments with 4,4'- diisothiocyano-2,2/-disulphonate stilbene demonstrate that the two active-site lysine residues are within 12 Å of each other (1 Å = 10 -10 m).

scholarly journals Deamidation and glycation of a Bacillus licheniformis α-amylase during industrial fermentation can improve detergent wash performance

Amylase ◽  
2021 ◽  
Vol 5 (1) ◽  
pp. 38-49
Author(s):  
Connie Pontoppidan ◽  
Svend G. Kaasgaard ◽  
Carsten P. Sønksen ◽  
Carsten Andersen ◽  
Birte Svensson
Keyword(s):  
Bacillus Licheniformis ◽  
Mutational Analysis ◽  
Peptide Mapping ◽  
Substrate Binding ◽  
Carbohydrate Binding ◽  
Surface Binding ◽  
Lysine Residues ◽  
Tandem Mass Spectrometric ◽  
Binding Cleft ◽  
Household Detergents

Abstract The industrial thermostable Bacillus licheniformis α-amylase (BLA) has wide applications, including in household detergents, and efforts to improve its performance are continuously ongoing. BLA during the industrial production is deamidated and glycated resulting in multiple forms with different isoelectric points. Forty modified positions were identified by tandem mass spectrometric peptide mapping of BLA forms separated by isoelectric focusing. These modified 12 asparagine, 9 glutamine, 8 arginine and 11 lysine residues are mostly situated on the enzyme surface and several belong to regions involved in stability, activity and carbohydrate binding. Eight residues presumed to interact with starch at the active site and surface binding sites (SBSs) were subjected to mutational analysis. Five mutants mimicking deamidation (N→D, Q→E) at the substrate binding cleft showed moderate to no effect on thermostability and k cat and K M for maltoheptaose and amylose. Notably, the mutations improved laundry wash efficiency in detergents at pH 8.5 and 10.0. Replacing three reducing sugar reactive side chains (K→M, R→L) at a distant substrate binding region and two SBSs enhanced wash performance especially in liquid detergent at pH 8.5, slightly improved enzymatic activity and maintained thermostability. Wash performance was most improved (5-fold) for the N265D mutant near substrate binding subsite +3.

Allosteric behaviour of 1:5 hybrids of mutant subunits of Clostridium symbiosum glutamate dehydrogenase differing in their amino acid specificity

2001 ◽  
Vol 360 (3) ◽  
pp. 651-656 ◽  
Author(s):  
Arun GOYAL ◽  
Xing-Guo WANG ◽  
Paul C. ENGEL
Keyword(s):  
Amino Acid ◽  
Glutamate Dehydrogenase ◽  
Cysteine Residue ◽  
The Other ◽  
Wild Type Enzyme ◽  
Triple Mutant ◽  
Subunit Stoichiometry ◽  
Clostridium Symbiosum ◽  
Mutant Forms ◽  
Fold Activation

Hybrid hexamers were made by refolding mixtures of two mutant forms of clostridial glutamate dehydrogenase. Mutant Cys320Ser (C320S) has a similar activity to the wild-type enzyme, but is unreactive with Ellman's reagent, 5,5′-dithiobis(2-nitrobenzoate) (DTNB). The triple mutant Lys89Leu/Ala163Gly/Ser380Ala (K89L/A163G/S380A), active with norleucine but not glutamate, is inactivated by DTNB, since the amino acid residue at position 320 is a cysteine residue. The chosen ratio favoured 1:5 hybrids of the triple mutant and C320S. The renatured mixture was treated with DTNB and separated on an NAD+–agarose column to which only C320S subunits bind tightly. Fractions were monitored for glutamate and norleucine activity and for releasable thionitrobenzoate to establish subunit stoichiometry. A fraction highly enriched in the 1:5 hybrid was identified. Homohexamers (C320S with 40mM glutamate and 1mM NAD+ at pH8.8, or K89L/A163G/S380A with 70mM norleucine and 1mM NAD+ at pH8.5) showed allosteric activation; succinate activated C320S approx. 50-fold (EC50 = 70mM, h = 2.4), and glutarate gave approx. 30-fold activation (EC50 = 35mM, h = 2.3). For the triple mutant, corresponding values were 80mM and 2.2 for succinate, and 75mM and 1.7 for glutarate, but maximal activation was only about 2-fold. In the 1:5 hybrid, with only one norleucine-active subunit per hexamer, responses to glutarate and succinate were still co-operative, and activation was more extensive than in the triple mutant homohexamer. A single norleucine-active subunit can thus respond co-operatively to a substrate analogue at the other five inactive sites. On the other hand, similar hyperbolic dependence on the norleucine concentration for the hybrid and the triple mutant homohexamer reflected the inability of C320S subunits to bind norleucine. With glutamate at pH8.8, an h value of 3.6 was obtained for the 1:5 hybrid, in contrast with an h value of 5.2 for the C320S homohexamer. The ‘foreign’ subunit evidently impedes inter-subunit communication to some extent.

scholarly journals Crystal structure of a chimaeric bacterial glutamate dehydrogenase

2016 ◽  
Vol 72 (6) ◽  
pp. 462-466 ◽  
Author(s):  
Tânia Oliveira ◽  
Michael A. Sharkey ◽  
Paul C. Engel ◽  
Amir R. Khan
Keyword(s):  
Crystal Structure ◽  
Glutamate Dehydrogenase ◽  
Rigid Bodies ◽  
Nucleotide Binding Domain ◽  
Oxidative Deamination ◽  
E Coli ◽  
Cofactor Binding ◽  
Signature Sequences ◽  
Clostridium Symbiosum ◽  
Domain I

Glutamate dehydrogenases (EC 1.4.1.2–4) catalyse the oxidative deamination of L-glutamate to α-ketoglutarate using NAD(P)+as a cofactor. The bacterial enzymes are hexameric, arranged with 32 symmetry, and each polypeptide consists of an N-terminal substrate-binding segment (domain I) followed by a C-terminal cofactor-binding segment (domain II). The catalytic reaction takes place in the cleft formed at the junction of the two domains. Distinct signature sequences in the nucleotide-binding domain have been linked to the binding of NAD+versusNADP+, but they are not unambiguous predictors of cofactor preference. In the absence of substrate, the two domains move apart as rigid bodies, as shown by the apo structure of glutamate dehydrogenase fromClostridium symbiosum. Here, the crystal structure of a chimaeric clostridial/Escherichia colienzyme has been determined in the apo state. The enzyme is fully functional and reveals possible determinants of interdomain flexibility at a hinge region following the pivot helix. The enzyme retains the preference for NADP+cofactor from the parentE. colidomain II, although there are subtle differences in catalytic activity.

scholarly journals An evolutionary analysis identifies a conserved pentapeptide stretch containing the two essential lysine residues for rice L-myo-inositol 1-phosphate synthase catalytic activity

PLoS ONE ◽  
2017 ◽  
Vol 12 (9) ◽  
pp. e0185351 ◽  
Author(s):  
Papri Basak ◽  
Susmita Maitra-Majee ◽  
Jayanta Kumar Das ◽  
Abhishek Mukherjee ◽  
Shubhra Ghosh Dastidar ◽  
...  
Keyword(s):  
Catalytic Activity ◽  
Evolutionary Analysis ◽  
Inositol 1 ◽  
Lysine Residues ◽  
Essential Lysine Residues ◽  
Phosphate Synthase

Alteration of the quaternary structure of glutamate dehydrogenase from Clostridium symbiosum by a single mutation distant from the subunit interfaces

1997 ◽  
Vol 25 (5-6) ◽  
pp. 417-422 ◽  
Author(s):  
Jonathan L. E. Dean ◽  
H. Cölfen ◽  
Stephen E. Harding ◽  
David W. Rice ◽  
P. C. Engel
Keyword(s):  
Glutamate Dehydrogenase ◽  
Quaternary Structure ◽  
Single Mutation ◽  
Clostridium Symbiosum
Sign in / Sign up

Export Citation Format

Share Document