The protein Glucose-6-Phosphate Dehydrogenase is an enzyme involved in the metabolic pathways of the majority of organisms. Leuconostoc mesenteroides is a Bacilli Gram-positive bacterium that expresses this enzyme.
Function
G6PD plays an important role in the metabolism of L.mesenteroides.
L.mesenteroides is a facultactively anaerobic micro-organism which metabolizes glucose to generate lactic acid, ethanol but also carbon dioxyde.
This glucose metabolic process (glycolysis and pentose phosphate pathway) is catalysed by G6PD. During this process, NADH is synthesised and used in the heterolactic fermentation and the biosynthesis of fatty acids.
The protein G6PD also has a role in protecting cells from destruction as it produces the co-factor NADPH which plays a role in protecting cells from reactive oxygen species [1].
Genomic context
It is coded by the G6PD gene (1461 nucleotides)[2].
It has in total 2 chains. In the link below, these two chains are represented by one unique sequence entity.
External resource : click on [1] to see the graphical representation on the RCSB website.
Catalytic activity
D-glucose 6-phosphate + NAD+ → 6-phospho-D-glucono-1,5-lactone + H+ + NADH[3]
KM=114 µM for G6PD (with NADP), KM=69 µM for G6PD (with NAD),
KM=8.0 µM for NADP, KM=160 µM for NAD.
Its regulation depends on the concentration of substrate and coenzyme, rate limiting step in pentose phosphate pathway[4].
Optimum pH for its activity is 5.4 - 8.9.
Evolutionary conservation
The different structures conserved evolutionary can be observed according to the scale following.
Check, as determined by ConSurfDB.
Mutations
Mutagenesis of this enzyme induces catalytic activity loss: more than 200 mutations have been identified.
A mutation in a nucleotide in the sequence coding for G6PD leads to disruption of the normal expression of the enzyme, or to a disruption in the amino acid structure of the enzyme which leads to a loss or decrease of catalytic activity toward its substrate.
The most common mutations in the amino acids sequence found that induce a loss of catalytic activity are a substitution of the bold amino acids by another one[5]:
MVSEIKTLVT FFGG T GDLAK R TK LYPSVFNL YKKGYLQKHF AIVGTA RQ AL NDDEFKQLVR DSIKDFTDDQ AQAEAFIEHF SYRAHDVTDA ASYAVLKEAI EEAADKFDID GNRIFYMSVA PRFFGTIAKY LKSEGLLADT GYNRLMIEK P FGTSYDTAAE LQNDLENAFD DNQLFRI DHY LG K EMVQNIA ALRFGNPIFD AAWNKDYIKN VQVTLSEVLG VEERAGYYDT AGALLDMIQN H TMQIVGWLA MEKPESFTDK DIRAAKNAAF NALKIYDEAE VNKYFVRAQY GAGDSADFKP YLEELDVPAD SKNNTFIAGE LQFDLPRWEG VPFYVRSGKR LAA K QTRVDI VFKAGTFNFG SEQEAQEAVL SIII D PKGAI ELKLNAKSVE DAFNTRTIDL GWTVSDEDKK NTPEP Y ERMI HDTMNGDGSN FADWNGVSIA WKFVDAISAV YTADKAPLET YKSGSMGPEA SDKLLAANGD AWVFKG.
This sequence being the normal protein sequence found in L. mesenteroides.
Structural highlights
Glucose-6-Phosphate Dehydrogenase is formed of a homodimer, so a dimer of two identical subunit.
Secondary structure
A subunit contains 485 residues where 285 residues are in secondary structure. 93 residues are involved in 15 β-sheet strands and 192 in 17 helices.
Tertiary structure
Each is composed of 2 domains, .
Each monomer is composed of a small domain localized in the amino terminal part (residues 1-177) which constitute the coenzyme binding domain and a larger domain in the carboxyl terminal part (residues 178-485).[6]
The coenzyme binding domain binds the NAD or NADP which participes in the dehydrogenation of G6P.
It is defined by a typical β-α-β dinucleotide-binding fold corresponding to a Rossman fold.
Only 17 residues over the total of 177 are strictly conserved some of them are involved in turns between some β strands and helices and the three last one of the domain are the first three residues of a strictly conserved nine-residue peptide.
Arg46 is strcilty conserved and involved in the binding with the 2'-phosphate of NADP. Gln47 could interact both with the 2'-phosphate of NADP or with the 2'-hydroxyl of NAD.
The carboxyl terminus domain is defined by a β+α particular fold which has created his own fold family the G6PD-like. It is composed of a large essentially antiparallel curved nine-stranded β-sheet with 11 helices and remain well ordered to the carboxy-terminal residue. It is essential in the activity of the enzyme because it ensure the formation of the tertiary and the quaternary structure.
At the boundary between the two domains some helices of the carboxy terminus domain interacts with multiples β strands and helices of the coenzyme binding domain ensuring the cohesion of the enzyme monomer.
There is a pocket where the phosphate of the substrate binds. Residues involved in the contact with the phosphate are His178, Glu147, Lys 148, Tyr 415 and Ile176.
Asp177 and His240 are two basic residues conserved and localized in the binding pocket which could act as base for the deshydrogenation reaction. Site-directed mutagenesis has shown that H240N mutant have a lower activity than the wild-type enzyme. So it has been deduced that His240 is involved as the base of the reaction.
Quaternary structure
Dimer
The dimer is very extend compared to the monomer with a size of 112Å.
The enzyme is found into a dimeric form in vivo. The two subunits interface is made by the contact between the two antiparallels β-sheets of the carboxyl terminus domain. Their interaction is crucial and form a β-half-barrel (β-Barrel ) on one side of the protein which is closed on the other side by helices of each monomer. The majority of dimer contacts are hydrophobic with hydrophobic residues in the inside of the β-half-barrel but there are three salt bridges, especially a conserved one between Glu183 and Lys386 ; Lys 32 with Asp 390 and Arg395 with Asp 407, and two main chain hydrogen bonds which participates to the cohesion of the structure. Glu183 is the last residue of the nine-residue conserved peptide.
The active site of the enzyme is contained in each monomer but the dimeric form is necessary to the biological activity indeed it confers the stability in aqueous medium.
