UDP-Galactose 4-Epimerase (3KO8)
from Pyrobaculum calidifontis
Created by: David Chu
UDP-galactose 4-epimerase (GalE, PDB
ID: 3KO8) is the enzyme responsible for catalyzing the interconversion of
UDP-galactose and UDP-glucose, a crucial step in the metabolism of galactose.
Specifically, this enzyme regenerates UDP-glucose from UDP-galactose in order
to maintain the production of glucose-1-phosphate, a substrate for the
energy-producing process of glycolysis (1). Studying the mechanism underlying
GalE’s function is important to understanding fully how galactose is
metabolized in humans. This understanding can give great insight into solving
any potential problems that may arise as a result of mutations that hinder the
ability of GalE to function properly.
GalE has a molecular weight of
34355.59 daltons and an isoelectric point of 6.55 and consists of a single
312-residue polypeptide chain (2). The enzyme is normally found in cells as a homodimer with each constituent bound via its α4 and α6 alpha helices. Each constituent of the dimer has an amino-terminal coenzyme-binding domain which forms a Rossmann-fold-motif and a carboxyl-terminal catalytic domain with each domain consisting of multiple alpha helices and beta sheets.
Focusing on the coenzyme-binding
function of the enzyme, this domain contains the two alpha helices responsible
for dimerization (α4 and α6) as well as a site for NAD-binding. The NAD-binding
involves hydrogen bonding between Asp-51, Glu-88, and N6 of the adenine base,
and between Glu-88, water, and N7 of the same adenine base. The hydroxyl groups
of the ribose component of adenine hydrogen bond with the side chains Asp-31, Asn-32, and Ser-34, and with the amide-bond hydrogens of Ser-34, Ser-35, and
Gly-36. The phosphate component of adenine hydrogen bonds with the side chains
of Ser-35 and His-174, two water molecules, and the amide-bond hydrogen of Phe-11.
The nicotinamide phosphate hydrogen bonds with the amide-bond hydrogen of Ile-12
and two water molecules, while the hydroxyl groups of the nicotinamide ribose
hydrogen bond with Tyr-137, Lys-141, and one water molecule. Given the
extensive hydrogen bond network described above, NAD is tightly bound to GalE
as a prosthetic group and the removal of NAD leads to the denaturation of GalE.
In particular, Asn-32, Ser-34, Ser-35, and Gly-36 participate in a binding loop that binds the adenine ribose of NAD tightly. During the conversion of
UDP-galactose to UDP-glucose, the NAD cofactor is reduced to NADH as a hydride
transfer occurs between NAD and UDP-galactose. This same hydride is then
returned to the opposite face of the oxidized UDP-galactose once it has been
rotated within the active site to convert it to UDP-glucose.
GalE is very stable at
high temperatures due to the large number of hydrophobic interactions between
the two monomers in its dimer form, particularly around the dimerization alpha
helices. Specifically, there exist 111 hydrophobic interactions within the
dimer. Specifically, Pro-82, Ile-83, Phe-86, Val-90, Val-91, Phe-94, Pro-133,
Val-136, Ala-143, Val-146, Met-147, Leu-154, and Phe-155 of the two alpha
helices participate in hydrophobic interactions. In particular, Phe-86 and Phe-94 of each α4 helix in each monomer form aromatic pairs.
The PSI-BLAST program
and the Dali server were used to find proteins with similar primary structures
and tertiary structures, respectively. The PSI-BLAST program takes the sequence
of a protein and compares it to sequences of other proteins to generate an E
value that is based on the number of differences between the sequence of
interest and the comparisons. A lower E value indicates greater similarity with
a threshold of 0.05 used to determine significantly similar protein sequences.
The Dali server calculates the similarities in intramolecular distances of two
proteins to generate a Z-score. A higher Z-score indicates that two proteins
have similar tertiary structures with a threshold of 2 used to determine
significantly similar proteins. Using these resources, GalE was compared to GalE found in humans (PDB ID: 1EK6) which had a Z-score of 35.3 (an E value
could not be found for this protein) and to GDP-mannose-3’,5’-epimerase (GME, PDB
ID: 2C54) found in Arabidopsis thaliana which had an E value of 1e-67 and a
Z-score of 34 (3-4).
Human GalE is also
found as a dimer and is also broken into an amino-terminal domain and a carboxyl-terminal
domain each comprised of multiple alpha helices and beta sheets. However, the
carboxyl-terminal domain of human GalE is more involved in the binding of NAD. At
the same time, human GalE does not have as extensive a hydrogen bond network as
P. calidifontis GalE, as only eleven
residues are directly involved in hydrogen bonding to NAD. These eleven
hydrogen bonds include the side chains of Asp-33, Asn-37, Asp-66, Tyr-157, and Lys-161, and the amide-bond hydrogens of Tyr-13 and Ile-14. It is apparent that
the locations of the hydrogen bonds differ greatly from that of P. calidifontis GalE. Hydrophobic
interactions also do not play as large a role in human GalE which explains its
relatively lower thermostability. While there are in total nineteen hydrogen
bonds formed on NAD, these bonds are not just between NAD and the protein; they
are also between NAD and water molecules. As a result, while NAD cannot be
removed from P. calidifontis GalE
without the enzyme denaturing, it can be readily removed from human GalE (5).
GME is an enzyme that catalyzes
the epimerization of the 3’ and 5’ positions of GDP- α –D-mannose to yield GDP-β-L-galactose
as part of vitamin C synthesis in plants. This enzyme also exists as a dimer
and contains a Rossmann fold domain that binds NAD similar to P. calidifontis GalE. However, this Rossmann fold is slightly modified with 7 parallel β-strands in its β-sheet surrounded by three α-helices on each face which assist in the binding of NAD. The
adenine ring of NAD is also bound to a loop structure that assists in tight
binding of NAD. A notable difference between GME and P. calidifontis GalE is the mechanism by which they carry out their
epimerization reactions. While P.
calidifontis GalE involves the oxidation of galactose followed by rotation
and then reduction, GME makes use of an acid/base pair involved in oxidation
followed by decarboxylation (6).
Notably, GME and P. calidifontis GalE are both
structurally similar to GalE found in Escherichia coli (PDB ID: 1XEL) which has a Z-score of 34.8 (no E value could be
determined) (4). Comparing human GalE to that found in E. coli, both are dimers that are split into an amino-terminal
domain dedicated to NAD binding and a carboxyl-terminal domain involved in the
catalyzing the epimerization reaction. The two enzymes also have extensive
hydrogen bonding networks that bind NAD. A major difference between these two
enzymes lies in the fact that the constituents of the dimer of P. calidifontis GalE are rotated to
differing degrees resulting in a very different arrangement of the dimerization
helices (α4 and α6). This differing arrangement strengthens the hydrophobic interactions between the two monomers of the dimer for P. calidifontis GalE but not for E. coli GalE. Note that the offset between the pairs of alpha helices for E. coli is greater than the offset between the pairs of alpha helices for P. calidifontis. This offset weakens the hydrophobic interactions between the two monomers of E. coli GalE. Even with this difference, E. coli also denatures when NAD is removed reinforcing the fact
that its hydrogen bonding network is similar to that of P. calidifontis GalE (7).
Also similar to P. calidifontis GalE is GalE found in Trypanosoma brucei (PDB ID: 1GY8), which
has an E value of 7e-75 and a Z-score of 35.7 (3-4). T. brucei GalE has a similar hydrogen bonding pattern as P. calidifontis GalE with Ser-33, Val-35,
and Gly-36 as well as Tyr-11 and Ile-12 which are similar amino acid residues
located in similar positions; notably, Gly-36 is conserved in both P. calidifontis GalE and T. brucei GalE. The mechanism of
epimerization is also conserved in both of these enzymes as each oxidizes
galactose, rotates it, and then reduces it again to its new form using NAD as a
hydride acceptor and donor during the hydride transfer (8).