Glycerol-3-phosphate dehydrogenase (PDB ID: 2QCU) from Escherichia coli Created by: Luke Cavanah
Sn-glycerol-3-phosphate dehydrogenase (GlpD; PDB ID: 2QCU) is a wild-type monotopic membrane enzyme
found in Escherichia coli (E. coli). GlpD is an oxidoreductase within
the inner membrane of E. coli and has implications for respiration,
glycolysis, and phospholipid biosynthesis (1-2). The biological significance of
GlpD is supported by its role in the three aforestated vital metabolic
processes, multi-level regulation, and high number of homologs found across
nearly all organisms (2-3).
GlpD
is one of the main dehydrogenases of the electron transport chain (ETC), where
it catalyzes the oxidation of glycerol-3-phosphate (G3P) to dihydroxyacetone
phosphate (DHAP) and the reduction of flavin adenine dinucleotide (FAD) to FADH2
(2). Subsequently, GlpD passes electrons on to ubiquinone (UQ) in the ETC
(2,4). Together, ETC and chemiosmosis generate the most ATP during cellular
respiration, reinforcing GlpD’s biological importance (4).
GlpD
was expressed in E. coli (1-2). The protein was crystallized through
vapor diffusion sitting drop and hanging drop methods (2). β-octylglucoside
(βOG) was used as detergent in expression, purification, and the detergent
reconstitution process of GlpD (2). Structural data was obtained through x-ray
diffraction. Phosphate ion, ethylene glycol, tris(hydroxyethyl)aminomethane,
and imidazole are ligands present in the crystal structure that were used
merely to induce crystallization, and serve no biological function in this
biomolecule. βOG is another ligand present in the crystal structure, which has
no biological function in GlpD (1-2).
GlpD has a molecular weight of 113483.07 Da,
and an isoelectric point of 7.03 (5). GlpD has two identical subunits (1-2).
The primary structure of GlpD consists of 1002 residues between its two
subunits. Though the subunits consist of both hydrophobic and hydrophilic amino
acids, hydrophobic residues greatly predominate (1-2). The high hydrophobic
content suits its location in the highly nonpolar cell membrane (2).
The secondary structure of each subunit is approximately 23% β-sheet (26 strands,
119 residues), 36% α-helix (25 helices, 184 residues), with the remaining
percentage being random coils and 3/10 helices (1). Both parallel and
anti-parallel β-sheet are present. Hydrogen bonding between carbonyl oxygen
atoms and amide nitrogen atoms in the peptide backbone provide significant
stabilization to GlpD’s structure and neutralization of the polar N-H and C=O
functions of the peptide backbone (2).
GlpD
contains two important domains: a soluble extramembranous C-terminal domain and
a N-terminal FAD-binding site (2). All of GlpD is represented in the published
structure, and the entire structure was crystallized (1-2). Residues 1-388 make
up the N-terminal domain, and residues 399-501 make up the C-terminal domain;
no residues are unresolved (2).
In
regard to tertiary structure of GlpD as described by secondary-structure elements, the β-sheets tend to predominate toward
the periphery of the protein, and the α-helices are prevalent throughout all
parts of the protein (1). The base of GlpD that is located in the plasma
membrane is distinctly positive. Electrostatic attraction between the positively
charged regions of GlpD and the negatively charged phospholipid head groups
serves to anchor GlpD in the fluid membrane. The cap domain is distinctly
negative, which is hypothesized to be the UQ-binding surfaces (2-3).
Due
to GlpD’s homodimeric nature, GlpD possesses quaternary structure (1-2). Dimer
formation is structurally important because it reduces the
surface-area-to-volume ratio, thus enabling burying of the hydrophobic
residues; this hydrophobic collapse is especially important because it is of
GlpD’s high hydrophobic content. Furthermore, burying of the hydrophobic
residues is entropically favorable, providing significant structural stability.
The dimer structure also allows protection of the active site because the
monomeric units enclose the site where dehydrogenation occurs. The apolar
microenvironment created by the dimer structure is required for catalysis (2).
Phosphoenolypyruvate
(PEP), glyceric acid 2-phosphate (2-PGA), and glyceraldehyde-3-phosphate (GAP)
are three substrates that complex to GlpD, and DHAP is a product that complexes
to GlpD (1-2). Hydrogen-bonding interactions between PEP, 2-PGA, GAP, and DHAP
define the active site. Ser-46, Leu-48, His-50, Leu-355, and Thr-356 interact with FAD, so substrates only bind at one face of the isoalloxazine ring. Arg-316
mediates the binding of the phosphate of the substrate analogues. Binding of
the phosphate of the substrate aligns the substrate for dehydrogenation. Arg-54,
Tyr-55, Thr-270, Thr-271, Asp-272, Arg-317, and Arg-332 mediate binding of
substrates. Arg-317 likely catalyzes the initial deprotonation of the hydroxyl
group bonded to C2 of G3P due to its proximity. The hydrogen bonding between
the guanidino nitrogen atoms of Arg-317 to the oxygen atoms of the phosphate
Asp-272 likely increase the basicity of Arg-317, thus facilitating the initial
deprotonation of G3P. Following dehydrogenation, hydride transfer occurs from
C2 of G3P to N5 of FAD. Leu-355 and especially Lys-354 stabilize the reduced
flavin formed after the hydride transfer occurs. His-233 likely acts as a
general base catalyst. FAD is the only prosthetic group present in GlpD. No
associated metal ions appear to be required for GlpD activity (2).
Position-specific-iterated Basic
Local Alignment Search Tool (PSI-BLAST) is a program that identifies proteins
with similar primary structure as the sequence queried. PSI-BLAST calculates an
E value for the proteins identified with similar sequence to the query by
examining the total sequence similarity and assigning gaps. Sequence homology
is inversely related to the E value, and gaps are directly related to the E
value. An E value of less than .05 suggests the protein has a similar sequence
to that of the query (6). Flavoenzyme PA-4991 (PDB: 5EZ7), typically derived
from Pseudomonas aeruginosa, and GlpD have a comparative E value of 3•10-20,
indicating highly similar primary structures (6-7).
The Dali server compares tertiary structures of proteins and calculates the differences in intramolecular distances by a statistical method known as the “sums-of-pairs” method. The Dali server then assigns a Z-score for the proteins identified to have similar tertiary structures to the query. A Z-score greater than 2 suggests the protein has a similar tertiary structure to that of the query. Flavoenzyme PA-4991 and GlpD have a comparative Z-score of 23.2, indicating highly similar tertiary structures (8).
Both GlpD and flavoenzyme PA-4991
are part of the family of FAD-dependent oxidoreductases, and thus they both
contain the FAD-binding domain and a FAD ligand (2, 3, 9). Additionally, both
GlpD and flavoenzyme PA-4991 contain three of the same active-site residues:
His-53, Arg-316, and Lys-345. Both proteins also share the structural feature
of an isoalloxazine ring (2, 9).
Despite
the structural similarities between GlpD and flavoenzyme PA-4991 above, there
are many structural and functional differences as well. In terms of primary
structure, flavoenzyme PA-4991 contains only one subunit, which has 392
residues (7, 9). The secondary structure of flavoenzyme-4991 contains a
significantly lower proportion of α-helices, and a slightly higher proportion
of β-sheets: flavoenzyme is 26% helical and 27% β-sheet (7). One major
difference in the tertiary structures of GlpD and flavoenzyme PA-4991 is the
structure of their active sites: the re face of the isoalloxazine ring
is accessible from the solvent in flavoenzyme PA-4991, unlike in GlpD (2, 9). Flavoenzyme
PA-4991 only has one chain, and GlpD has two; thus flavoenzyme PA-4991 lacks
quaternary structure. Another notable structural difference between the two
proteins is flavoenzyme PA-4991 has mercury (II) ion ligate to the structure,
when GlpD does not (1, 2, 7, 9). The differences between GlpD and flavoenzyme
PA-4991 manifest as functional changes, too, as illustrated by the lack of
enzymatic activity of flavoenzyme PA-4991 when DHAP is the substrate, and their
difference in substrate specificity (9).
Ultimately, the biological
significance of GlpD in metabolism is due to its involvement in the
dehydrogenation of G3P (2). GlpD’s unique primary, secondary, tertiary, and
quaternary levels of structure enable it to be a successful monotopic-membrane
oxidoreductase (2). The transfer of electrons from GlpD to UQ is not
well-understood, particularly UQ’s docking site. Future directions include
investigating UQ’s docking site on GlpD.