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INTRODUCTION |
Mammals, birds, and yeast produce fatty acids via a FAS-I system,
a biosynthetic pathway in which each enzymatic activity resides on the
single polypeptide chain of a very large multifunctional enzyme (1-3).
In contrast, most plants and bacteria utilize a FAS-II system in which
each of the enzymatic activities corresponds to individual polypeptides
(4-6). The best characterized FAS-II system is that of
Escherichia coli, which includes
-ketoacyl-ACP synthases
(FabB, FabF, and FabH), a
-ketoacyl-ACP reductase (FabG),
-hydroxyacyl-ACP dehydrases (FabA and FabZ), and an enoyl-ACP reductase (currently known as FabI and formerly known as EnvM). The
E. coli FAS-II system initializes fatty acid biosynthesis by
condensing acetyl and malonyl substrates, and then it continues the
process by elongating the resulting product through the consecutive addition of C2 units, until the fatty acyl chain reaches a length of
about 16 carbons.
Some bacteria, such as mycobacteria, possess both a FAS-I and a FAS-II
system (7). The mycobacterial FAS-I system displays a bimodal
distribution of products centered on C16 and C24-C26 (7, 8). This
FAS-II system prefers C16 as a starting substrate (7) and can extend up
to C56 (9), indicating that the mycobacterial FAS-II system utilizes
the products of the FAS-I system as primers to extend fatty acyl chain
lengths even further. The longer chain products of the FAS-II system
are the precursors of mycolic acids. Mycolic acids are long chain
-alkyl-
-hydroxy fatty acids, which are a major component of
mycobacterial cell walls (10, 11).
Isoniazid, a drug used as a first-line antibiotic for the treatment of
Mycobacterium tuberculosis infections for the past 40 years,
is known to inhibit mycolic acid biosynthesis (12-15). More recently,
isoniazid was shown to prevent radiolabel from being incorporated into
the chain extension of C24-C26 fatty acyl substrates (16-20),
suggesting that the target of isoniazid action is within the
mycobacterial FAS-II system. Using a genetic approach to isolate the
isoniazid target, a single open reading frame was identified (referred
to as inhA) in which transformation of the wild-type gene
carried on a multicopy plasmid or allelic exchange with a single amino
acid substitution mutant (Ser94 to Ala) was sufficient to
confer isoniazid resistance in Mycobacterium smegmatis
(21).
The protein encoded by the inhA gene, referred to as InhA,
has a similar amino acid sequence to two previously characterized enoyl-ACP reductases, namely FabI from E. coli (28%
identity) (22-25), and ENR1
from Brassica napus (oilseed rape) (23% identity) (26, 27). Further analysis revealed that InhA catalyzes the
NADH-dependent reduction of the trans double
bond between positions C2 and C3 of fatty acyl substrates (28). In
addition, InhA prefers fatty acyl substrates of C16 or greater,
consistent with its being a member of the mycobacterial FAS-II system
(28).
The connection between isoniazid action and InhA inhibition is somewhat
complicated. Isoniazid is a pro-drug that must be converted, via a
mycobacterial catalase-peroxidase (KatG), into an activated form of the
drug (29-32) (for recent reviews, see Refs. 33-35). The activated
form of isoniazid (suspected to be an isonicotinic-acyl radical)
becomes covalently attached to the nicotinamide ring of the NADH bound
within the active site of InhA, creating an NADH adduct that acts as a
very tightly bound inhibitor (36). In contrast, the S94A mutation of
InhA, which is resistant to the effects of isoniazid, has a decreased
affinity for NADH (28, 37).
The World Health Organization has estimated that one-third of the
world's population, nearly 2 billion people, is infected with
tuberculosis. Tuberculosis kills more adults now than all other
infectious diseases combined, and it is the leading cause of death in
HIV-positive individuals. Data from the World Health Organization-sponsored Global Project on Anti-tuberculosis Drug Resistance Surveillance indicates that 36% of tuberculosis patients are infected with a strain that is resistant to traditional antibiotics (isoniazid or rifampicin). Even though mutations within the
inhA gene are known to facilitate isoniazid resistance (21,
38-41), InhA remains a good candidate for drug design. Several reasons for this are as follows: (i) the vast majority of the mutations found
in isoniazid-resistant clinical isolates are associated with the
isoniazid activator (the KatG catalase-peroxidase) (38-41); (ii) only
one enoyl-ACP reductase is found in M. tuberculosis, unlike
some of the other enzymes of bacterial FAS-II systems (42); and (iii)
the longer substrate chain length specificity of InhA distinguishes it
from the enoyl-ACP reductases from other sources, such as the enoyl-ACP
reductase component of the human FAS-I system.
To facilitate the drug design process and increase our understanding of
the mechanism of an enoyl-ACP reductase reaction, full characterization
of the active site of InhA is now available. In an initial report, the
crystal structure of InhA in complex with NADH was determined at 2.5-Å
resolution (37), which revealed the putative location of the fatty acyl
substrate binding pocket, lying adjacent to the bound NADH (28). We
describe here the crystal structure of InhA in complex with
NAD+ and a C16 fatty acyl substrate
(trans-2-hexadecenoyl-(N-acetylcysteamine)-thioester). The substrate contains a fatty acyl chain of 16 carbons, with a
trans double bond between positions C2 and C3
(trans-2-hexadecenoyl moiety), which is attached through a
thioester linkage to a small portion of the phosphopantetheine arm of
the acyl carrier (N-acetylcysteamine moiety). The chemical
formula is
CH3-(CH2)12-CH=CH-CO-S-CH2-CH2-NH-CO-CH3. This compound is not a natural biological substrate of InhA; however, it and long chain fatty acyl derivatives of coenzyme A are functional substrates in vitro.
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EXPERIMENTAL PROCEDURES |
Synthesis of C16 Fatty Acyl
Substrate--
Trans-2-hexadecenoyl-(N-acetylcysteamine)-thioester
was prepared in two stages. In the first step,
trans-2-hexadecenoic acid was activated by reaction with 1 equivalent of ethyl chloroformate and 1 equivalent of
diisopropylethylamine in tert-butyl methyl ether. The
reaction mixture was stirred at room temperature overnight and then
filtered and concentrated, giving the mixed anhydride as an oil. In the
second step, the thioester was formed by the coupling of
N-acetylcysteamine with the mixed anhydride. To the oil was
added a solution of N-acetylcysteamine in ethanol, ethyl acetate, and 0.3 M aqueous sodium bicarbonate (1:1:1
v/v/v). After the reaction mixture was stirred for 4 h at room
temperature, the solvents were removed by rotary evaporation. The
product was purified by flash chromatography on silica gel using
hexane, ethyl acetate, and acetic acid (75:25:1 v/v/v) as the eluting
agent. The product was obtained in 37% overall yield and the structure was confirmed by 1H NMR spectroscopy.
Crystallization of the Ternary Complex--
Expression of the
M. tuberculosis inhA gene within an E. coli host,
purification of the resulting soluble InhA protein, and production of
hexagonal crystals (space group P6222) containing InhA in
complex with NADH have been reported previously (28, 37). Monoclinic
crystals (space group C2) of the ternary complex (InhA,
NAD+, and C16 fatty acyl substrate) were produced under new
conditions, using the hanging drop vapor diffusion method. The drop was
created by combining an equal amount of well solution (10%
polyethylene glycol-4000, 6% Me2SO, 100 mM
ammonium acetate, and 100 mM ADA, pH 6.8) with a
concentrated protein mixture. The protein mixture was produced by first
solubilizing the C16 fatty acyl substrate in Me2SO (600 µM stock solution) and then slowly adding 1 ml of stock
solution at room temperature to 100 ml of a dilute solution of InhA
(0.1 mg/ml = 3 µM) in the presence of 600 µM NAD+. The final molar ratio of the mixture
was 1:200:2 for InhA, NAD+, and C16 fatty acyl substrates,
respectively, and the final concentration of Me2SO was 1%
(v/v). The mixture was concentrated at 4 °C with an Amicon
Centricon-10 until the concentration of InhA reached 10 mg/ml.
X-ray Diffraction Data Collection--
X-ray diffraction data
were collected to 2.8-Å resolution at room temperature using a
MacScience DIP2030 image plate detector with double-focusing mirrors
coupled to a Rigaku x-ray generator utilizing a copper rotating anode
(CuK
wavelength = 1.54 Å). A 0.0075-mm nickel filter was
placed in line before the mirrors, and the diameter of the x-ray beam
collimator was 0.5 mm. The detector was placed 230 mm from the crystal
with no offset in the 2
angle. Each frame of data was exposed for 15 min and consisted of a 1.5° rotation of the crystal. The data from
two crystals (size 0.4 × 0.4 × 0.1 mm) were processed and
merged using the DENZO/Scalepack software package (43), and a 3
cutoff was applied to the final structure factors. Data collection
statistics are listed in Table I.
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Table I
X-ray diffraction data statistics
Completeness = number of
Fobserved/number of
Fexpected) × 100; Rsym = |I I |/ I) × 100; I/ I = (I/ I)/number of I; R factor = ( (| Fobserved Fcalculated|)/ (Fobserved)) × 100; Rfree = R value of portion of
data omitted from model refinement. I, intensity; F,
amplitude.
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Initial Phase Determination and Model Refinement--
All
calculations were performed with the XPLOR software package (44). A
single subunit of InhA (protein coordinates only) derived from the
hexagonal crystal form (Protein Data Bank entry 1eny) was used as a
search model with the molecular replacement technique. Six subunits
were found in the asymmetric unit of the monoclinic crystal form,
corresponding to a solvent content of 53%. Four of the subunits form a
homotetramer based on noncrystallographic (local) 2-fold symmetry axes,
and the remaining two subunits form a second homotetramer based on a
combination of both noncrystallographic (local) and crystallographic
2-fold symmetry axes.
Subsequent to rigid body refinement (R factor = 30.2%,
Rfree = 38.3%), noncrystallographic
(NCS) restraints were applied to both the atomic positions and
individual temperature factors. After simulated annealing refinement
(R factor = 25.6%, Rfree = 36.0%), the NAD+ and C16 fatty acyl substrate could be
identified in a difference Fourier (Fo-Fc)
electron density map, confirming the correctness of the molecular
replacement solution. Subsequent to conventional positional refinement
in the presence of NAD+ and the C16 fatty acyl substrate
(R factor = 23.4%, Rfree = 35.6%), several rounds of conventional positional refinement were
performed during the addition of 682 ordered water molecules
(R factor = 21.7%, Rfree = 34.4%). Model refinement statistics are listed in Table
II, and an omit electron density map
(XPLOR, Ref. 44) covering the NAD+ and C16 fatty acyl
substrates is shown in Fig. 2A.
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Table II
Model refinement statistics
r.m.s. deviation from ideal stereochemistry: bond lengths = 0.014 Å; bond angles = 2.0°; dihedral angles = 25.7°; improper
angles = 1.7°.
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Analytical Size-exclusion Chromatography--
The quaternary
structure of InhA was investigated by determining the molecular weight
of InhA in aqueous solution using analytical size-exclusion
chromatography. A prepacked Pharmacia Superdex-200 HR-10/30 column
(separation range 10-600 kDa) was equilibrated in 150 mM
NaCl and 50 mM HEPES, pH 7.5. The flow rate was set at 0.25 ml/min and controlled by a BioCAD SPRINT system from PerSeptive Biosystems (Cambridge, MA). The column was calibrated by individually applying a ~5 mg/ml solution of each standard protein obtained from a
Pharmacia gel-filtration calibration kit to the column and then
calculating a standard line by plotting the resulting elution volumes
versus known molecular mass information. An ~5 mg/ml
solution of InhA was applied to the column, and the molecular mass of
InhA was estimated by comparing the value of its column elution volume
with the standard line.
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RESULTS AND DISCUSSION |
Overall Tertiary and Quaternary Structure of InhA--
InhA is a
member of the short chain dehydrogenase/reductase (SDR) family of
enzymes (45, 46). The main characteristic of this family is a
polypeptide backbone topology in which each subunit consists of a
single domain with a central core that contains a Rossmann fold
supporting an NADH binding site. Within InhA, several
-helices and
-strands of the Rossmann fold extend beyond the NADH binding site,
creating a deep crevice for the fatty acyl substrate (28, 37). Part of
this extension, referred to here as the "substrate binding loop"
(residues 196-219), consists of two perpendicular
-helices that
form one side of the fatty acyl substrate binding crevice (Fig.
1).

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Fig. 1.
Polypeptide backbone of InhA. A,
single subunit of InhA shown in a cyan ribbon,
except for the substrate binding loop (residues 196-219), which is
shown in an orange ribbon. The bound NAD+ is
represented as a yellow CPK model, and the bound C16 fatty
acyl substrate,
trans-2-hexadecenoyl-(N-acetylcysteamine)-thioester,
is represented as a red CPK model. B,
homotetramer (four identical subunits) of InhA, in which each subunit
is shown as a different colored ribbon (green, purple, cyan,
and magenta). The substrate binding loops, bound
NAD+, and bound C16 fatty acyl substrates are shown with
the same coloring as in A. Three perpendicular 2-fold
symmetry axes intersect in the center of the homotetramer, creating a
molecule with internal 222 symmetry. C, superposition of the
backbone of E. coli short chain substrate enoyl-ACP
reductase (FabI) (Protein Data Bank entry 1dfg) (dark blue)
with that of M. tuberculosis long chain substrate enoyl-ACP
reductase (InhA) (green) demonstrates that the location of
the substrate binding loop (yellow, InhA; cyan,
FabI) differs between the two structures. Panels A and
B were produced using the MOLSCRIPT program (55) coupled to
the RASTER-3D program (56), and panel C was produced using
the SPOCK program (57).
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Analytical size-exclusion chromatography demonstrates that InhA is a
homotetramer in aqueous solution. The crystal structure of InhA
revealed that the homotetramer is a molecule possessing internal 222 symmetry. Three perpendicular two-fold symmetry axes intersect in the
center of the molecule, creating a molecule where each subunit is
related to every other subunit by one of the perpendicular 2-fold
symmetry axes (Fig. 1B). According to the DSSP program (47)
(and the color code of Fig. 1B), the amount of buried
surface area between subunit 1 (green) and subunit 2 (purple) is 1550 Å2 (or ~12% of a single
subunit), between subunit 1 and subunit 3 (cyan) is 700 Å2 (or ~5% of a single subunit), and between subunit 1 and subunit 4 (magenta) is 1500 Å2 (or ~12% of a single
subunit). Therefore, the total buried surface area for any given
subunit is 3750 Å2 (or ~29%), indicating that a fairly
large portion of each subunit of the homotetramer is involved in
intramolecular contacts. Furthermore, the organization of the InhA
homotetramer is identical to that of the homotetramers found in the
crystal structures of two other enoyl-ACP reductases, namely E. coli FabI (1.6 Å r.m.s. deviation in
-carbon position over 848 residues) (48) and B. napus ENR (a 1.6-Å r.m.s. deviation
in
-carbon position over 632 residues) (49).
Location and Conformation of the InhA-bound C16 Fatty Acyl
Substrate--
Within the orientation of Fig. 1A, the bound
NAD+ sits on top of the shelf created by the Rossmann fold,
and the C16 fatty acyl substrate sits on top of the NAD+
and is held in place by the substrate binding loop. The InhA-bound C16
fatty acyl substrate folds into a general "U-shaped" conformation (Fig. 2). This conformation is not
unusual for protein-bound fatty acids. It has been seen before in the
crystal structure of human muscle fatty acid binding protein in complex
with several different long chain fatty acids (50).

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Fig. 2.
Conformation of bound substrate and shape of
binding crevice. A, atomic coordinates of the
InhA-bound NAD+ and C16 fatty acyl substrate,
trans-2-hexadecenoyl-(N-acetylcysteamine)-thioester,
superimposed onto the final NCS-averaged simulated annealing "omit"
electron density (44) map contoured at 0.8 . The different atom
types are represented by unique colors: carbon (gray),
oxygen (red), nitrogen (blue), sulfur
(yellow), and phosphorus (magenta). The C16 fatty
acyl substrate folds into a general U-shaped conformation, with the
trans double bond located directly over the nicotinamide
ring of NAD+. B, solvent-accessible surface of
the M. tuberculosis long chain substrate enoyl-ACP reductase
(InhA) binding crevice shown in black dots. A portion of the
bound NAD+ (nicotinamide, nicotinamide ribose, and
phosphates) is shown in green, and the bound C16 fatty acyl
substrate is shown in red. C, solvent-accessible
surface of the E. coli short chain substrate enoyl-ACP
reductase (FabI) binding crevice shown in black dots. A
portion of the bound NAD+ (nicotinamide, nicotinamide
ribose, and phosphates) is shown in green, and the bound
benzodiazaborine drug is shown in cyan (Protein Data Bank
entry 1dfg). The C16 fatty acyl substrate from the InhA ternary complex
is superimposed onto the FabI surface. This figure was produced using
the SPOCK program (57).
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The fatty acyl substrate binding crevice of InhA has an oval-shaped
cavity with the approximate dimensions 16 × 13 × 7 Å. One
side of the cavity is completely open and exposed to solvent (Fig. 2,
B and C, major portal on right side),
whereas the other side contains a small opening (Fig. 2, B
and C, minor portal on upper left side). The
N-acetylcysteamine portion of the substrate faces the major
portal, which is consistent with the requirement that this region of
the natural substrate should face outward, toward the solvent, when
attached to the acyl carrier protein.
The substrate binding crevice of InhA must accommodate fatty acyl
substrates that are longer than C16 in order to produce the precursors
of mycolic acids (28). The major and minor portals may provide some
assistance with substrate binding. For example, the
-end of the
fatty acyl substrate (position C16) faces the major portal, which could
allow chain extensions beyond C16 (of natural substrates) to interact
with the acyl carrier protein. Alternatively, the upper edge of the
U-shaped turn (positions C8-C10) faces the minor portal, which could
permit the fatty acyl chain to thread out through the minor portal and
then loop back in, so that the
-end of the fatty acyl chain is still
anchored inside the binding crevice.
The trans double bond, between positions C2 and C3 of the
fatty acyl chain, faces the closed end of the substrate binding crevice
(Fig. 2B, lower left) and is located directly adjacent to
the nicotinamide ring of NAD+. A comparison of the position
of the nicotinamide ring between the binary complex (InhA and NADH)
(37) and the ternary complex (InhA, NAD+, and C16 fatty
acyl substrate) shows no significant difference (only an 0.7-Å r.m.s.
deviation over 9 atoms), indicating that NAD+ is an
appropriate model for NADH at this resolution. Furthermore, the C3
position of the bound fatty acyl chain is closer (4.8Å) than the C2
position (5.1Å) to the C4 position of the nicotinamide ring of
NAD+. These distances are consistent with the previous
proposal that hydride ion transfer is directed from the C4 position of
the nicotinamide ring of NADH to the C3 position of the fatty acyl
chain during substrate catalysis (28).
The C2 and C3 positions of the fatty acyl chain contain the flat
(dihedral ~180°) trans double bond, whereas positions
C4-C8 form the turn of the U-shaped conformation (Fig. 2A).
Considering that the trans double bond is so close to the
turn, it is tempting to speculate that during substrate catalysis, the
active site may impose a slightly tighter U-shaped conformation upon
the substrate by adding a twist to the substrate double bond. This
requirement could facilitate the conversion from planar carbons to
tetrahedral carbons through conformational strain of the
trans double bond.
Specific Interactions between Bound Substrate and InhA Active
Site--
Using a 1.4-Å radius probe, the Connolly solvent accessible
surface area (51) of the fatty acyl substrate binding crevice is
approximately 670 Å2, with 72% of the area covered by
nonpolar atoms and the remaining 28% of the area covered by polar
atoms (Fig. 2B). This surface incorporates one buried water
molecule, which is within hydrogen bonding distance of the fatty acyl
substrate thioester sulfur. All hydrogen bonds and van der Waals
contact distances between the C16 fatty acyl substrate and the InhA
active site are listed in Table III.
The fatty acyl chain portion of the substrate is completely surrounded
by the side chains of hydrophobic residues (Fig.
3). The majority of these hydrophobic
side chains reside on the substrate binding loop (Ala198,
Met199, Ala201, Ile202,
Leu207, Ile215, Leu218). Additional
hydrophobic residues, having side chains that surround the fatty acyl
chain but are not part of the substrate binding loop, include
Met103, Phe149, Met155,
Tyr158, and Met161. The side chain of
Phe149 is located near the nicotinamide ring of
NAD+ and appears to help guide the turn of the fatty acyl
substrate U-shaped conformation.

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Fig. 3.
Stereo view of the active site of InhA.
A selection of amino acid residues is shown within the interior of the
substrate binding crevice. The fatty acyl chain of the bound C16
substrate is completely surrounded by hydrophobic residues, the
majority of which reside on the substrate binding loop
(Met103, Phe149, Tyr158,
Lys165, Thr196, Met199,
Leu207, Ile215). This figure was produced using
the SPOCK software (57).
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There is only one direct hydrogen bond between the fatty acyl substrate
and the InhA protein, namely between the thioester carbonyl oxygen and
the side chain hydroxyl oxygen of Tyr158 (Fig.
4A), as Tyr158 is
conserved in both the bacterial FabIs and plant ENRs, this interaction
is likely to be a key feature of fatty acyl substrate binding common to
other enoyl-ACP reductases.

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Fig. 4.
Active site hydrogen bonds and protein
conformational change. A, hydrogen bonds between the
active site of InhA and the bound C16 fatty acyl substrate are shown as
black lines drawn between interacting atoms, with the
distances between labeled in angstroms. Each atom type is represented
by a unique color: carbon (green), oxygen (red),
nitrogen (blue), sulfur (yellow), and phosphorus
(magenta). B, comparison of the positions of the
substrate binding loop and the side chain of Tyr158 before
(cyan) and after (red) binding of the C16 fatty
acyl substrate. Upon substrate binding, the substrate binding loop
shifts 4.0 Å to the left and the side chain of
Tyr158 rotates 60° to the right, widening the
substrate binding crevice. This figure was produced using the
InsightII© software (BIOSYM/Molecular Graphics Simulations,
1995).
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Two additional hydrogen bonds occur between the fatty acyl substrate
and NAD+. These are between the amide nitrogen of the
N-acetylcysteamine portion of the substrate and a phosphate
oxygen of NAD+ and between the substrate thioester carbonyl
oxygen and the 2'-hydroxyl oxygen of the nicotinamide ribose of
NAD+. Furthermore, there is an additional hydrogen bond
between the substrate thioester sulfur and an ordered water molecule
held by a phosphate oxygen of NAD+ and the side chain of
Thr196 (Fig. 4A).
Substrate Binding Loop Length Determines Substrate Binding Crevice
Depth--
An interesting correlation between substrate binding loop
length and fatty acyl substrate chain length specificity can be inferred from amino acid sequence alignment of the known FAS-II enoyl-ACP reductases. Enoyl-ACP reductases that prefer shorter chain
substrates (C2-C16) have shorter substrate binding loops (13 residues)
(such as E. coli FabI and B. napus ENR), whereas longer substrate binding loops are seen in those enzymes that use
longer chain substrates (C16-C56). (23 residues) (such as M. smegmatis and M. tuberculosis InhA) (Fig.
5). This finding suggests that the size
of the substrate binding loop is a primary determinant of the enzymes
ability to distinguish between shorter versus longer chain
substrates.

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Fig. 5.
Sequence alignment of FAS-II system enoyl-ACP
reductases. The amino acid sequence information was obtained from
GenBankTM, using the following accession numbers for the
bacterial FabIs (E. coli, X78733; Salmonella
typhimurium, M31806; and Helicobacter pylori,
AE000539), the plant ENRs (Brassica napus, S60064;
Arabidopsis thaliana, Y13860; and Nicotiana
tabacum, Y13862), and the mycobacterial InhAs (Mycobacterium
avium, AF002133; Mycobacterium bovis, U41388;
Mycobacterium smegmatis, U02530; and M. tuberculosis, U02492). The numbering above the
alignment corresponds to the M. tuberculosis InhA sequence
and the lower-case letters below the alignment mark the
location of -helices (h) and -strands (s).
The substrate binding loop of the long chain substrate mycobacterial
InhAs (residues 196-219) is larger than that of the short chain
substrate bacterial FabIs and plant ENRs.
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Superposition of the crystal structure of an E. coli FabI
ternary complex (FabI, NAD+, and a benzodiazaborine drug)
(48) with that of the M. tuberculosis InhA ternary complex
(InhA, NAD+, and C16 fatty acyl substrate) demonstrates
that there is a significant difference between these two enzymes with
respect to the location of their substrate binding loops (Fig.
1C). The longer substrate binding loop of InhA creates a
substrate binding crevice with more depth than that of FabI. The
distance from the bottom of the C16 fatty acyl substrate to the top of
the substrate binding loop is 18 Å for the InhA ternary complex, and
it is only 10 Å for FabI superimposed onto the InhA ternary complex.
The crystal structure of the E. coli FabI binary complex
(FabI and NAD+) indicates that the substrate binding loop
is disordered in the absence of a fatty acyl substrate (48). Upon
binding of a benzodiazaborine inhibitor, the shorter substrate binding
loop is ordered, and the substrate binding loop makes direct contact
with the bound inhibitor (48). The shape of the FabI substrate binding
crevice, which results from the ordered substrate binding loop, is
similar to InhA, in that both a major and minor portal are retained
(Fig. 2C). Interestingly, even though a fatty acyl substrate
is not present, the resulting binding crevice is continuous from one portal to the other, appearing as an open vertical tube, which lies
adjacent to the nicotinamide ring of NAD+. When the InhA
ternary complex (InhA, NAD+, and C16 fatty acyl substrate)
is superimposed onto the FabI ternary complex (FabI, NAD+,
and a benzodiazaborine drug), the thioester and trans double bond portion of the C16 fatty acyl substrate overlap with the diazaborine drug, whereas the turn of the fatty acyl substrate (positions C4-C8) fits into the open vertical tube (Fig.
2C).
The position of the shorter FabI substrate binding loop occludes the
majority of the fatty acyl chain beyond C8. If this conformation of the
substrate binding loop is indicative of how FabI interacts with fatty
acyl substrates, then the substrates would not be accommodated in a
U-shaped conformation, and chain extensions beyond C8 would most likely
be directed out the minor portal. This situation would limit the
ability of FabI to bind only shorter chain fatty acyl substrates.
In contrast, the geometry of the
-helices of the longer substrate
binding loop of InhA would sterically hinder the loop from folding
downward into the substrate binding cavity, leaving a larger opening
for the fatty acyl substrate. The longer substrate binding loop of InhA
would be prevented from making proper contact with shorter chain fatty
acyl substrates and therefore would provide InhA with selection
criteria against shorter chain substrates. The deeper substrate binding
crevice of InhA would only be appropriate for binding of longer chain
fatty acyl substrates, consistent with the substrate specificity of the
enzyme and the role InhA plays in mycolic acid biosynthesis (28).
Conformational Changes within InhA upon Substrate Binding--
A
comparison of the structure of the binary complex (InhA and NADH) (37)
with that of the ternary complex (InhA, NAD+, and C16 fatty
acyl substrate) clearly identifies two major conformational changes in
the protein upon fatty acyl substrate binding. The substrate binding
loop (residues 196-219) was shifted 4.0 Å, and the side chain of
Tyr158 rotated by 60° (Fig. 4B). Together
these changes act to widen the substrate binding cavity to accommodate
the bound C16 fatty acyl substrate.
The substrate binding loops are located on the outer surface of the
homotetramer (Fig. 1B), and widening of the substrate binding cavity is accomplished when the loops push outward toward the
solvent. In addition, no hydrogen bonds are broken during this
conformational change. Furthermore, the loops are not involved in
contacts between subunits of the homotetramer, and loop movement does
not disrupt organization of the homotetramer (0.4-Å r.m.s. deviation
in
-carbon position over 972 residues between the binary and ternary
complexes when the substrate binding loops are omitted from
consideration). This lack of major structural reorganization indicates
that the substrate binding loop is somewhat flexible and can move
independently and therefore may provide the active site with additional
adaptability to accommodate substrates of varying chain length during
the fatty acyl chain elongation process.
SDR Family Catalytic Triad and Mechanism of InhA Catalysis--
In
addition to FAS-II system enoyl-ACP reductases, numerous steroid
dehydrogenases have been shown to be members of the SDR family (45,
46). The 7
-hydroxysteroid dehydrogenase (7
-HSDH), catalyzes the
NAD+-dependent dehydrogenation of the hydroxyl
group at position seven on the steroid skeleton of bile acids (52).
Amino acid sequence alignment of M. tuberculosis InhA with
E. coli 7
-HSDH shows 22% identity, and superposition of
their crystal structures shows that they share a very similar backbone
topology (1.5-Å r.m.s. deviation in
-carbon position over 187 residues for a single subunit). In addition, the bile acid substrate
binds to E. coli 7
-HSDH in nearly the same location that
the C16 fatty acyl substrate binds to InhA (53). The SDR family
catalytic triad of E. coli 7
-HSDH is
Ser146-Tyr159-Lys163. The tyrosine
and serine residues directly interact with the bile acid substrate
hydroxyl group (produce oxo group), whereas the lysine residue
interacts with the 3'-hydroxyl group of the nicotinamide ribose of the
substrate NAD+ (product NADH) (Fig.
6A).

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Fig. 6.
SDR family catalytic triad.
A, schematic representation of the active site of the
E. coli 7 -HSDH in complex with a bile acid product (7-oxo
group) and NADH product (Protein Data Bank entry 1fmc). The SDR family
catalytic triad is
Ser146-Tyr159-Lys163. The
dashed lines represent hydrogen bonds, and the numeric
values are their distances in angstroms. B, M. tuberculosis enoyl-ACP reductase (InhA) in complex with a C16
fatty acyl substrate and NAD+. The structurally analogous
SDR family catalytic triad of InhA is
Phe149-Tyr158-Lys165. This figure
was produced using Chemistry 4-D Draw© (ChemInnovation Software, San
Diego, CA, 1995).
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The tyrosine and lysine residues of the SDR family catalytic triad are
strictly conserved and therefore mark the location of a consensus
sequence (Tyr-X-X-X-Lys), where
separation between the tyrosine and lysine can vary from three to seven
residues (54). Among the various crystal structures of members of the SDR family, the location of the lysine residue is nearly identical, whereas the position of the tyrosine residues has a tendency to vary
somewhat among different SDR family members (46). This trend suggests
that the function of the lysine residue is to hold the NADH in place.
The function of the tyrosine residue is to directly interact with the
substrate, and that variation of the position of the tyrosine residue
among different SDR family members allows the tyrosine residue to
complement and accommodate different types of substrates. The
structurally analogous SDR family catalytic triad of InhA is
Phe149-Tyr158-Lys165 (Fig.
6B). The fact that the side chain of Tyr158
interacts with the C16 fatty acyl substrate thioester carbonyl oxygen
and that the side chain of Lys165 interacts with the
3'-hydroxyl oxygen of the nicotinamide ribose of NAD+ is
consistent with interactions in other SDR family members.
The proposed mechanism of how InhA catalyzes the reduction of the
trans double bond between positions C2 and C3 of the fatty acyl substrate consists of the formation of an enolate intermediate through the direct transfer of a hydride ion from NADH to position C3
of the substrate, which is followed by protonation of position C2 (28).
In the past, it has been proposed that the side chain of
Lys165 stabilizes the enolate intermediate (28). However,
in light of the above SDR family comparison and the fact that the side chain hydroxyl oxygen of Tyr158 forms a direct hydrogen
bond with the fatty acyl substrate thioester carbonyl oxygen, we
propose that the side chain of Tyr158 assumes this role. In
addition, the crystal structure of the InhA ternary complex suggests
that the 2'-hydroxyl oxygen of the nicotinamide ribose of NADH may
assist in the stabilization of the enolate intermediate because it
forms a hydrogen bond to the fatty acyl substrate thioester carbonyl
oxygen as well (Fig. 4A).
The role of the remaining residue of the catalytic triad
(Ser146 in E. coli 7
-HSDH and
Phe149 in M. tuberculosis InhA) is more
ambiguous. In terms of the SDR family catalytic triad, this residue is
the only component that distinguishes a dehydrogenase (7
-HSDH) from
a reductase (InhA). In terms of function, the main feature that
distinguishes these two types of enzymes is the position on the
substrate at which hydride transfer occurs (carbonyl carbon for the
dehydrogenase and enoyl carbon for the reductase). Furthermore, the
fatty acyl substrate of InhA also contains a thioester carbonyl carbon
that needs to be protected from being reduced. Because
Phe149 of InhA is fairly close (4 Å) to the nicotinamide
ring of NAD+, it is tempting to speculate that this
aromatic residue may help direct hydride ion transfer to the correct
location on the fatty acyl substrate by acting as an intermediate that
passes the hydride ion from NADH to the substrate. The importance of
the aromatic character of residue 149 is conserved in either a
phenylalanine (mycobacterial InhAs) or a tyrosine (bacterial FabIs and
plant ENRs).
The source of protons for reduction at position C2 of the fatty acyl
substrate is not clear from our crystal structure. There are no
polar/ionizable residues close enough to position C2 to be potential
candidates, suggesting that the proton may in fact come from the
solvent. The location of the C2 position of the fatty acyl substrate,
at opposite ends of the major and minor solvent portals (Fig.
2B), eliminates these portals as a possible sources of
proton donation. However, there is a hydrogen-bonded network of ordered
water molecules that leads from the closed end of every substrate
binding crevice (Fig. 2B, lower left) to the center of the
homotetramer. The side chain of Phe149 acts as a barrier
between the active site and the water channel. Rotation of the side
chain of Phe149 would, however, result in exposure of the
water channel to the active site. Within the crystal structure of an
InhA binary complex (InhA and isonicotinic-acyl-NADH) (36), the bound
form of the drug isoniazid causes the side chain of Phe149
to move away from the water channel, indicating that rotation of the
side chain of Phe149 is sterically possible.