Cofactor specificity motifs and the induced fit mechanism in
Class I ketol-acid reductoisomerases
Jackson K.B. Cahn
1,*
,
Sabine Brinkmann-Chen
1,*
,
Thomas Spatzal
*,†,‡
,
Jared A. Wiig
§
,
Andrew R. Buller
*
,
Oliver Einsle
‡,
‖
,
Yilin Hu
§
,
Markus W. Ribbe
§,¶
, and
Frances H. Arnold
2,*
*
Division of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena,
CA 91125, USA
†
Howard Hughes Medical Institute, California Institute of Technology, Pasadena, CA 91125, USA
‡
Institut für Biochemie, Albert-Ludwigs-Universität Freiburg, 79104 Freiburg, Germany
§
Department of Molecular Biology and Biochemistry, University of California, Irvine, CA 92697
‖
BIOSS Centre for Biological Signaling Studies, Albert-Ludwigs-University Freiburg, 79104
Freiburg, Germany
¶
Department of Chemistry, University of California, Irvine, CA 92697
Abstract
Although most sequenced members of the industrially important ketol-acid reductoisomerase
(KARI) family are Class I enzymes, structural studies to date have focused primarily on the Class
II KARIs, which arose through domain duplication. Here, we present five new crystal structures of
Class I KARIs. These include the first structure of a KARI with a 6-residue
β
2
α
B (cofactor
specificity determining) loop and an NADPH phosphate binding geometry distinct from that of the
7- and 12-residue loops. We also present the first structures of naturally occurring KARIs that
utilize NADH as cofactor. These results show insertions in the specificity loops that confounded
previous attempts to classify them according to loop length. Lastly, we explore the conformational
changes that occur in Class I KARIs upon binding of cofactor and metal ions. The Class I KARI
structures indicate that the active sites close upon binding NAD(P)H, similar to what is observed
in the Class II KARIs of rice and spinach and different from the opening of the active site
observed in the Class II KARI of
E. coli.
This conformational change involves a decrease in the
bending of the helix that runs between the domains and a rearrangement of the nicotinamide
binding site.
2
To whom correspondence should be addressed (fha@che.caltech.edu)
1
These authors contributed equally to this work
Coordinates and structure factors have been deposited in the Protein Data Bank with accession numbers 4TSK, 4XDY, 4XDZ, 4XEH,
and 4XIY, as shown in Table 1.
Author Contributions
: Jackson Cahn and Sabine Brinkmann-Chen conceived the project; Sabine Brinkmann-Chen prepared and
crystallized three proteins, Jackson Cahn one, and Jared Wiig and Thomas Spatzal one; Jackson Cahn solved the protein crystal
structures, one in collaboration with Thomas Spatzal and one with Andrew Buller; Yilin Hu and Markus Ribbe provided guidance to
Jared Wiig, Oliver Einsle to Thomas Spatzal, and Frances Arnold to Jackson Cahn, Sabine Brinkmann-Chen, and Andrew Buller;
Sabine Brinkmann-Chen measured the thermostability of one of the KARIs; Jackson Cahn analyzed the crystal structures and
prepared the figures; Jackson Cahn, Sabine Brinkmann-Chen, Andrew Buller, and Frances Arnold wrote the manuscript.
HHS Public Access
Author manuscript
Biochem J
. Author manuscript; available in PMC 2016 June 15.
Published in final edited form as:
Biochem J
. 2015 June 15; 468(3): 475–484. doi:10.1042/BJ20150183.
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Keywords
Crystal structure; cofactor binding; conformational change; KARI; AHAIR
Introduction
The bifunctional enzyme ketol-acid reductoisomerase (KARI, EC 1.1.1.86, also known as
acetohydroxyacid isomeroreductase (AHIR or AHAIR)), catalyzes the second step in the
biosynthesis of the branched-chain amino acids (BCAAs) valine, leucine, and isoleucine [
1
].
The enzyme converts 2-(
S
)-acetolactate (2SAL) into (
R
)-dihydroxyisovalerate (RDHIV) in
an ordered two-step reaction, where a Mg
2+
-dependent alkyl migration is followed by
reduction using a hydride from NAD(P)H (Figure 1a) [
1
]. This unusual reaction has inspired
much work to characterize the order of binding, metal ion requirements, and NAD(P)H
specificity [
1
-
7
].
Because animals lack the BCAA pathway, there is a sizable market for these amino acids
produced in microorganisms for human and animal dietary supplements [
8
-
11
], and
inhibitors of KARIs such as
N
-hydroxy-
N
-isopropyloxamate (IpOHA) are an active area of
research as potential herbicides and antibiotics [
12
-
16
]. Beyond this, the BCAA pathway has
been re-engineered in microbes for production of isobutanol, a promising second-generation
biofuel and chemical feedstock [
17
].
KARIs are divided into two classes based on their length and oligomerization state. Class I
KARIs include all fungal KARIs and are
∼
340 amino acid residues in length. Class II
KARIs include all plant KARIs and are
∼
490 residues long. Bacterial KARIs can be either
Class I or Class II [
18
]. KARIs are composed of two types of domains, an N-terminal
Rossmann domain and one or two C-terminal knotted domains. Two intertwined knotted
domains are required for function, and in the short-chain or Class I KARIs, each polypeptide
chain has one knotted domain. As a result, dimerization of two monomers forms two
complete KARI active sites (Figure 2a). In the long-chain or Class II KARIs, a duplication
of the knotted domain has occurred, and, as a result, the protein does not require
dimerization to complete its active site. As first proposed by Ahn and co-workers [
18
], this
domain duplication suggests that Class II KARIs evolved from a primordial Class I KARI.
This pair of knotted domains is extensively intertwined to form a figure-of-eight knot.
Dimerization of the Class I KARIs also forms this knot, albeit with an additional
disconnection due to being comprised of two chains. The Class II KARI from spinach was
the first deeply knotted protein identified [
19
] and remains the most deeply embedded knot
observed in a protein crystal structure [
20
].
Class I and Class II KARIs are also distinguished by the length of the Rossmann fold's
β
2
α
B-loop, which binds the adenine ribose of NAD(P)(H) and has been shown to be the
primary determinant of whether an enzyme prefers NADH or NADPH [
2
,
21
]. From a set of
558 Class II KARI sequences downloaded from Pfam [
22
], all but 20 have a 12-residue
β
2
α
B-loop. In contrast, only five of 2,860 Class I KARIs do. Class I KARIs instead favor 6-
and 7-residue loops (Supplemental Information 1). Prior to this study, no KARI from the
significant proportion of Class I homologs having a 6-residue
β
2
α
B-loop had been
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crystallized, nor had any of the Class I KARIs with 12-residue
β
2
α
B-loops. This loop, which
we will hereafter term the “specificity loop,” has been the target of our efforts to engineer
the cofactor specificity of KARIs [
21
,
23
]. The lack of crystal structures for KARIs having
6-residue loops, however, prevented a thorough understanding of the loop structure-function
relationship.
The other major gap in our knowledge of KARIs is how the Class I enzymes change
conformation upon binding the Mg
2+
ions, substrate, and cofactor. Two crystallographic
studies by Guddat and co-workers on substrate-induced rearrangements of Class II KARIs
reported different mechanisms of induced fit in plant KARIs [
24
] and
E. coli
KARI [
25
].
Though both enzymes show an ordered binding in which Mg
2+
and NADPH bind before
substrate [
1
,
26
], the structural effects of binding are dramatically different. In the plant
enzymes, the hinge between the Rossmann domain and the knotted domain moves freely in
the apo state, an observation supported by H-D exchange mass spectroscopy [
5
]. Binding of
magnesium and NADPH draws the domains together, closing the active site, and rearranges
the
α
3+1 helix to preorganize the enzyme for substrate binding. In contrast, in the bacterial
enzyme the active site is closed in the apo crystal structure, but binding of magnesium and
NADPH opens the interface between the domains and allows the substrate to bind. Nothing,
however, is known about the conformational changes in Class I KARIs, which may be
complicated by the dimeric nature of the protein.
In this study, we present five new Class I KARI structures from four previously un-
crystallized enzymes. Three of these structures give insight into the unexpected diversity of
the specificity loop. With the other two structures, we demonstrate that the mechanism of
induced fit in the bacterial Class I KARIs we studied involves closure of the interdomain
hinge, coupled with a rearrangement in the nicotinamide amide-binding portion of the active
site. This behavior is opposite that observed in the bacterial Class II KARI previously
described by Wong
et al
. [
25
] and more closely resembles the behavior of the plant KARIs
[
24
]. Taken together, these structures enhance our understanding of the structural and
functional diversity of this enzyme class.
Experimental
Cloning, Expression, and Purification of KARIs
Cloning, expression and purification of Aa_KARI (
Alicyclobacillus acidocaldarius
),
Ua_KARI (Uncultured archaeon), and Ia_KARI (
Ignisphaera aggregans
) were described
previously [
2
,
21
]. Av_KARI was isolated from
Azotobacter vinelandii
strain AvOP (ATCC
BAA-1303). Cells growth and Av_KARI purification by anion exchange and gel filtration
chromatography was performed as published earlier [
27
].
Crystallization and Data Collection of Aa_KARI, Ua_KARI, and Ia_KARI
N
-hydroxy-
N
-isopropyloxamate (IpOHA) was prepared as described [
21
]. High-throughput
screening of crystallization conditions was conducted at the Beckman Molecular
Observatory at the California Institute of Technology. For Aa_KARI in the presence of
NADPH, the best condition was 1M sodium potassium tartrate, 200 mM sodium chloride,
100 mM imidazole pH 8. For Ia_KARI in the presence of NADPH and with IpOHA as
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inhibitor, the best condition was a 0.1 M bis-tris, pH 6, solution with 22% polyethylene
glycol monomethylether 5000. The apo crystal of Ia_KARI was obtained from the same
conditions, but with 200 μM NADH instead of NADPH. For Ua_KARI in the presence of
NADH and with IpOHA as inhibitor, the best condition was a 0.1 M bis-tris, pH 5, solution
with 20% w/v polyethylene glycol 1500 as precipitant. For cryo-protection, the crystals were
soaked in a mother liquor containing 25% glycerol prior to flash-freezing in liquid nitrogen.
Diffraction data were collected using a Dectris Pilatus 6M detector on beamline 12-2 at the
Stanford Synchrotron Radiation Laboratory SSRL at 100 K. Diffraction datasets were
integrated with
XDS
[
28
] and scaled using SCALA [
29
].
Crystallization and Data Collection of Av_KARI
Av_KARI was crystallized under strictly anaerobic conditions using 0.25 M NaCl, 28 %
PEG3350 and 0.1 M Bis-Tris, pH 5.5. Partial dehydration of the observed crystals was
achieved by equilibrating the crystals against 40 % PEG 3350 for 12 h, followed by
subsequent flash-freezing in liquid nitrogen. Diffraction experiments were carried out at
100K at the Swiss-Light-Source PXI beamline X06SA (Paul-Scherrer-Institute, Villigen/
Switzerland). Data were integrated using iMOSFLM [
30
] and scaled in SCALA [
29
].
Structure solution
For Aa_KARI_holo the structure of
S. exigua
KARI (Se_KARI_holo, PDB code 4KQW)
was used for molecular replacement with MOLREP [
31
]; Ia_KARI_holo and
Ua_KARI_holo used the structure of the DDV mutant of that enzyme (Se_KARI_DDV,
PDB code 4KQX) and molecular replacement was performed using Phaser [
32
]. For
Ia_KARI_apo, the structure of Ia_KARI_holo was used for molecular replacement with
Phaser; the structure was split into two PDB files containing the Rossmann and knotted
domains, which were fit as separate ensembles. Molecular replacement for Av_KARI_met
used Pa_KARI_apo (PDB code 1NP3) [
18
] for molecular replacement with MOLREP, with
determination and refinement of iron-sites performed using SHELX [
33
]. After molecular
replacement, several iterations of automated refinement with Refmac5 [
34
] (CCP4 suite
[
35
]) and manual refinement in Coot were performed [
36
].
Structure analysis
For the whole-enzyme alignments (i.e. Figure 3a), the alignment was performed using
PyMOL [
37
]; for the domain-specific alignments, LSQ superposition was performed using
Coot [
36
]. For analysis of helix bending, the HELANAL-Plus web server [
38
] was used,
with the helices of each structure assigned by STRIDE. From the first half of the
α
1-helix
(that is, before the break in helicity) the l, m, n direction cosines of the first and last i/i-3 pair
were extracted, and the angle between them was computed.
Ia_KARI thermostability determination
T
50
is defined as the temperature at which 50% of the initial activity is retained after 1 h
incubation. Thirty-μL aliquots of purified enzyme were transferred to PCR tubes. Each tube
was assigned a specific incubation temperature on the block of an Eppendorf master cycler
PCR machine. The measurements were conducted in duplicates. The tubes were incubated
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in their slots for 1 h, and then quenched on ice. Residual activity was determined with the
activity assay described previously [
2
].
Results and Discussion
We present five crystal structures representing four Class I KARIs, with resolutions of 1.15–
2.50 Å (Table 1). We previously described three of these KARIs in our work on cofactor
specificity engineering. The cofactor specificity of Aa_KARI from
Alicyclobacillus
acidocaldarius
was inverted by the introduction of three mutations in its specificity loop
[
21
]. Ua_KARI from an uncultured organism and Ia_KARI from
Ignisphaera aggregans
were correctly predicted to utilize NADH, based on their loop sequences [
2
]. The fourth
KARI, from
Azotobacter vinelandii,
crystallized serendipitously as an impurity of another
protein purified from
A. vinelandii
lysate. Table 1 places these enzymes and the structures
reported here in the context of the previously available KARI structural data.
For some proteins, including Ia_KARI in this study, multiple structures are available with
different bound cofactors, metals, and substrate analogues. Refinement statistics of the
structures reported here are provided in Supplemental Information 2. All of these KARIs
have the same fold as previously solved Class I KARIs (Figure 2). Their active sites,
particularly the metal-binding sites, are also in agreement with those of previous structures
(see,
e.g.
Biou et al. [
39
]).
Various nomenclatures have been used to discuss the structures of KARIs. In this paper, we
will refer to the N-terminal domain as the Rossmann domain and the C-terminal domain as
the knotted domain. The topology of the Class I enzyme is shown and labeled in Figure 2b.
Secondary structural elements are numbered starting from the first
β
-strand that makes up
the canonical Rossmann fold; secondary structure prior to this strand is not conserved. In the
Rossmann domain,
β
-strands are numbered 1 to 8, and
α
-helices are assigned the letters
from A to G. In the knotted domain, which is entirely
α
-helical, helices are assigned
numbers from 1 to 8. Loops between secondary structural elements are named based on the
flanking secondary structural elements, as in
β
2
α
B-loop or
α
3
α
4-loop. We refer to the
β
2
α
B-loop of the Rossmann domain as the specificity loop. Supplemental Information 3,
which has an alignment of the KARI sequences discussed herein, is also annotated with the
secondary structure labels used. In Class I KARIs, the secondary structural elements of the
dimeric partner are denoted with an apostrophe, as in
α
3
′
-helix. In Class II KARIs, the
duplicated elements are denoted with ‘+1’, as in
α
3+1-helix. Lastly, we refer to the proteins
themselves with two-part names (i.e. Aa_KARI) and the structures with three-part names
(Aa_KARI_holo) indicating their complexation state.
Structural diversity of the specificity loop: structure of Aa_KARI's 6-residue specificity
loop
Until recently, KARI family enzymes were believed to be exclusively NADPH-dependent
[
2
]. Because NADH utilization is advantageous in the industrial production of amino acids
and biofuels [
21
,
23
], engineering KARI cofactor specificity has been of considerable
interest. Previous studies by our group aimed at reversing KARI cofactor specificity focused
extensively on the specificity loop of the Rossmann domain due to its key role in binding the
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adenine ribose of the cofactor. Structures of enzymes with a 12-residue specificity loop [
25
]
and multiple structures with 7-residue specificity loops [
21
,
39
,
40
] with bound NADPH
were available. However, no structure of a KARI having a 6-residue specificity loop was
available, with or without NADPH. In a previous study, we found that whereas similar
mutations reversed the specificity of KARIs with 7- and 12-residue specificity loops, a
modified approach was required for the 6-residue specificity loop enzymes [
21
].
Ambiguities in the alignments of the different length of the loop sequences limited our
ability to propose a structural model to explain the different sets of mutations (see the
alignment in Figure 3). For that reason, we selected for structural analysis the KARI from
A.
acidocaldarius
(Aa_KARI), a mildly thermophilic enzyme that had been engineered for
NADH preference in our previous study [
21
]. Aa_KARI crystallized readily, and we
obtained a crystal that diffracted to 2.5 Å.
The overall structure of Aa_KARI_holo is very similar to that of the other Class I KARIs
that have been published, with RMSDs of 0.48 Å and 1.56 Å against Se_KARI_holo [
21
]
and Pa_KARI_apo [
18
], respectively. Figure 4 shows the specificity loop of Aa_KARI_holo
(part c) as well as other KARIs, including ones that will be discussed later in this paper.
While the side-chains of three residues of the 7- and 12-member specificity loops form
hydrogen bonds to the NADP(H) phosphate (e.g. Arg58, Ser61, and Ser63 in
Se_KARI_holo), only two residues do so for the 6-member specificity loop of
Aa_KARI_holo, Arg48 and Ser52. However, the rearrangement of the loop brings the
α
B-
helix closer to the adenine ring, allowing the terminal serine residue's side chain to hydrogen
bond not only with the phosphate but also with O2
′
and O3
′
of the ribose, creating a
hydrogen bond between the backbone N-H group of Ser52 and the phosphate oxygen. This
interaction between the phosphate and the dipole of the
α
B-helix has not been observed
previously in a KARI.
In our previous study we found that reversing the cofactor specificity of KARIs having 7-
and 12-residue specificity loops from NADPH to NADH required installation of aspartate
residues at the ultimate and antepenultimate loop positions [
21
]. Existing structures showed
that these two residues provide the primary hydrogen bonding interactions with the NADPH
phosphate [
21
,
25
]. The 6-residue specificity loops, in contrast, only required a single
aspartate along with mutation of the arginine to proline at the second position of the loop to
switch cofactor preference to NADH. Having obtained the structure of the first KARI with a
6-residue specificity loop, we can now explain why. Only the ultimate residue of
Aa_KARI_holo's specificity loop forms a hydrogen bond with the phosphate. There is no
residue corresponding to the interaction provided by the antepenultimate position.
Presumably to compensate for this missing interaction, the contact between the arginine at
the second position of the loop and the phosphate is more extensive, such that mutation of
this residue is also critical in order to abolish phosphate binding and switch cofactor
specificity.
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Structural diversity of the specificity loop: structure of the naturally NADH-preferring
Ua_KARI
In the past, it had been assumed that all KARIs were specific for NADPH [
23
]. However,
we recently showed that some native KARIs display strong preference for NADH or at least
exhibit equal activity on NADPH and NADH [
2
]. The KARI with the greatest preference for
NADH (in terms of catalytic efficiency,
k
cat
/
K
M
) [
2
] came from an uncultured archaeon
(Ua_KARI) and was also one of the only five known Class I KARIs to possess a 12-residue
specificity loop (Supplemental Information 1). However, we could not predict the
arrangement of the residues around the NADH based on sequence alone, because the loop
sequence was so different from any previously crystallized KARI (Figure 3). It also did not
allow us to effectively compare it to the recipe we proposed previously for engineering
naturally NADPH-preferring KARIs to use NADH. For this study, we were able to obtain a
crystal of Ua_KARI bound to NADH, which diffracted to 1.54 Å.
Ua_KARI_holo possesses the same overall KARI fold as other Class I KARIs, with a 0.62
Å RMSD to Se_KARI_holo. However, the specificity loop of Ua_KARI_holo differs
significantly from any other crystallized KARI (Figure 4d). Glu46, the first residue of the
β
2
α
B-loop, lies along the N3 edge of the adenine moiety and forms a bi-dentate interaction
with the O2
′
hydroxyl of the ribose. This residue is structurally homologous to the
conserved leucine that makes a similar interaction with the adenine in other KARIs. The
position of this glutamate is stabilized by a hydrogen bond interaction with Asn55.
In most KARIs, an arginine in the position immediately following the conserved leucine
forms a cation-pi interaction with the adenine and a salt bridge with the phosphate of the
cofactor [
18
]. In Ua_KARI_holo, the arginine is replaced by Leu49, which removes the
positive charge. Additionally, the specificity loop is extended by five residues that
accommodate the structural differences between arginine and leucine. In
E. coli
KARI,
which also has a 12-residue loop, the five additional residues form a short helix inserted
after the arginine. In Ua_KARI_holo, by contrast, there are three residues before Leu49 and
two after. This leads to a reorientation of the specificity loop (Figure 4d). As with the
canonical NADPH-binding 7- and 12-residue specificity loops, Ua_KARI_holo's specificity
loop has two additional residues that provide contacts to the (phospho-) ribose. Ser57 at the
end of the specificity loop is highly conserved and, as in Aa_KARI_holo, forms hydrogen
bonds with both O2
′
and O3
′
of the ribose. Though there is no phosphate to hydrogen bond
to its backbone amine, this backbone nitrogen hydrogen bonds to the side-chain of another
residue, Asn55. The increased size of Asn55 over the consensus serine at this position
allows it to make contact with O2
′
rather than the absent phosphate, and the nitrogen forms a
hydrogen bond with Glu46, as previously noted.
The lack of a short helix within the loop suggests that the ancestral Ua_KARI did not have a
canonical 12-residue specificity loop. Ua_KARI may have evolved from a KARI possessing
a shorter loop. This is supported by the fact that the KARIs with the sequences most similar
to Ua_KARI (other than a 98% identical one from another uncultured archaeon) both had 6-
residue specificity loops, as discussed previously [
2
].
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Structure of Ia_KARI: an unusual
β
2
α
B-loop in a bi-specific KARI
The
I. aggregans
KARI from our previous study (Ia_KARI) displayed sub-micromolar
K
M
values for both NADH and NADPH [
2
], and measurements carried out for this study
confirmed that it is highly thermostable, with half-maximal residual activity (T
50
) at 95 °C.
We were unable to obtain a structure of Ia_KARI bound to NADH, but did succeed in co-
crystallizing it with NADPH and the transition state analogues
N
-isopropyloxamate (Figure
1). The ligand's electron density, however, indicated that it had been reduced at the
N
position (cause unknown) and a water molecule had taken the place of the missing -OH
group (Supplemental Information 4).
Because Ia_KARI_holo has a 7-residue specificity loop, we had expected the second loop
residue, a glutamate, to make contact with the face of the adenine ring and the
antepenultimate and ultimate residues to bind the phosphate. Surprisingly, the binding mode
for NADP closely resembles that of the 6-residue loop from Aa_KARI_holo, with a single
serine residue at the ultimate position of the loop hydrogen bonding with both the O3
′
hydroxyl and the phosphate (Figure 4e). Furthermore, the glutamate at the second position
of the loop faces away from the cofactor entirely, while the arginine at the third position
forms the typical packing interaction against the adenine ring. Without an NADH co-crystal
structure, it is difficult to speculate what functional role this glutamate serves or how it
contributes to the enzyme's cofactor bi-specificity. However, it is possible that a
rearrangement of the loop might allow it to occupy the position of either of the conserved
residues flanking it. Such rearrangements of adenosine ribose-binding loops have been
observed in bi-specific glucose-6-phosphate dehydrogenase [
41
], xylose reductase [
42
],
glyceraldehyde-3-phosphate dehydrogenase [
43
], and coenzyme A-disulfide reductase [
44
].
It is interesting that both of the naturally NADH-utilizing KARIs, Ua_KARI and Ia_KARI,
had amino acid insertions relative to other structurally similar enzymes. Insertions and
deletions in genes represent a major source of sequence and function variation in natural
evolution [
45
,
46
], but are rarely used in protein engineering and directed evolution. In our
previous work, classifying the binding mode based on the length of the specificity loop was
the first step to identifying a protein engineering strategy for engineering KARI cofactor
specificity [
21
]. These results, however, demonstrate that loop length may be an insufficient
indicator of binding geometry.
Conformational changes in Class I KARIs: induced fit in Ia_KARI
The Ia_KARI structures with and without bound metals or ligands allowed us to observe the
changes in the protein structure upon the binding of NADPH, metals, and
N
-
isopropyloxamate. As in the plant and
E. coli
Class II KARIs, the two domains of Ia_KARI
undergo a rigid body movement, where only the angle between them changes significantly.
With metals, cofactor, and inhibitor bound, the Rossmann domains have moved closer
together, sealing off the active sites and generating a “closed” state. Either domain of the
ligand-bound protein can be superimposed on its respective apo-protein domain, but the full
structures do not superimpose. Figure 5 shows a plot of the C
α
-C
α
distances along the length
of the chain for Ia_KARI_holo and Ia_KARI_apo, aligned by Rossmann domain (blue) or
knotted domain (red). With either alignment a distinct jump in the C
α
-C
α
distances indicates
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a “hinge point” at Glu189. Glu189 is on the
α
1-helix of the knotted domain rather than the
β
8
α
1-loop between the domains, which stays rigid with respect to the N-terminal domain. In
the ligand-bound structure, the
α
1-helix is less bent, with an angle of 4.7° versus 8.7° in the
apo structure, as measured using HELANAL-Plus [
38
]. Figure 6 shows this movement
structurally (a and b) and quantitatively (c) for Ia_KARI as well as other structure pairs to be
discussed later. However, there is no significant rearrangement in the helix to explain this
change in curvature. The changes are subtle and cumulative at the various backbone
positions. Only a handful of residues in the protein change rotamer state between the two
crystals, most notably His108, Lys131, Asp191, and Glu195, all of which are involved in the
binding of the two Mg
2+
ions. Similarly, Arg49 and Arg56, which are involved in binding
the NADP(H) cofactor, reorient slightly.
Three regions have notably non-rigid-body behavior: the N-terminus of the
α
A-helix
(including the latter half of the Rossmann-identifying GxGxxG motif [
47
]), the
β
6
α
F-loop
and the N-terminus of the
α
F-helix, and the
α
3
α
4-loop. Indeed, in the ligand-bound
structure, the
α
A-helix and
β
6
α
F-loop interact with the
α
3-helix not of their own chain but
of their dimeric partner. This interaction makes up the opposite side of the active site from
the Asp191/Glu195/
α
1 face closer to the hinge. A rearrangement of the
α
3+1-helix was
previously identified in the plant KARIs and was thought to be involved in binding IpOHA
and “capping” the active site [
24
]. In the Ia_KARI_apo structure, a combination of this
rearrangement and an increase in the
α
1-helix curvature means that the interaction between
these regions is absent. The distance between the alpha carbons of Gly134 (on the
α
F-helix)
and Ala250
′
(on the dimeric partner's
α
3-helix) goes from 11.8 Å in the apo structure to 6.6
Å in the ligand-bound structure.
Critically, this closure is centered around the nicotinamide moiety of NADPH, in particular
the C7N amide. As shown schematically in Figure 7a, the side-chain of Gln28 hydrogen
bonds to the backbone amine of Gly134 in the absence of the cofactor. With cofactor bound,
the terminal amide is inserted into this gap, with O7N hydrogen bonding to the backbone
amine and Gln28 flipped out of the way. The N7N nitrogen of the amide recruits the
α
3
′
-
helix, specifically the backbone carbonyl of Ala250
′
, as well as the side-chain of Ser27.
Ser27 hydrogen-bonds to the cofactor, Gly134, and Ala250
′
, holding the complex together
and “capping” the active site. Supplemental Information 5 shows this motion in the
structure.
Conformational changes in Class I KARIs: induced fit in other class I KARIs
From this pair of structures, it was unclear to what extent the gross conformational change
was driven by the smaller-scale rearrangements related to metal binding, or cofactor
binding, or some combination thereof. However, we were also able to obtain a structure of
another KARI, from
A. vinelandii
, with metal ions (Mg
2+
and Fe
2+
) bound in the active site,
but without a cofactor or a substrate analogue. Because Av_KARI_met is from a mesophile
and possesses a 7-residue NADPH-dependent
β
2
α
B-loop, we have compared it not to
Ia_KARI, but to the structures of
Pseudomonas aeruginosa
KARI (Pa_KARI_apo), which
was crystallized in the apo form [
18
], and
Slackia exigua
(Se_KARI_holo), which was
crystallized with cofactor, substrate analogue, and metals [
21
]. Additionally, Av_KARI has
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higher sequence identity (Supplemental Information 6) to these than to Ia_KARI. Below,
residues will be discussed using the numberings in Av_KARI; the corresponding
numberings in Pa_KARI and Se_KARI are given in Supplemental Information 7.
The structure of Av_KARI_met aligns very well with that of Pa_KARI_apo, deviating only
in the
β
2
α
B-loop and at the C-terminus. The former deviation is likely a result of increased
flexibility in this loop due to the lack of a cofactor. Both structures are in an “open” state,
with their active sites exposed to solvent. Se_KARI_holo is in the “closed” state, and to
align Av_KARI_met to Se_KARI_holo, we were forced to align the N-termini and C-
termini separately, as in Figure 5. As with Ia_KARI, it is clear that each domain is separate
and rigid, with a hinge between them, and again this hinge is the
α
1-helix. This provides an
unambiguous answer to the question above: the bending of the
α
1-helix is driven not by the
binding of metal ions to the acidic residues on this helix, but by the subsequent binding of
the cofactor.
It is worth noting that of the six metal-binding residues, only Lys130, Asp190, and Glu226
′
undergo rotamer changes upon metal binding that are much more subtle than the changes
observed with Ia_KARI. The residues of Pa_KARI_apo are therefore more preorganized for
metal binding, although it is not clear whether this is an artifact of crystallization or a
function of the protein structure. Nevertheless, this is further evidence that metal ion binding
is not a driver of a larger rearrangement.
The gross conformational change between Av_KARI_met and Se_KARI_holo closely
resembles that observed in Ia_KARI, as does the correlated reorganization of the
α
3
′α
4
′
-
loop and the
α
F-helix around the nicotinamide amide group. The reorganization in
Av_KARI_met/Se_KARI_holo, however, involves a more intricate hydrogen bond network
(Figure 7b, Supplemental Information 8). In place of Ala250
′
, Av_KARI has Ser249
′
, the
side-chain of which is capable of hydrogen bonding. Likewise, Pro135 of the
α
F-helix,
which did not participate in the rearrangement is replaced with His134, is reoriented upon
cofactor binding and hydrogen bonds to the side-chains of both Ser249
′
and Ser26.
Furthermore, Gln27, instead of hydrogen-bonding to the backbone amine of Gly133 in the
cofactor-free structure, hydrogen-bonds to the carbonyl of Ala131. This hydrogen bond is
not interrupted by the binding of the cofactor. A different network of interactions is present
in Pa_KARI_apo, but inspection of the electron density unambiguously shows this to be a
modeling error (Supplemental Information 9).
It is interesting that this mechanism (open apo state and closed cofactor-bound state)
resembles that of the plant Class II KARIs rather than the one from the bacterial Class II
KARIs. The plant KARIs also undergo rearrangement of the
α
3+1-helix upon binding of
NADPH, though the plant KARI conformational change is more complex, involving a
reorganization of a long C-terminal tail – absent in Class I KARIs – upon cofactor binding
[
24
]. In contrast,
E. coli
KARI adopts an open conformation after binding of cofactor and
substrate, the reverse of what is observed in the plant KARIs and the bacterial Class I
KARIs discussed in this paper. Nonetheless, the conformational change in
E. coli
KARI also
involves rearrangements centered around the nicotinamide amide. In
E. coli
KARI, the
α
2+1- and
α
3+1-helices interact with the
β
6
α
F-loop in the absence of cofactor; the binding
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of nicotinamide into the active site collapses the
β
6
α
F-loop (as briefly discussed by Wong
et
al.
[
25
]) and breaks this interaction (Supplemental Information 10). Until more structural
information is available, it is impossible to say whether
E. coli
KARI is unique in this regard
or what evolutionary pathway led it to have this induced fit mechanism opposite that of the
other KARIs.
Conclusions
Understanding and controlling the cofactor specificity of KARI enzymes has been a goal for
protein engineers for nearly two decades [
48
]. These efforts have, by necessity, relied
heavily on incomplete structural data to guide mutagenesis strategies. With the addition of
the structures described in this paper to those previously available, we now have
representatives of all known canonical KARI cofactor specificity loop lengths. These data
show that the 6-residue specificity loops use a distinct binding arrangement for the
phosphate of NADPH, providing a clear structural explanation for results from previous
cofactor engineering efforts [
21
]. Furthermore, structures of two of the naturally NADH-
utilizing KARIs show that insertions near the N-terminus of the specificity loop likely
determine cofactor specificity, whereas the laboratory engineering of NADH preference
involved specific loop substitutions.
These structures also provide insight into the large conformational change associated with
KARI cofactor binding and catalysis. The rearrangement observed in these Class I KARIs
facilitates hydrogen bond interactions with the nicotinamide group and effectively closes the
enzyme active site through motion of the
α
1-helix. This geometry resembles that observed
in plant KARIs [
24
], but is distinct from that of
E. coli
KARI, which goes from a closed
conformation to an open one upon cofactor binding [
25
]. The principal study on order of
ligand binding in KARIs was conducted using
E. coli
KARI [
1
], raising the question of
whether the kinetic mechanism for ordered binding has been maintained throughout
divergent evolution. The structures provided in the current study answer several outstanding
questions about the structural and functional diversity of KARIs and bring us closer to a
comprehensive understanding of this enzyme class.
Supplementary Material
Refer to Web version on PubMed Central for supplementary material.
Acknowledgments
We thank Dr. Jens Kaiser and Dr. Pavle Nikolovski for their continued support.
Funding
: This publication was supported by the Gordon and Betty Moore Foundation through GBMF2809 to the
Caltech Programmable Molecular Technology Initiative, by the Resnick Sustainability Institute at Caltech
(J.K.B.C.), and by NIH Grant GM 67626 (M.W.R.). The Molecular Observatory is supported by the Gordon and
Betty Moore Foundation, the Beckman Institute, and the Sanofi-Aventis Bioengineering Research Program at
Caltech.
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Summary
The Class I KARI subfamily has received less attention than the smaller Class II
subfamily, leaving several gaps in our structural knowledge. We present five new
structures that elucidate basis of cofactor specificity and ligand-induced conformational
changes.
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Figure 1.
a) The two-step reaction catalyzed by KARI. b) The four substrate analogues which have
been crystallized in the active site of KARIs (including in this study). PDB ligand IDs are
shown in parentheses.
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Figure 2.
a) The crystal structure of the Ia_KARI_holo dimer showing bound NADPH and inhibitor
(white) and Mg
2+
ions (cyan). One monomer is shown in grey; the other is shown in green
(Rossmann domain) and magenta (knotted domain). b) The topology of Class I KARIs is
illustrated schematically, with cyan helices and red
β
-strands; the knotted domain has blue
helices. Also shown is the knotted domain of the dimeric partner (magenta), which has been
truncated for simplicity. The figure was constructed using TopDraw [
50
].
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Figure 3.
A structure-guided alignment of the specificity loops (including four residues on each side)
of some of the KARIs discussed in this paper, annotated with the length, the class of the
KARI, the kingdom of origin, and the cofactor preference. Residues contacting the
phosphate are highlighted in orange.
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Figure 4.
The
β
2
α
B-loops of a) Se_KARI_holo (PDB 4KQW) [
21
], b) Ec_KARI_holo (PDB 3ULK)
[
25
], c) Aa_KARI_holo, d) Ua_KARI_holo, e-f) Ia_KARI_holo and _apo. Polar interactions
(i.e. hydrogen bonds) of 3.5 Å or less to the 2
′
and 3
′
moieties of the ribose are shown with
dashed lines, as are selected interactions between amino acid residues.
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Figure 5.
C
α
-C
α
distances for residues of Ia_KARI_holo aligned against Ia_KARI_apo. Because of
the rigid body movement of the interdomain hinge, the Rossmann (blue) and knotted (red)
domains have been aligned separately. The nonaligned domains are shown in a lighter color.
Points of interest are numbered as follows: 1)
α
A-loop, 2) Specificity loop, 3)
β
6
α
F-loop, 4)
Hinge, 5)
α
3-helix.
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Figure 6.
a) An alignment between the apo and holo dimers of Ia_KARI. The structures are aligned
based on their rightmost Rossmann domains. The apo structure is shown in green and cyan,
and the holo structure is shown in magenta and tan. b) The Rossmann domains and
α
l-
helices of the apo (green) and holo (magenta) crystals of Ia_KARI, showing the change in
the bend of the first half of the
α
1-helix, c) Quantitative analysis of the bending of the first
half of the
α
1-helix. Error bars show standard deviations of the values for multiple chains in
the asymmetric unit of the crystal. Bars are denoted with an ‘O’ or a ‘C’ to designate open
and closed states of the active site, as assigned visually. Ec_KARI is a clear outlier, both
because it goes from a closed to open state upon binding cofactor, and because there is no
corresponding change in the
α
1-helix bending.
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Figure 7.
Hydrogen bonds involved in the recruitment of the
α
3
′α
4
′
-loop upon cofactor binding in (a)
Ia_KARI (b) and and Av_KARI_met and Se_KARI_holo. R = adenosine diphosphate ribose
phosphate.
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