The plant pathogen enzyme AldC is a long-chain aliphatic aldehyde dehydrogenase

Article abstract of DOI:10.1074/jbc.RA120.014747

Aldehyde dehydrogenases are versatile enzymes that serve a range of biochemical functions. Although traditionally considered metabolic housekeeping enzymes because of their ability to detoxify reactive aldehydes, like those generated from lipid peroxidation damage, the contributions of these enzymes to other biological processes are widespread. For example, the plant pathogen Pseudomonas syringae strain PtoDC3000 uses an indole-3-acetaldehyde dehydrogenase to synthesize the phytohormone indole-3-acetic acid to elude host responses. Here we investigate the biochemical function of AldC from PtoDC3000. Analysis of the substrate profile of AldC suggests that this enzyme functions as a long-chain aliphatic aldehyde dehydrogenase. The 2.5 ? resolution X-ray crystal of the AldC C291A mutant in a dead-end complex with octanal and NAD¹ reveals an apolar binding site primed for aliphatic aldehyde substrate recognition. Functional characterization of site-directed mutants targeting the substrate- and NAD(H)-binding sites identifies key residues in the active site for ligand interactions, including those in the “aromatic box” that define the aldehyde-binding site. Overall, this study provides molecular insight for understanding the evolution of the prokaryotic aldehyde dehydrogenase superfamily and their diversity of function.

Full text of DOI:10.1074/jbc.RA120.014747

ARTICLE
The plant pathogen enzyme AldC is a long-chain aliphatic
aldehyde dehydrogenase
Received for publication, June 15, 2020, and in revised form, August 11, 2020 Published, Papers in Press, August 12, 2020, DOI 10.1074/jbc.RA120.014747
1
,2,‡
1,‡
1
1
1
1
Soon Goo Lee1
, Kate Harline
, Orchid Abar , Sakirat O. Akadri , Alexander G. Bastian , Hui-Yuan S. Chen ,
1 1 1 1
1
Michael Duan , Caroline M. Focht , Amanda R. Groziak , Jesse Kao , Jagdeesh S. Kottapalli , Matthew C. Leong ,
1
1
1
1
1
1
1
Joy J. Lin , Regina Liu , Joanna E. Luo , Christine M. Meyer , Albert F. Mo , Seong Ho Pahng , Vinay Penna ,
1
1
1
1
2
1,3
Chris D. Raciti , Abhinav Srinath , Shwetha Sudhakar , Joseph D. Tang , Brian R. Cox , Cynthia K. Holland
,
1
1
1,4
1
1,
Barrie Cascella , Wilhelm Cruz , Sheri A. McClerkin , Barbara N. Kunkel , and Joseph M. Jez *
1
2
From the Department of Biology, Washington University in St. Louis, St. Louis, Missouri, USA, the Department of Chemistry and
Biochemistry, University of North Carolina–Wilmington, Wilmington, North Carolina, USA, the Department of Biology, Williams
College, Williamstown, Massachusetts, USA, and the Department of Molecular Genetics and Cell Biology, University of Chicago,
3
4
Chicago, Illinois, USA
Edited by Chris Whitfield
Aldehyde dehydrogenases are versatile enzymes that serve a be encoded by multiple genes in the genome of a given species
range of biochemical functions. Although traditionally consid- (9, 10) (Fig. 1).
ered metabolic housekeeping enzymes because of their ability
to detoxify reactive aldehydes, like those generated from lipid
peroxidation damage, the contributions of these enzymes to
other biological processes are widespread. For example, the
plant pathogen Pseudomonas syringae strain PtoDC3000 uses
an indole-3-acetaldehyde dehydrogenase to synthesize the
phytohormone indole-3-acetic acid to elude host responses.
Here we investigate the biochemical function of AldC from
PtoDC3000. Analysis of the substrate profile of AldC suggests
that this enzyme functions as a long-chain aliphatic aldehyde
dehydrogenase. The 2.5 Å resolution X-ray crystal of the AldC
Aldehyde dehydrogenases are generally associated with the
detoxification of aldehydes, which are highly reactive com-
pounds generated through cellular metabolism (11). For exam-
ple, these enzymes can scavenge aldehydes, such as malondial-
dehyde resulting from lipid peroxidation, and convert them to
a less chemically reactive carboxylic acid (12). Aldehyde dehy-
drogenases and their biochemical functions are also linked to a
wide variety of biochemical processes ranging from ethanol
metabolism via oxidation of acetaldehyde into acetate (13–15),
polyamine metabolism (16), and plant cell wall ester biogenesis
(17, 18) to protective cellular responses to stresses such as
1
C291A mutant in a dead-end complex with octanal and NAD
reveals an apolar binding site primed for aliphatic aldehyde sub- dehydration, osmotic shock, and temperature changes (19, 20).
strate recognition. Functional characterization of site-directed Recent work has also linked aldehyde dehydrogenase activity to
mutants targeting the substrate- and NAD(H)-binding sites the synthesis of indole-3-acetic acid, the primary plant hor-
identifies key residues in the active site for ligand interactions,
including those in the “aromatic box” that define the aldehyde-
binding site. Overall, this study provides molecular insight for
understanding the evolution of the prokaryotic aldehyde dehy-
drogenase superfamily and their diversity of function.
mone auxin, in the plant pathogenic microbe Pseudomonas
syringae strain PtoDC3000 (21).
To suppress host defenses and promote diseases develop-
ment, Pseudomonas syringae produces a variety of virulence
factors, including phytohormones or chemical mimics of hor-
mones, to manipulate hormone signaling in its host plants (22–
2
4). P. syringae and many other plant-associated microbial
In all organisms, the diversification of large superfamilies of
enzymes provides a foundation for the evolution of biochemical
capacity and the ability to metabolize varied small molecules
pathogens can synthesize the major auxin indole-3-acetic acid
(
(
IAA), whose production is implicated in pathogen virulence
21, 25–29). PtoDC3000 was shown to synthesize IAA using an
(
1). Typically, across a superfamily, the core chemical mecha-
uncharacterized pathway requiring indole-3-acetaldehyde de-
hydrogenase activity (21). Previously, a mutation in Azospiri-
lum brasilense (aldA) was identified that decreased IAA pro-
duction and was linked to a gene and annotated as encoding
an aldehyde dehydrogenase (30). Bioinformatic analysis of
PtoDC3000 identified a set of aldehyde dehydrogenases
nism is retained with substrate profiles becoming either speci-
alized or promiscuous, depending on evolution and the meta-
bolic needs of the organism (2). Classic examples of enzyme
superfamilies found across all kingdoms include the cyto-
chromes P450, alcohol dehydrogenases, aldo-keto reduc-
tases, and aldehyde dehydrogenases (3–10). For example,
aldehyde dehydrogenases are NAD(P)(H)-dependent enzymes
that metabolize a wide range of aldehydes to their correspond-
ing carboxylic acids in prokaryotes and eukaryotes and tend to
(
AldA–C) sharing 30–40% amino acid identity with each
other. Subsequent metabolic, biochemical, and in planta
analyses of AldA (UniProt: PSPTO_0092), AldB (UniProt:
PSPTO_2673), and AldC (UniProt: PSPTO_3644) demon-
strated that AldA functions as an indole-3-acetaldehyde de-
hydrogenase, is essential for IAA synthesis, and contributes
to virulence of PtoDC3000 (21). AldB may also contribute to
T‡ his article contains supporting information.
These authors contributed equally to this work.
*
For correspondence: Joseph M. Jez, jjez@wustl.edu.
13914 J. Biol. Chem. (2020) 295(40) 13914–13926
©
2020 Lee et al. Published under exclusive license by The American Society for Biochemistry and Molecular Biology, Inc.

Analysis of AldC from P. syringae DC3000
Figure 1. Overall reaction catalyzed by NAD(P)(H)-dependent aldehyde dehydrogenases.Ă
IAA synthesis but is not as metabolically or kinetically effi- P. fluorescens, and P. savastanoi (Fig. 2B and Table S1). AldC
cient as AldA for IAA production (21). Analysis of AldC belongs to a clade consisting of aldehyde dehydrogenases,
indicates that it lacks a role in IAA synthesis (21); however, which share ;90% sequence identity, from various plant patho-
its potential biochemical role in PtoDC3000 was not fully genic Pseudomonas spp.: P. syringae, P. viridiflava, P. savasta-
examined.
noi, and P. amygdali in the P. syringae phylogenetic group.
Here we investigate the substrate profile of AldC from None of AldC-related enzymes from Pseudomonas had previ-
PtoDC3000, which suggests that this enzyme functions as a ously been experimentally characterized, and their substrates
long-chain aliphatic aldehyde dehydrogenase. The 2.5 Å resolu- and functions had yet to be described.
tion X-ray crystal of the AldC C291A mutant in complex with
To determine the substrate preference of AldC, a panel of 23
1
octanal and NAD and biochemical analysis of a set of site- molecules (Table S2), including short- to long-chain aliphatic
directed mutants provide insight on substrate recognition in aldehydes (acetaldehyde, propionaldehyde, butyraldehyde,
this enzyme. Comparison of the three-dimensional structures valeraldehyde (pentanal), isovaleraldehyde, 3-methylcroto-
naldehyde, hexanal, trans-2-hexen-1-al, heptanal, octanal,
nonanal, trans-2-nonenal), betaine aldehyde, and aromatic
aldehydes (benzaldehyde, m-anisaldehyde, p-anisaldehyde,
phenylacetaldehyde, cinnamaldehyde, hydrocinnamalde-
hyde, coniferylaldehyde, sinapaldehyde, 4-pyridinecarbox-
aldehyde, and indole-3-acetaldehyde) was used to screen for
enzymatic activity. Spectrophotometric assays of AldC
identified aliphatic aldehydes of 5–9-carbon length, as well
as hydrocinnamaldehyde and 4-pyridinecarboxyaldehyde,
as substrates (Fig. 3A) with octanal having the highest spe-
cific activity (Fig. 3B). These data suggest that short 2–4-
carbon aldehydes, branched aliphatic aldehydes, and larger
aromatic aldehydes are poor substrates for AldC. To evalu-
ate the nicotinamide cofactor preference of AldC, the activ1-
ity of the enzyme was tested with octanal and either NAD
of AldA and AldC from PtoDC3000 reveals the sequence and
structural changes that lead to distinct substrate profiles of
these aldehyde dehydrogenases.
Results and discussion
Biochemical analysis of AldC as a long-chain aliphatic
aldehyde dehydrogenase
Previously, three aldehyde dehydrogenases (AldA–C) from
the plant pathogen P. syringae strain PtoDC3000 were identi-
fied and examined for their contribution to the synthesis of
IAA and virulence (21). Each protein was expressed and puri-
fied to homogeneity for enzyme assays using indole-3-acetalde-
hyde as a substrate (21). Unlike AldA and AldB, which func-
tioned as physiological tetramers, AldC was shown to be a
homodimer (21). Steady-state kinetic parameters for AldA
using indole-3-acetaldehyde showed this enzyme to be 130-
and 710-fold more efficient than AldB and AldC, respec-
tively (21).
1
1
or NADP , which showed a distinct preference for NAD
Fig. 3C).
Steady-state kinetic analysis of AldC with valeraldehyde,
(
hexanal, heptanal, octanal, and nonanal indicates that the 8-
carbon substrate (i.e. octanal) is the preferred aliphatic alde-
hyde substrate (Table 1). Although the kcat values of AldC vary
Bioinformatic analysis predicted that AldC contains the
PF00171 domain, a signature domain of the aldehyde dehydro-
33
482
genase superfamily, ranging from Ser to Ile (Fig. 2A). Spe- less than 3-fold between these substrates, the Km value for
cifically, the 4 catalytic residues (Asn , Glu , Gly , and octanal is the lowest (1.2 mM) with that of valeraldehyde as the
159
257
288
2
91
1
155
Cys ) and 19 residues in the NAD -binding site (Ile
–
,
highest (48.8 mM). The overall effect is a marked difference in
1
59
182
219
233
236
239
242
257
Asn , Lys , Gly , Ile –Ser , Ala , Leu , Glu
catalytic efficiency (kcat/K ) between octanal and the other ali-
m
2
58
259
291
391
393
Leu , Gly , Cys , Glu , and Phe ) define the aldehyde phatic substrates ranging from use of nonanal exhibiting a 10-
dehydrogenase consensus sequence motifs of AldC. The NCBI fold reduction in kcat/Km to ;80-fold less efficient use of the
Conserved Protein Domain Family server places AldC in the 5-carbon substrate valeraldehyde. Generally, the aromatic alde-
cd01738/ALDH_CddD_SSP0762 family, 1 of 42 aldehyde dehy- hyde substrates hydrocinnamaldehyde and indole-3-acetalde-
drogenase families found in the Pseudomonas (31, 32).
hyde were poorly used by AldC with catalytic efficiencies
The Pseudomonas orthologous groups classification system comparable with valeraldehyde and 4-pyridinecarboxaldehyde
in the Pseudomonas Genome Database (RRID:SCR_006590) used with a kcat/Km value 6-fold lower than octanal (Table 1).
found orthologs of AldC (group ID POG018413) from 93 Pseu- Overall, the biochemical analysis of AldC suggests that this
domonas species and strains, including P. aeruginosa, P. putida, enzyme functions primarily as a long-chain aliphatic
J. Biol. Chem. (2020) 295(40) 13914–13926 13915

Analysis of AldC from P. syringae DC3000
Figure 2. Domain architecture and phylogeny of AldC from P. syringae strain PtoDC3000. A, the UniProt aldehyde dehydrogenase (ALDH) domain
(
(
green), NAD(P)(H)-binding site (blue triangles), and catalytic residues (red triangles) of PtoDC3000 AldC (WP_011104646.1) were predicted using InterPro79.0
59). B, the PtoDC3000 AldC sequence was used as a BLAST query to identify ortholog group members of aldC (group ID POG018413) from the Pseudomonas
Orthologous Groups classification system in the Pseudomonas Genome Database (RRID:SCR_006590) (Table S1). Amino acid sequences from 93 Pseudomonas
species strains were multialigned using Clustal Omega (60). The phylogenetic tree was generated with MEGAX (61) with evolutionary relationships inferred
using the maximum likelihood method and JTT matrix-based model (62). A clade consisting of aldehyde dehydrogenases from plant pathogenic Pseudomonas
species and strains is highlighted (green) with AldC from PtoDC3000 indicated.
aldehyde dehydrogenase (Fig. 3D). Moreover, conservation 21). The AldC C291A point mutant was generated by PCR mu-
of related homologs in 93 different Pseudomonas species tagenesis, and the resulting protein was expressed, purified,
and strains (Fig. 2B) indicates a likely conserved function and crystallized with the goal of obtaining a dead-end complex
1
across these organisms.
of the enzyme with octanal and NAD bound in the active site.
The 2.5 Å resolution X-ray crystal structure of the AldC
1
(
C291A)·octanal·NAD complex was solved by molecular
Three-dimensional structure of AldC C291A mutant in
complex with octanal and NAD
replacement using the three-dimensional structure of P. syrin-
gae AldA as a search model (21) (Table 2). Each of the two
1
To understand how AldC recognizes aliphatic aldehyde sub- unique protein chains in the asymmetric unit form a corre-
strates, crystals of the AldC C291A mutant were grown in the sponding physiological dimer through crystallographic symme-
1
presence of octanal and NAD . The analogous cysteine to try (Fig. 4A). The secondary structure features and domains of
2
91
Cys
of AldC in aldehyde dehydrogenases, including AldA the AldC monomer are similar to those of other aldehyde dehy-
from P. syringae, is the essential catalytic residue (9, 10, 14–16, drogenase family members (Fig. 4, B and C). The N-terminal
13916 J. Biol. Chem. (2020) 295(40) 13914–13926

Analysis of AldC from P. syringae DC3000
Figure 3. Screening of AldC with aldehyde substrates. A, screening of aldehyde substrates. Assays were performed as described under “Experimental pro-
1
cedures.” Enzymatic activity was measured spectrophotometrically (A340 nm) in assays with a fixed concentration of NAD (2 mM) and each aldehyde (5 mM).
Spectrophotometric absorbance changes versus time are shown for AldC-catalyzed conversion of hydrocinnamaldehyde, octanal, nonanal, 4-pyridinecarbox-
yaldehyde, hexanal, 2-hexanal, valeraldehyde, isovaleraldehyde, and phenylacetylaldehyde. B, comparison of AldC specific activity for substrate aldehydes. Av-
erage values 6 S.D. (n = 3) are shown. C, AldC nicotinamde cofactor preference. Spectrophometric data for assays of AldC using octanal (5 mM) and either
1
1
NAD and NADP (each at 2 mM) is shown. D, summary of aldehyde substrates and potential reactions catalyzed by AldC.
Rossmann-fold domain contains a central b-sheet (b9-b8-b7- these enzymes from widely varied organisms (Fig. S1). Amino
b10-b11) surrounded by a-helices to form the NAD(H)-bind- acid sequence comparisons of these proteins highlight how the
ing site (Fig. 4, B and C, blue). The C-terminal region consists catalytic cysteine and residues of the NAD(H)-binding site are
of a mixed a/b domain, which includes the catalytic cysteine highly conserved with major variations in the substrate-binding
residue and forms the aldehyde-binding site (Fig. 4, B and C, site leading to functional differences in these proteins (Fig. S2).
red). An interdomain linker region (Fig. 4, B and C, green) con-
Although the specific determinants of oligomerization in
nects the N- and C-terminal domains of AldC. A small three- aldehyde dehydrogenases can be highly variable (38), the X-ray
stranded b-sheet domain (Fig. 4, B and C, gold) facilitates crystal structure of AldC provides possible insight on its dimer
oligomerization.
structure compared with the tetramer reported for AldA (21).
The overall fold of AldC shares structural similarity with The P. syringae AldA tetramer is formed from a dimer of
multiple aldehyde dehydrogenases, including P. syringae AldA, dimers with an extensive interaction surface, shown in Fig. 5 (A
from a variety of microbes and eukaryotes (Fig. S1 and Table and B) with the oligomerization region highlighted in the close-
S3). Overlays of the AldC structure with twelve aldehyde, reti- ups. Fig. 5 (C and D) shows the same region of the AldC dimer
nal, betaine aldehyde, and indole-3-acetaldehyde (i.e. AldA) de- for comparison. Multiple amino acid differences between AldA
hydrogenases (14, 15, 21, 33–37), which are related by 38–46% and AldC in the oligomerization domain alter both the electro-
amino acid identity, show the conservation of the three-dimen- static surface charge, which is strongly basic in AldA, and the
sional fold with 0.7–1.2 Å RMSD for 400–450 C -atoms in surface topology, especially in the positioning of b5, b6, and the
a
J. Biol. Chem. (2020) 295(40) 13914–13926 13917

Analysis of AldC from P. syringae DC3000
Table 1
occupy two separate pockets, which are at the opposite ends of
a ;45-Å-long tunnel (Fig. 6B). The Rossmann fold of the NAD
(H)-binding domain provides extensive polar and apolar inter-
Steady-state kinetic parameters of AldC with aldehyde substrates
Assays were performed as described under “Experimental procedures.” The aver-
age values 6 S.D. (n = 3) are shown. Assays with varied NAD used octanal as the
substrate.
1
1
actions that position the nicotinamide ring of NAD in prox-
imity to the C291A point mutation, which would be the invari-
ant catalytic cysteine in WT AldC (Fig. 6C). The nicotinamide
ring is mainly held in place by van der Waals contacts with
Substrate
K
cat
K
m
K
cat/K
21 21
m
2
1
min
mM
M
s
Valeraldehyde/pentanal
Hexanal
Heptanal
34.3 6 4.2
22.9 6 1.0
22.1 6 1.5
67.4 6 1.4
27.7 6 5.0
16.8 6 1.6
38.7 6 1.9
3.6 6 0.2
48.8 6 17.1
16.4 6 2.3
7.4 6 2.0
1.2 6 0.1
4.9 6 2.3
14.2 6 3.1
4.4 6 0.6
1.3 6 0.3
0.5 6 0.1
12
23
258
419
456
Leu , Leu , and Phe and a critical hydrogen bond from
49
2
58
Octanal
924
94
the backbone carbonyl of Leu to the NH group of the cofac-
2
Nonanal
Hydrocinnamaldehyde
-Pyridinecarboxaldehyde
Indole-3-acetaldehyde
NAD (octanal)
1
tor carboxamide. The nicotinamide-ribose of NAD estab-
20
147
14
4
lishes multiple polar interactions with the backbone carbonyl
a
2
35
391
of Gly and the carboxylate side chain of Glu . In addition,
1
30.9 6 0.4
1,050
1
a
the adenine ring of NAD is mainly stabilized by multiple van
der Waals interactions with Pro , Ile , Leu , and Val
Steady-state kinetic parameters of AldC with indole-3-acetaldehyde were previously
reported (21).
216
233
242
243
,
along with a hydrogen bond with a water molecule in an apolar
pocket. As with nicotinamide-ribose binding, polar interactions
Table 2
182
between the adenine-ribose ring and the side-chains of Lys
and Glu contribute to NAD binding. Interaction of Glu
Summary of crystallographic statistics for AldC structures
185
1
185
1
Crystal
AldC (C291A)·octanal·NAD
C222
with the 2’-hydroxyl-group of the adenine-ribose is the key
structural determinant of the cofactor specificity, as observed
in other aldehyde dehydrogenases (39). AldC is not able to
accommodate the 29-phosphate of NADP(H) sterically and
electrostatically, which is consistent with its preference for
NAD(H) as cofactor (39). Sequence comparison of AldC and
structurally related aldehyde dehydrogenases (Fig. S2) under-
scores the conservation of the NAD(H)-binding site across
these enzymes from distantly related organisms.
Space group
Cell dimensions (Å)
a
b
c
1
92.19
92.17
231.4
Data collection
Wavelength
Resolution range (highest shell)
Reflections (total/unique)
Completeness (highest shell)
0.979 Å
39.6–2.52 Å (2.54–2.52 Å)
116,137/32,956
97.8% (99.1%)
12.8 (1.8)
,
I/s. (highest shell)
a
Rsym (highest shell)
6.0% (52.9%)
In contrast to the NAD(H)-binding site, apolar interactions
dominate the octanal binding in the hydrophobic substrate-
binding pocket (Fig. 6, B and C). A cluster of aromatic residues
Refinement
b
c
R
cryst /Rfree
17.1%/21.9%
7,180
197
106
No. of protein atoms
No. of waters
No. of ligand atoms
RMSD
Bond lengths (Å)
Bond angles (°)
Average B-factor (Å )
Protein
Water
Ligand
Stereochemistry (%)
Favored
Allowed
1
60
163
450
456
468
(Trp , Tyr , Trp , Phe , and Tyr ) and two apolar
114
118
residues (Met and Leu ) provide the hydrophobic environ-
ment that accommodates octanal and other aliphatic alde-
hydes. As described for other aldehyde dehydrogenases (40–
42), the substrate-binding site forms an “aromatic box” for
adaptable apolar ligand interaction.
0.008
0.93
2
45.9
43.7
55.9
1
In the AldC(C291A)·octanal·NAD complex, the 8-carbon
95.7
4.3
0
chain of octanal extends toward the solvent exposed, but highly
apolar, opening of the aldehyde-binding site. This places the re-
Outliers
a
b
c
159
R
R
R
sym = SjI
h
2 ,I
2 ,F
h
.j/SI
h
, where ,I
o
h
. is the average intensity over symmetry.
, where summation is over the data used for refinement.
active aldehyde group, which hydrogen bonds with Asn and
cryst = SjF
o
c
.j/SF
291
the backbone nitrogen of Ala , in proximity to the C4 position
free is defined the same as Rcryst but was calculated using 5% of data excluded from
refinement.
of the nicotinamide ring that undergoes hydride transfer in the
chemical reaction and the location of the catalytic cysteine.
b5-b6 loop of the oligomerization domain of the two proteins. Multiple sequence alignment of AldC and other aldehyde dehy-
drogenases (Fig. S2) shows that the residues forming the alde-
hyde-binding site are highly variable, which account for sub-
strate preferences in these enzymes.
Molecular docking of the valeraldehyde (pentanal), hexanal,
heptanal, nonanal, hydrocinnamaldehyde, and 4-pyridinecar-
boxaldehyde (Fig. S3) provides insight on likely binding modes
for each substrate that place the reactive aldehyde near the cat-
alytic center of AldC and the aliphatic or aromatic portions of
each into the apolar tunnel leading toward the solvent opening.
Although the size of the site would allow short-chain aliphatic
In addition, the length of the b5-b6 loop differs between AldA
1
43
153
138
142
(
Ala –Val ) and AldC (Val –Thr ), which contributes
to alterations in the interaction region (Fig. 5, B and D, and Fig.
S2, orange box). In AldC, these changes appear to alter what
would be the corresponding tetramer (i.e. dimer–dimer) inter-
face of AldA but may only be one contributing factor to differ-
ent oligomerization.
Active site structure of AldC
1
Unambiguous electron densities for NAD and octanal substrates, such as acetaldehyde, propionaldehyde, and butyral-
define how these ligands bind to the AldC C291A mutant and dehyde, to bind, the region of the substrate-binding site where
indicate the location of the substrate and cofactor-binding sites these molecules would fit is more polar than the largely hydro-
1
(
Fig. 6A). Forming a dead-end complex, NAD and octanal phobic region of the tunnel accessible to longer-chained
13918 J. Biol. Chem. (2020) 295(40) 13914–13926

Analysis of AldC from P. syringae DC3000
Figure 4. Overall structure of the AldC C291A mutant structure. A, the dimeric structure of AldC dimer is shown as a ribbon diagram. Secondary structure
features of monomer A are colored, as follows: a-helices and b-strands are shown as gold cylinders and blue arrows, respectively. The ribbon diagram of mono-
mer B is colored gray with the N and C termini labeled. Ligands are shown as space-filling models. B, domain organization of the AldC monomer. The color
scheme for the AldC monomer shows the NAD(H)-binding domains in blue, the catalytic domain in red, the interdomain linker in green, and the oligomeriza-
1
tion domain in gold. The positions of octanal and NAD (space-filling models) are indicated. C, topology of AldC showing the four domains described in B with
the same coloring and secondary structure features numbered.
aliphatic substrates (i.e. heptanal, octanal, and nonenal), which coli and purified using nickel-affinity and size-exclusion chro-
is consistent with the biochemical assays of AldC. This suggests matographies. The solubility and purity of each mutant was
that substrate preference is governed by van der Waals contacts comparable with WT protein. Under standard assay condi-
tions, enzymatic activities of WT AldC, the AldC C291A mu-
tant (as described above), and the additional 31 point mutants
were tested with fixed concentrations of octanal (5 mM) and
that maximize nonpolar surface–ligand interactions.
Site-directed mutagenesis of AldC active site residues
1
NAD (2 mM) using up to 100 mg of each protein (Fig. 7). The
To examine the contribution of active site residues, a series
of site-directed mutants targeting residues in the NAD(H)-
initial enzyme activity screening assays showed that mutation
of the catalytic cysteine (C291A) and certain residues in the
NAD(H)-binding site (K182A, K182Q, T234V, E257A, and
E391A), and octanal-binding site (N159A and L419A) resulted
in enzymes with less than 1% of WT specific activity. For
example, no activity was detected with the N159A, K182A, and
C291A mutants.
1
58
182
185
234
236
257
binding site (i.e. Trp , Lys , Glu , Thr , Ser , Glu
,
3
91
114
118
and Glu ) and the octanal-binding site (i.e. Met , Leu
,
,
1
60
163
164
285
292
419
450
456
Trp , Tyr , Gln , Arg , Ser , Leu , Trp , Phe
4
68
and Tyr ) were generated. All of the 31 AldC point mutants
M114A, L118A, W158A, W158H, N159A, W160A, W160Y,
Y163A, Q164A, Q164N, K182A, K182Q, E185A, E185Q,
(
Kinetic analysis of mutants with changes in the NAD(H)-
T234A, T234V, S236A, S236T, E257A, E257Q, R285A, binding site of AldC reveals critical residues for biochemical
S290A, S292A, E391A, E391D, L419A, L419V, W450A, function. As noted above, low activity of the K182A, K182Q,
F456A, F456W, and Y468A) were expressed in Escherichia T234V, E257A, and E391A mutants did not allow for accurate
J. Biol. Chem. (2020) 295(40) 13914–13926 13919

Analysis of AldC from P. syringae DC3000
Figure 5. Oligomerization interface differences in AldA (tetramer) and AldC (dimer) from P. syringae DC3000. A, electrostatic surface charge of AldA
dimer. Electrostatic surface charge was generated using the APBS plugin in PyMOL (Schrödinger) with red (acidic residues) and blue (basic residues). The box
corresponds to the oligomerization interface for forming a homotetramer. B, ribbon diagram of monomer A and surface view of monomer B of AldA. The
box corresponds to the oligomerization interface for forming a homotetramer. The a-helix and b-sheets for oligomerization are colored blue and gold,
respectively. C, electrostatic surface charge of AldC dimer. Coloring is as in A. D, ribbon diagram of monomer A and surface view of monomer B of
AldC. Coloring is as in B.
158
234
236
determination of their kinetic parameters (Fig. 7); however, the
Substitutions of Trp , Thr , and Ser in the NAD(H)-
other mutant proteins with changes in the NAD(H) site were binding site also reduced activity. The loss of steric bulk with
1
82
analyzed (Table 3). Mutation of Lys
(K182A and K182Q) the W158A mutant, which may change how the pyrophosphate
1
removes interaction of the lysine side-chain with a hydroxyl backbone of NAD binds in the site, resulted in a 10-fold
group of the adenine ribose of NAD . Similarly, alanine substi- decrease in kcat/K with NAD , but the W158H mutant mod-
tutions of Glu and Glu severely disrupted AldC activity estly reduced efficiency 3-fold (Table 3). Mutation of Thr
1
1
m
2
57
391
234
,
(
Fig. 7), whereas the E257Q and E391D mutations, which intro- which is positioned with its methyl group to provide surface
duced subtler alterations, displayed comparable or less than a contacts to the nicotinamide ring and its hydroxyl group ori-
1
168
2
(
-fold change in catalytic efficiency (i.e. k /K ) with NAD
ented toward the amine of Lys (Fig. 6), led to a slight 5-fold
cat
m
257
1
Table 3). Loss of the Glu side-chain would remove an inter- reduction of kcat/Km with NAD , but nearly inactive protein
action that helps position the amide group of the nicotinamide with a valine mutation (T234V). It is possible that local confor-
and changes at this residue could alter the orientation of the mational changes impact the positioning of the nicotinamide
nicotinamide group for hydride transfer (Fig. 6). Similar effects ring to impact activity of this mutant protein. The kinetic pa-
1
have been observed in other NAD(P)(H)-dependent enzymes rameters of S236A and S236T mutations with NAD were con-
3
91
(
43). Mutation of Glu to alanine eliminates interaction of the sistent with the loss of a hydrogen bond or changes in the side-
carboxylate group to the nicotinamide ribose group, which chain position for interaction with the pyrophosphate bridge of
1
can be partially complemented by an aspartate, as seen in the NAD (Table 3).
1
59
E391D mutant (Table 3). The effects of mutating the third
Within the octanal-binding site (Fig. 6), mutations of Asn
glutamate in the NAD(H)-binding site (i.e. Glu ), which Trp , Ser , Leu , and Phe were the most disruptive with
,
1
85
160
292
419
456
1
interacts with the adenine ribose group of NAD , are compa- other changes having more modest effects (Table 3). These res-
rable with changes of the other two acidic residues, but not as idues are located closer to the catalytic site of AldC than those
severe. The E185A mutant displayed a 21-fold decrease in residues with lesser impact on AldC activity. The N159A and
1
kcat/K with NAD , and the E185Q mutant is similar to that L419A mutants lacked significant activity, as noted above.
m
of WT AldC (Table 3).
Although the W160A, S292A, L419V, and F456A mutants
13920 J. Biol. Chem. (2020) 295(40) 13914–13926

Analysis of AldC from P. syringae DC3000
1
1
Figure 6. Active site of AldC. A, electron density for octanal (left panels) and NAD (right panels) in the AldC(C291A)·octanal·NAD structure is shown as a
1
o c
2F 2 F omit map (1.6 s). B, surface view of the AldC active site. Octanal (substrate) and NAD (cofactor) are shown as stick models. The active site tunnel is
shown as a surface view from the exterior into the catalytic site and the same view rotated 90°. Hydrophobicity was calculated based on the Eisenberg hydro-
phobicity scale in PyMOL. The darkest red indicates the strongest hydrophobicity, with white indicating the most polar. C, amino acid residues forming interac-
1
1
tions with octanal and NAD in the AldC active site. Side chains of residues interacting with octanal (green) and NAD (blue) are shown as stick renderings.
Two water molecules interacting with the cofactor are shown as red spheres. Hydrogen bond interactions between the amino acid residues and ligands are
shown as yellow dotted lines. The C291A mutation is labeled in red.
Figure 7. Comparison of WT and mutant AldC specific activities. Mutants proteins are grouped and colored by active site location, as follows: blue, NAD
(
H)-binding site; and green, octanal-binding site. Standard assay conditions were used as described under “Experimental procedures.” Average values 6 S.D.
21 21
(n = 3) are plotted relative to WT specific activity (1.02 6 0.08 mmol min mg protein ).
292
displayed activity in the initial screen experiment (Fig. 7), the in the undetected activity of this mutant. Ser is also posi-
estimated Km values for these proteins with octanal exceeded tioned in proximity to the reactive aldehyde group of octanal
the solubility limit of this substrate (;10 mM) in assays, which with mutation to an alanine, resulting in a 30-fold decrease in
prevented accurate assessment of kinetic parameters. The specific activity (Fig. 7).
159
160
N159A mutant abrogates the hydrogen bond between Asn
Trp provides surface contacts along the central portion of
and the carbonyl of octanal; the loss of this interaction likely octanal carbon chain with the W160A mutation significantly
alters the position of the reactive group for catalysis and results altering the topology of the binding tunnel. Replacement of
J. Biol. Chem. (2020) 295(40) 13914–13926 13921

Analysis of AldC from P. syringae DC3000
Table 3
Steady-state kinetic analysis of mutant AldC. The protein and location of the mutation are indicated. Enzyme assays were performed as
described under “Experimental procedures.” Average values 6 S.D. (n = 3) are shown.
1
Octanal
NAD
Protein
Ligand site
K
cat
K
m
Kcat/K
m
K
cat
K
m
Kcat/K
m
21
21 21
21
21 21
min
mM
M
s
min
mM
M
s
WT
67.3 6 1.4
2.6 6 0.1
13.7 6 0.5
4.0 6 0.2
79.5 6 2.5
21.6 6 1.2
7.0 6 0.4
3.0 6 0.2
9.8 6 1.0
25.3 6 2.4
22.0 6 1.0
17.0 6 1.7
63.7 6 4.5
70.2 6 8.7
76.3 6 17.6
66.0 6 7.9
16.5 6 0.5
2.8 6 0.1
9.8 6 1.0
140 6 33
8.4 6 0.6
1,210 6 93
340 6 44
927
127
284
15
30.9 6 0.4
5.4 6 0.1
6.9 6 0.5
5.2 6 0.1
15.0 6 0.2
6.8 6 0.7
6.4 6 0.2
7.3 6 0.4
15.7 6 4.5
10.3 6 3.0
11.7 6 0.5
11.0 6 0.4
6.1 6 0.6
18.8 6 5.3
29.2 6 5.3
24.8 6 1.5
11.8 6 0.5
7.5 6 0.2
5.1 6 0.3
2.9 6 0.1
8.6 6 0.3
490 6 25
643 6 57
273 6 38
1,900 6 120
236 6 14
594 6 217
1,420 6 143
2,200 6 294
287 6 170
248 6 183
543 6 80
298 6 34
516 6 26
244 6 162
416 6 213
258 6 38
434 6 66
1,220 6 81
805 6 167
622 6 5
1,050
140
406
46
W158A
W158H
E185A
E185Q
T234A
S236A
S236T
E257Q
E391D
M114A
L118A
W160Y
Y163A
Q164A
Q164N
R285A
S290A
W450A
F456W
Y468A
NAD(H)
NAD(H)
NAD(H)
NAD(H)
NAD(H)
NAD(H)
NAD(H)
NAD(H)
NAD(H)
Octanal
Octanal
Octanal
Octanal
Octanal
Octanal
Octanal
Octanal
Octanal
Octanal
Octanal
804 6 117
4,380 6 419
1,620 6 150
1,520 6 300
5,190 6 689
527 6 187
818
237
22
1,060
191
75
95
55
530 6 258
308
154
301
262
491
179
309
485
250
56
912
692
360
615
197
1,280
1,170
1,600
453
102
106
78
2,730 6 743
1,220 6 218
1,080 6 432
2,160 6 472
6,540 6 1,580
4,110 6 2,250
2,270 6 819
1,100 6 128
830 6 180
220 6 90
5,420 6 2,680
2,520 6 548
742
431
56
743 6 81
193
1
60
Trp with a tyrosine resulted a less than 2-fold change in cata- environmental conditions (i.e. severe nutrient limitation,
419
lytic efficiency (Table 3). Leu is also positioned at the inter- extreme temperatures, high salinity, and low oxygen or water
section of the NAD(H) and octanal-binding sites and forms van availability), they also evolved metabolic diversity and plasticity
291
der Waals contacts with Ala in the AldC structure. Changes to use a variety of nutrient sources (i.e. carbon, nitrogen, and
of this residue to either alanine (L419A) or valine (L419V) may sulfur), to detoxify toxic organic chemicals, and to produce
alter the orientation of the catalytic sulfhydryl required for the multiple specialized metabolites, including polymers and small
conversion of octanal to its carboxylic acid. In the AldC crystal molecule compounds (31, 44). In particular, P. syringae, a spe-
4
56
468
structure, Phe p-stacks with Tyr , which forms an interac- cies that includes many plant pathogenic strains, developed
1
63
450
tion network with Tyr
and Trp
(Fig. 6). Mutation of diverse bacterial virulence mechanisms to survive in the
4
56
Phe to an alanine (F456A) also increases the apparent K of adverse environmental conditions of the phyllosphere (45).
m
octanal, as described above, which likely results from altered Further, recent studies suggest that PtoDC3000 uses several
packing along the aldehyde-binding site of the F456A mutant. strategies to manipulate the auxin biology in its host plants to
In contrast, the F456W mutant results in a modest 2-fold promote pathogenicity, including using an indole-3-acetalde-
decrease in kcat/K with octanal (Table 3).
hyde dehydrogenase for IAA synthesis (21). Recently, a genus-
wide study identified a total of 6,510 aldehyde dehydrogenase
sequences in 258 Pseudomonas strains belonging to 46 dif-
ferent species, but only a limited number of newly identified
Pseudomonas aldehyde dehydrogenases have been bio-
chemically characterized (32).
In contrast to the better-studied aromatic aldehyde dehydro-
genases identified from various Pseudomonas species (46–48),
which have been studied because of their ability to catabolize
and biodegrade a range of compounds, including napthalene-
derived molecules, and metabolic aldehyde dehydrogenases
linked to the osmoprotectant betaine and growth in biofilms
m
160
468
163
450
The opposites of Trp , Tyr , Tyr , and Trp are inter-
connected through a series of hydrogen bonds to form one of
the walls of the octanal-binding site (Fig. 6). Disruptions in this
network using the Y468A, Y163A, and W450A mutants
resulted in 20-, 5-, and 9-fold decreases in catalytic efficiency
with octanal (Table 3). It appears that removal on one of these
aromatic side chains alters AldC activity but is not sufficient to
significantly disrupt substrate binding and catalysis. Alanine
substitutions of the three residues situated toward the solvent
114
118
entrance of the aldehyde-binding site, Met , Leu , and
2
85
Arg , have only slight 3–4-fold reductions in catalytic effi-
ciency compared with WT AldC (Table 3), which is consistent
with the interior hydrophobic surface as the major determinant
of substrate interaction. Two other residues in the octanal-
(49–51), less is understood about the biochemistry of aliphatic
aldehyde dehydrogenases in these microbes. Here we compre-
hensively examined and identified AldC from PtoDC3000 as a
long-chain aliphatic aldehyde dehydrogenase using an inte-
grated approach combining phylogenetic, crystallographic, and
biochemical analyses.
1
64
290
binding site, Gln and Ser , also play lesser roles in substrate
binding, as suggested by the modest effects of the Q164A and
Q164N mutations and the S290A mutant (Table 3).
The biochemical activity of AldC from PtoDC3000 is consist-
ent with the traditional role of aldehyde dehydrogenases
Conclusion
The genus Pseudomonas is one of the most ubiquitous and as metabolic clean-up enzymes that convert reactive aldehydes
complex among the Gram-negative bacteria (22). Because into less active carboxylates (11, 12); however, growing
many Pseudomonas species evolved to grow under unfavorable evidence suggests that these enzymes may have broader
13922 J. Biol. Chem. (2020) 295(40) 13914–13926

Analysis of AldC from P. syringae DC3000
contributions to the specialized environments that different imidazole. AldC was further purified by size-exclusion chro-
Pseudomonas species occupy. For example, recent work iden- matography with a Superdex 200 16/60 size-exclusion col-
tified AldA as an indole-3-acetaldehyde dehyrogenase umn (GE Healthcare) equilibrated in 25 mM Hepes (pH 7.5)
involved in the synthesis of the phytohormone IAA in the and 100 mM NaCl. Fractions corresponding to the purified
2
1
plant pathogen P. syringae DC3000 and demonstrated a role
for this protein in virulence of the pathogen in plant leaves
AldC protein were pooled and concentrated to ;9 mg ml
.
Protein concentrations were determined using the Bradford
method with BSA as a standard.
(
21). Given the growth environment of this pathogenic Pseu-
domonas species in the interior of plant leaves, it is possible
that aliphatic molecules in the apoplast could contribute to
energy sources for the microbe or to the synthesis of surfac-
tants that help establish microbial colonization in the host
plant (52–54). The physiological contribution of AldC to the
life cycle of PtoDC3000 and related plant pathogenic or plant-
associated species and strains remains to be evaluated but
Enzyme assays and steady-state kinetic analysis
Enzymatic activity of WT and mutant AldC was measured
21
21
by monitoring NADH formation (e340 nm = 6220 M cm
00-ml volume) at A340 nm using an EPOCH2 microplate spec-
trophotometer (BioTek). The substrate and cofactor screening
;
1
raises the question of how these microbes develop host rela- experiments were performed at 25 °C in a standard assay mix of
tionships in the natural environment. Overall, this study pro- 100 mM Tris·HCl (pH 8.0) and 100 mM KCl with a fixed con-
1
vides molecular insight for understanding the evolution of the centration of NAD (2 mM) and aldehyde (5 mM). The panel of
prokaryotic aldehyde dehydrogenase superfamily and for the aldehydes and their chemical structures used for substrate
potential development of inhibitors for the pathogenic Pseu- screening is shown in Table S2. Steady-state kinetic parame-
domonas species, which could be useful for pathogen control ters of WT and mutant AldC were determined at 25 °C in a
1
in agriculture.
standard assay mix with either fixed NAD (2.0 mM) and
varied aldehyde (i.e. valeraldehyde, hexanal, heptanal, octa-
nal, nonanal, hydrocinnamaldehyde, or 4-pyridinecarboxal-
dehyde; 0.01–10 mM) or with fixed octanal (5 mM) and var-
Experimental procedures
Cloning and site-directed mutagenesis
1
1
ied cofactor (NAD or NADP ; 0.05–5 mM). Protein
amounts ranged from 0.1 mg for WT AldC to 100 mg for less
active AldC mutants. The resulting initial velocity data were
The pET28a-AldC construct was previously described (21).
Briefly, the full-length coding sequence of AldC (UniProt:
PSPTO_3644; GenBank : WP_011104646.1) was amplified
from P. syringae strain PtoDC3000 genomic DNA and cloned
into the pBlunt II-TOPO vector (Invitrogen). The AldC frag-
ment from the pBlunt II-TOPO-AldC was excised using NdeI
TM
fit to the Michaelis–Menten equation, v = (kcat[S])/(Km
1
[
S]), using SigmaPlot.
and XhoI and subcloned into pET-28a (Novagen). Site-directed Protein crystallography
mutants of AldC (M114A, L118A, W158A, W158H, N159A,
Protein crystals of the AldC C291A mutant were grown by
the hanging-drop vapor-diffusion method at 4 °C. Crystals of
W160A, W160Y, Y163A, Q164A, Q164N, K182A, K182Q,
E185A, E185Q, T234A, T234V, S236A, S236T, E257A, E257Q,
R285A, S290A, C291A, S292A, E391A, E391D, L419A, L419V,
W450A, F456A, F456W, and Y468A) were generated using the
QuikChange PCR method (Agilent Technologies) with the
pET28a-AldC construct as template. For protein expression,
each pET28a-AldC construct was transformed into E. coli BL21
21
the AldC C291A mutant (9 mg ml ) in complex with octanal
1
and NAD formed grew in drops of a 1:1 mixture of proteins
and crystallization buffer: 20% (w/v) PEG-1000, 100 mM
1
Tris·HCl (pH 7.0), 2 mM octanal, and 5 mM NAD . Crystals
were stabilized in cryoprotectant (mother liquor with either
3
0% (v/v) glycerol or 30% (v/v) ethylene glycol) before flash-
(
DE3) (EMD Millipore).
freezing in liquid nitrogen for data collection at 100 K. Diffrac-
tion data were collected at Beamline 19-ID of the Advanced
Photon Source at the Argonne National Laboratory HKL3000
was used to index, integrate, and scale the collected X-ray data
(55). Molecular replacement was used to solve the X-ray crys-
tal structure of AldC using PHASER (56) with the three-
dimensional structure of the AldA indole-3-acetaldehyde de-
hydrogenase from P. syringae, which shares 39% amino acid
identity with AldC, as a search model (Protein Data Bank code
Protein expression and purification
Transformed E. coli BL21(DE3) cells containing either WT
or mutant AldC construct were grown at 37 °C in Terrific
2
1
broth with 50 mg ml kanamycin until A600 nm = ;0.6–0.9.
After induction with 1 mM isopropyl-1-thio-b-D-galactopyra-
noside, the cells were grown at 18 °C overnight. Following cen-
trifugation (5,000 3 g for 30 min), the cell pellets were resus-
pended in 50 mM Tris, pH 8.0, 500 mM NaCl, 25 mM imidazole,
5
IUW) (21). COOT (57) and PHENIX (58) were used for itera-
1
0% (v/v) glycerol, and 1% (v/v) Tween 20. Following lysis by
sonication, the cell debris was removed by centrifugation
12,000 3 g for 45 min), and the supernatant was loaded onto a
tive rounds of model building and refinement, respectively.
Data collection and refinement statistics are summarized in
(
6
490
21
Table 2. The final model included Leu –Leu
of chain A,
Ni -nitriloacetic acid column (Qiagen). The column was
6
489
1
washed with 50 mM Tris, pH 8.0, 500 mM NaCl, 25 mM imidaz- Leu –Asp of chain B, 1 octanal, and 1 NAD in each chain,
ole, and 10% (v/v) glycerol to remove unbound proteins. Col- and 197 waters. Atomic coordinates and structure factors for
1
umn-bound AldC protein was eluted using 50 mM Tris, pH the AldC(C291A)·octanal·NAD complex were deposited in
8
.0, 500 mM NaCl, 25 mM imidazole, 10% glycerol, and 250 mM the RCSB Protein Data Bank (code 6X9L).
J. Biol. Chem. (2020) 295(40) 13914–13926 13923

Analysis of AldC from P. syringae DC3000
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Funding and additional information—This work was supported by
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part of Bio4522, an upper-level laboratory course at Washington
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Conflict of interest— The authors declare that they have no conflicts
of interest with the contents of this article.
1
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Abbreviations—The abbreviations used are: IAA, indole-3-acetic
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13926 J. Biol. Chem. (2020) 295(40) 13914–13926

The plant pathogen enzyme AldC is a long-chain aliphatic aldehyde
dehydrogenase
Soon Goo Lee, Kate Harline, Orchid Abar, Sakirat O. Akadri, Alexander G. Bastian,
Hui-Yuan S. Chen, Michael Duan, Caroline M. Focht, Amanda R. Groziak, Jesse Kao,
Jagdeesh S. Kottapalli, Matthew C. Leong, Joy J. Lin, Regina Liu, Joanna E. Luo,
Christine M. Meyer, Albert F. Mo, Seong Ho Pahng, Vinay Penna, Chris D. Raciti,
Abhinav Srinath, Shwetha Sudhakar, Joseph D. Tang, Brian R. Cox, Cynthia K.
Holland, Barrie Cascella, Wilhelm Cruz, Sheri A. McClerkin, Barbara N. Kunkel and
Joseph M. Jez
J. Biol. Chem. 2020, 295:13914-13926.
doi: 10.1074/jbc.RA120.014747 originally published online August 12, 2020
Access the most updated version of this article at doi: 10.1074/jbc.RA120.014747
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