Crystal Structure of GenD2, an NAD-Dependent Oxidoreductase Involved in the Biosynthesis of Gentamicin

Article abstract of DOI:10.1021/acschembio.9b00115

Gentamicins are clinically relevant aminoglycoside antibiotics produced by several Micromonospora species. Gentamicins are highly methylated and functionalized molecules, and their biosynthesis include glycosyltransferases, dehydratase/oxidoreductases, aminotransferases, and methyltransferases. The biosynthesis of gentamicin A from gentamicin A2 involves three enzymatic steps that modify the hydroxyl group at position 3″ of the unusual garosamine sugar to provide its substitution for an amino group, followed by an N-methylation. The first of these reactions is catalyzed by GenD2, an oxidoreductase from the Gfo/Idh/MocA protein family, which reduces the hydroxyl at the C3″ of gentamicin A to produce 3′′-dehydro-3′′-oxo-gentamicin A2 (DOA2). In this work, we solved the structure of GenD2 in complex with NAD+. Although the structure of GenD2 has a similar fold to other members of the Gfo/Idh/MocA family, this enzyme has several new features, including a 3D-domain swapping of two β-strands that are involved in a novel oligomerization interface for this protein family. In addition, the active site of this enzyme also has several specialties which are possibly involved in the substrate specificity, including a number of aromatic residues and a negatively charged region, which is complementary to the polycationic aminoglycoside-substrate. Therefore, docking simulations provided insights into the recognition of gentamicin A2 and into the catalytic mechanism of GenD2. This is the first report describing the structure of an oxidoreductase involved in aminoglycoside biosynthesis and could open perspectives into producing new aminoglycoside derivatives by protein engineering.

Full text of DOI:10.1021/acschembio.9b00115

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Article
Crystal Structure of GenD2, an NAD-Dependent
Oxidoreductase Involved in the Biosynthesis of Gentamicin
Natalia Cerrone de Araújo, Priscila dos Santos Bury, Mauricio Temotheo Tavares,
Fanglu Huang, Roberto Parise Filho, Peter Leadlay, and Marcio Vinicius Bertacine Dias
ACS Chem. Biol., Just Accepted Manuscript • DOI: 10.1021/acschembio.9b00115 • Publication Date (Web): 17 Apr 2019
Downloaded from http://pubs.acs.org on April 18, 2019
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Crystal Structure of GenD2, an NAD-Dependent Oxidoreductase Involved in the
Biosynthesis of Gentamicin
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Natalia Cerrone de Araújo1, Priscila dos Santos Bury1, Maurício Themoteo Tavares2,
Fanglu Huang3, Roberto Parise Filho2, Peter Leadlay3, Marcio Vinicius Bertacine
Dias1,4*
.
1Department of Microbiology, Institute of Biomedical Science, University of São Paulo,
Avenida Prof. Lineu Prestes 1374, 05508-900, São Paulo, Brasil.
2Department of Pharmacy, Faculty of Pharmaceutical Sciences, University of Sao Paulo,
Prof. Lineu Prestes Avenue 580, 05508-900, Sao Paulo, Brazil.
3Department of Biochemistry, University of Cambridge, 80 Tennis Court Road,
Cambridge, CB2 1GA, UK.
4 Department of Chemistry, University of Warwick, Coventry, CV4 7AL, UK
*Corresponding Author: mvbdias@usp.br
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Abstract. Gentamicins are clinically relevant aminoglycoside antibiotics produced by
several Micromonospora species. Gentamicins are highly methylated and functionalized
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molecules
and
their
biosynthesis
include
glycosyltransferases,
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dehydratase/oxidoreductases, aminotransferases, and methyltransferases. The
biosynthesis of gentamicin A from gentamicin A2 involves three enzymatic steps that
modify the hydroxyl group at position 3ʹʹ of the unusual garosamine sugar to provide its
substitution for an amino group, followed by an N-methylation. The first of these
reactions is catalyzed by GenD2, an oxidoreductase from the Gfo/Idh/MocA protein
family, which reduces the hydroxyl at C3ʹʹ of gentamicin A to produce 3ʹʹ-dehydro-3ʹʹ-
oxo-gentamicin A2 (DOA2). In this work, we solved the structure of GenD2 in complex
with NAD+. Although the structure of GenD2 has a similar fold to other members of
Gfo/Idh/MocA family, this enzyme has several new features, including a 3D-domain
swapping of two β-strands that are involved in a novel oligomerization interface for this
protein family. In addition, the active site of this enzyme also has several specialties
which are possibly involved in the substrate specificity, including a number of aromatic
residues and a negatively charged region, which is complementary to the polycationic
aminoglycoside-substrate. Therefore, docking simulations provided insights into the
recognition of gentamicin A2 and into the catalytic mechanism of GenD2. This is the first
report describing the structure of an oxidoreductase involved in aminoglycoside
biosynthesis and could open perspectives into producing new aminoglycosides
derivatives by protein engineering.
Keywords: gentamicins; oxidoreductase; domain swapping; crystal structure.
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INTRODUCTION
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Aminoglycosides are antibiotics that usually target the bacterial ribosome causing
decoding errors and consequently leading to inaccurate mRNA translation and/or
premature termination of protein synthesis1. Gentamicins are among the most clinically
relevant antibiotics of this class to treat severe infections caused by gram-negative
bacteria2. The commercial form of gentamicin is composed of a mixture of five different
compounds (gentamicin C1a, C2b, C2a, C2, and C1) that differ in the level of methylation
in their unusual sugar rings3. Gentamicins have also been demonstrated to have effects
on cancer cells by delaying the cell growth and inducing cell death, indicating that this
antibiotic has other pharmacological use4,5,6. In spite of their benefits, gentamicins,
similar to other aminoglycosides, also have several side effects, including reversible
nephrotoxicity and irreversible hearing loss7. Gentamicins are highly modified
trisaccharides that contains a central 2-deoxystreptamine (2-DOS) ring as an
aminocyclitol aglycone (Ring I), which is further decorated by a purpurosamine (ring II)
and garosamine (ring III) at positions C4 and C6, respectively8,9 (Figure 1). Gentamicins
are produced by several Micromonospora species, including M. echinospora10, and their
biosynthesis have been extensively studied3,8,10-11, although the last steps for the
gentamicin complex C are not yet completely understood12. A minimal set of genes
responsible for the biosynthesis of gentamicin A, including the biosynthetic
intermediates, was elucidated recently3. The biosynthesis of gentamicin A from
gentamicin A2 occurs in three steps, which involves the dehydrogenation of the 3ʹʹ-
hydroxyl group on the ring III by GenD2 using nicotinamide adenine dinucleotide
(NAD+) as a cofactor, the resulting C3ʹʹ-keto group is transaminated by GenS2 followed
by the methylation at the 3ʹʹ-amine by GenN, an N-methyltransferase3. (Figure 1). The
functions of these enzymes have been validated in vivo and in vitro3. The crystal structure
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of GenN has been solved and its catalytic mechanism was proposed recently13.
Interestingly, Huang et al.3 were not able to prove the activity of any of these enzymes
individually and the conversion of gentamicin A2 to gentamicin A was only possible
when the three enzymes were incubated together in the presence of NAD+, L-glutamate
and S-adenosyl methionine (SAM). It is possible to speculate that these enzymes might
form a protein complex13 or the activity of GenD2 and GenS2 could be inhibited by low
levels of their own products and the activity of GenN could restore the activity of two
upstream enzymes3. GenD2 is the 3ʹʹ-oxidoreductase that catalyzes the oxidation of the
C3ʹʹ-hydroxyl group of gentamicin A to produce DOA2, which is the substrate for the
transaminase GenS23. GenD2 only shows high sequence identity to Sis12 (97%), TobD2
(56.6%) and KanD2 (48.7%) involved in the biosynthesis of the aminoglycosides
sisomycin, tobramycin, and kanamycin, respectively14. On the other hand, these four
enzymes show modest similarity to other dehydrogenases or oxidoreductases, including
several ones that have been structurally characterized. GenD2 belongs to the
Gfo/Idh/MocA family of oxidoreductases/dehydratases based on its sequence, in which
the proteins have a fold composed of two domains, a Rossman-like or nucleotide binding
domain and an oligomerization C-terminal domain15. However, most of Gfo/Idh/MocA
family enzymes are involved in the oxidoreduction of monosaccharides. In contrast,
gentamicin A2 is a pseudotrisaccharide and consequently, GenD2 may have a number of
specializations for its substrate recognition. In addition, only a few oxidoreductases that
act on position C3ʹʹ-hydroxyl groups from sugars were so far characterized indicating a
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lacking on the understanding of these enzymes16,17
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Figure 1. Biosynthesis of gentamicin A from gentamicin A2. The ring I is the 2-DOS (2-
deoxystreptamine), II is the purpurosamine and, III is the garosimine. A2 (gentamicin
A2); DOA2 (3ʹʹ-dehydro-3ʹʹ-oxo-gentamicin A2); DAA2 (3ʹʹ-dehydro-3ʹʹ-amino-
gentamicin A2); A (gentamicin A). GenD2 is an oxidoreductase; GenS2 is an
aminotransferase and, GenN is an N-methyltransferase.
In order to understand the structure of GenD2 and provide insights into its substrate
specificity and catalytic mechanism, herein we have determined the 3D structure of the
C3ʹʹ-oxidoreductase GenD2 in complex with NAD+ and performed docking simulations.
In addition, we have confirmed the preference of GenD2 for NAD+ using Isothermal
Titration Calorimetry (ITC) and the stabilization of the GenD2 by different
aminoglycoside-like substrates through Differential Scanning Fluorimetry (DSF).
RESUTLS AND DISCUSSION
Overall Structure
GenD2 was successfully expressed in BL21(DE3) cells and the enzyme had about 95%
of purity after the gel filtration purification step, in which the enzyme eluted as a single
peak (Figure S1, Supporting Information (SI)). Crystals for the enzyme were obtained in
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several conditions, although the unique that was reproducible contained a high
concentration of sodium formate.
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GenD2 structure in complex with NAD+ was solved by molecular replacement using the
MrBUMP server18 at a resolution of 2.4 Å. Attempts to solve the structure using a single
model of oxidoreductases from Protein Data Bank were unsuccessful. GenD2 crystals
belong to the space group P 31 2 1 and have six protomers in the asymmetric unit, which
although the proteins eluted as a tetramer by analytical gel filtration (figure not shown),
these molecules do not form the canonical GFOR tetramer in the asymmetric unit.
However, this tetrameric quaternary structure could be obtained by applying the
symmetrical operations and this has a 222-point symmetry.
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All six protomers of the asymmetric unit have almost the entire polypeptide chain (340
residues), with exception of the last alanine, and the RMSD between them is about 0.3 Å,
indicating a rigid and restrained structure. Table 1 shows the X-ray data analyses,
refinement and stereochemistry quality. The sequence of GenD2 has modest similarity to
other oxidoreductase and dehydratase structures, with about 24% and 23% of identity
with Glucose-Fructose Oxidoreductase (GFOR) from Zymomonas mobilis19 and with
oxidoreductase YvaA from Bacillus subtilis (PDB entry 3GFG, data not published),
respectively (Figure 2). However, a search using the server DALI rendered the structure
of oxidoreductase domain from Kribbella flavida (PDB entry 4H3V, data not published)
and oxidoreductase WlbA from Bordetella pertussis17, with a Z-score of 37.5 and 36.7
and RMSD of about 2.0 Å.
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Table 1. Data Collection and Refinement Statistics
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Data Collection
PDB entry
Wavelength (Å)
Resolution range (Å)
Space group
PDB:
6NOR
0.976260
49.95 - 2.40 (2.49 - 2.40)
P 31 2 1
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Unit cell (Å)
124.0 124.0 272.6
1215767 (53058)
95397 (9371)
12.7 (11.4)
99.98 (99.86)
13.48 (3.38)
33.93
0.17 (1.05)
0.19 (1.13)
0.05 (0.32)
1 (0.78)
Total reflections
Unique reflections
Multiplicity
Completeness (%)
Mean I/sigma(I)
Wilson B-factor (Å2)
R-merge
R-meas
R-pim
CC1/2
Refinement
R-work
R-free
0.174 (0.224)
0.223 (0.287)
16099
15399
264
436
2049
0.009
1.17
96
0.49
4.12
33.7
33.7
34.40
33.40
Number of non-hydrogen atoms
macromolecules
ligands
water
Protein residues
RMS(bonds) (Å)
RMS(angles) (º)
Ramachandran favored (%)
Ramachandran outliers (%)
Clashscore
Average B-factor (Å2)
macromolecules
ligands
solvent
All number in parenthesis refer to the high-resolution shell.
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Figure 2. Sequence analysis of GenD2. Alignment of closest homologues of GenD2
rendered by DALI server20. Amino acids in red boxes are conserved in all sequences;
Amino acids in red and inside blue line boxes are conserved in most of the sequences;
Amino acids in black are not significantly conserved. The alignment was prepared using
ENDScript server21.
GenD2 protomers have a general folding similar to other NAD/NADP dependent
oxidoreductases as described previously for GFOR and dihydrodipicolinate reductase
(DHPR)19. GenD2 protomer has two domains, including an N-terminal domain, which is
a Rossmann fold or the nucleotide binding domain and a C-terminal domain characterized
to have a six-stranded β-sheet that participates in both active site and in oligomerization
and further two α-helices. The C-terminal β-sheet is predominantly hydrophobic, and it
is involved in the dimerization through of a back to back of a β-sheet from one protomer
with the β-sheet from the adjacent protomer forming a two-fold symmetry axis. The α-
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helices of the C-terminal domain is in another face of the C-terminal domain β-sheet and
form an α/β sandwich. The N-terminal Domain is located adjacently to the α-helices of
the C-terminal domain (Figure 3A). The superposition of GenD2 to other Gfo/Idh/MocA
protein family members indicated that N-terminal domain is structurally conserved, while
the C-terminal domain has more drastic conformational changes (Figure 3B). The most
prominent structural difference is in the region between the residues Gly261 to Pro283,
which includes the β-strands 7 and 8, that is flipped about 180 in comparison to other
oxidoreductase/dehydrase structures17,19,22. These two β-strands are involved in a 3D
domain swapping since they are involved in the 8-strand β-sheet commonly found in all
member of this protein class. At least by our knowledge, this is the first description of a
3D domain swapping into the Gfo/Idh/MocA protein family involving two β-strands to
form a quaternary structure. However, the description of swapping of other secondary
elements has been reported for other members of this protein class, including for GFOR
from Zymomonas mobilis19. The structure of this enzyme has an N-terminal arm which
extends to another protomer of the tetramer and a truncated protein missing the first 31
residues rendered only dimers instead of tetramers and has an impact in the binding of
co-factor, including the preference for NADP or NAD, indicating a key functional role of
this extension23. Thus, although 3D domain swapping has been related to have a possible
physiological relevance, including allosteric regulation, this role in GenD2 has not been
studied in this work. In contrast, in other NAD(P) dependent oxidoreductases, as GFOR
and WlbA, this region produces two extra strands of the C-terminal β-sheet to form the
intradomain eight-stranded β-sheet. In GenD2, this region is also participating in the C-
terminal domain β-sheet, but in an interdomain mode and consequently, it is involved in
the contact with C-terminal domain β-sheet of the adjacent protomer to form a continuous
β-sheet, with 6 β-strands from one monomer and 2 from the adjacent one. Unlike other
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members of Gfo/Idh/MocA protein family, which all strains of C-terminal domain form
a continuous and extensive 16-stranded β-sheet involving two protomers, the C-terminal
domain of each GenD2 protomer is not involved to produce a 16-stranded β-sheet and
rather than it is observed 4 individual 8-stranded β-sheet in the interface of the tetramer.
Due to that, the 8-stranded β-sheets of each protomer of this dimer interface seem to be
twisted about 30º in relation to each other in comparison to other proteins from the same
family (Figure 3C and S2). The tetramer of GenD2 form a homooligomer of about
72x95x50 Å indicating that the quaternary structure of the protein has an approximately
flattened shape (Figure 3D). The main contact for oligomerization is the C-terminal β-
sheet, which are back to back between two protomers with extensive hydrophobic
interaction and between the other two protomers through the 3D-domain swapping,
described above and which is involved in the inter 8-stranded β-sheet.
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Figure 3. Tertiary and quaternary structure of GenD2. (A) The monomer of GenD2. In
Green is represented the Nucleotide Binding Domain; In pink is represented the C-
terminal domain; and in blue the two β-strand involved in the 3D-domain swapping. (B)
The superposition of GenD2 with the two closest structural homologues from the
Gfo/Idh/MocA family. In a dashed orange circle is represented the two β-strand involved
in the domain swapping. GenD2 structure is in green; 3Q2I PDB entry (WlbA
dehydrogenase from Chromobacterium violaceum) is in pink, and 4H3V PDB entry
(oxidoreductase from Kribbella flavida) is in blue. (C) Dimer interface involving the 3D-
domain swapping in GenD2. (D) Quaternary structure of GenD2 in two different
perspectives with a rotation of 90º.
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Analysis of the potential electrostatic surface of GenD2 tetramer did not reveal any
evident surface for interaction with other enzymes involved in the modification of C3ʹʹ
position of ring III of gentamicin (Figure S3, SI). Attempts to copurify GenD2 with
GenS2 and GenN or to form the complex using cross-linkage assays failed. Whether the
activity of GenD2 is dependent on protein complex formation remains elusive.
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Cofactor preference of GenD2
In order to identify the preference of GenD2 to NAD+ or NADP+, we have measured the
KD using Isothermal Titration Calorimetry (ITC). GenD2 was titrated against both co-
factors, in which only NAD+ indicated interaction with the protein, while NADP+ did
not show any significant heat of interaction (Figure S4, SI)). The thermodynamic
parameters obtained in the presence of NAD+ is shown in Table S1 and the obtained KD
was about 7.1±0.1 µM, which is consistent with the in vitro enzymatic assays3 in which
GenD2 is a NAD-dependent oxidoreductase.
The crystal structure of GenD2-NAD+ complex was obtained by incubating the protein
in the presence of NAD+ during crystallization experiments. As expected, NAD+ binds
to GenD2 in the N-terminal domain, sitting on the strands 1345 of the Rossmann fold and
is highly buried in the protein between the N- and C-terminal domains (Figure 4A). The
adenine-ribose moiety of NAD+ is relatively exposed to the solvent and does not form
any hydrogen bond interaction with residues of the protein. The phosphoryl group is
hydrogen bonded to the main chain nitrogens of Phe14 and Met15 and nitrogen indole of
Trp163. The nicotinamide hydroxyl ribose groups hydrogen interacts with the main chain
carbonyl groups of Thr73 and Lys96 and the imidazole side chain of His78. Finally, the
nicotinamide group is positioned in the active site and interacts through a hydrogen bond
with the side chain of Glu95, Tyr293, Asn124, the main chain carbonyl group of Gly122
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and through a water molecule with the side chain of Glu95, Asn124, and Gln296 (Figure
4B).
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A
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Figure 4. NAD+ binding site on GenD2. (A) The binding site of NAD+ indicating that
NAD+ is sitting on the β1-β4 on GenD2 structure. (B) NAD+ binding site indicating the
residues that hydrogen interact with NAD+.
Insights into the substrate binding and catalytic mechanism
Our attempts to obtain a crystal structure of GenD2 in complex with gentamicin
homologues, kanamycin and tobramycin, through co-crystallization and soaking assays
failed to observe any electron density corresponding to these molecules at the active site.
However, we have performed a DSF assay using different aminoglycosides and most of
the tested molecules promoted an increase in the melting temperature of GenD2 ranging
from 4.5 °C for paromamine to 10 °C for gentamicin C, suggesting that interactions might
occur between GenD2 and these aminoglycosides (Figure S5 and Table S2, SI). The lower
increment of melting temperature observed for paromamine is expected since this
compound does not have the ring III and only 2-DOS (ring I) and ring II contribute to the
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increase in temperature, indicating a lower affinity of this ligand compared to the other
ligands used.
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Although various GFO/IDH/MocA family members have been structurally characterized,
only a limited number of enzymes that act on C3-hydroxyl groups of sugars have been
studied, including WlbA from Bordetella pertussis and Chromobacterium violaceum,
which catalyze the oxidation of UDP-N-acetyl-D-glucosaminuronic acid (UDP-
GlcNADcA) to UDP-2-acetamido-2-deoxy-3-oxo-D-glucuronic acid (UDP-3-keto-Glc-
NAcA)17. The catalytic mechanism of the enzymes in this class involves a NAD(P)(H)-
mediated hydride ion (H-) transfer from (dehydrogenases) or to the substrate
(reductases)15. GenD2 was proved to be a dehydrogenase acting on the gentamicin A2 at
C3ʹʹ position of ring III to produce DOA2 during the biosynthesis of gentamicin A.
However the activity of GenD2 was only observed in the presence of another two
enzymes, aminotransferase GenS2 and N-methyltransferase GenN11. Similar to most of
the other members of the Gfo/Idh/MocA protein family, the active site of GenD2 is a
solvent-exposed region near the nicotinamide moiety of NAD+ between the N- and C-
domain. In addition, GenD2 has the conserved motif EKP (Figure 2). The Lys96 in the
motif interacts with side chains of Asp177 and His181 and could be the catalytic triad as
suggested for other GFO/IDH/MocA family members19,24. Trp233 might also play a role
in coordinating the position of Asp177 via the interaction between the nitrogen in the
indole side chain to the side chain of Asp177 (Figure 5A). In addition, Lys96, as observed
in other members of Gfo/Idh/MocA protein family oxidoreductases, is located in the N-
terminal domain at end of β6. The superposition of GenD2 holoenzyme with WlbA
dehydrogenase holoenzyme from Chromobactrium violaceum in complex with UDP-
GlcNAcA suggests that the ring III of gentamicin A2 might adopt similar position and
interactions as the glucose moiety of UDP-GlcNAcA and the two enzymes could have
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similar catalytic mechanisms, although WlbA has an asparagine instead an aspartate in
the catalytic triad (Figure 5A). Lys96 could act as a catalytic base, abstracting a proton
from C3ʹʹ-hydroxyl group, and a hydride ion from C3ʹʹ oxygen could be transferred to the
C4 of the nicotinamide ring of NAD+. In addition, the position of the nicotinamide moiety
of NAD+ is also conserved in GenD2 indicating that the ring III of gentamicin A2 may
also bind at the re face of this moiety (Figure 5B). Residues Asp160 and Glu239 at the
active site pocket may also be involved in the substrate recognition through their
negatively charged side chains, which are complementary to the highly positively charged
aminoglycoside substrate (Figure 5C). Furthermore, Asn124, Thr156, and Tyr293 could
potentially form hydrogen bonds with the substrate. Finally, several hydrophobic amino
acids, Phe14, Phe125, Phe154, Phe164, Trp257, Trp274, and Trp287 could form π-
interaction with the substrate rings for the correct positioning at the catalytic cavity
(Figure 5D).
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B
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C
D
Figure 5. Hypothetical substrate binding site of GenD2. (A) The hypothetical catalytic
triad that could be involved in the oxidoreductase reaction. (B) Superposition of GenD2
(carbons in yellow) and WlbA dehydrogenase from Chromobacterium violaceum in
complex with NAD+ and UDP-GlcNAcA (carbons in white). The electrostatic potential
surface is from GenD2. (C) An electrostatic potential surface of GenD2 indicating the
highly negative charged hypothetical substrate binding site. (D) Hydrophobic residues
that could be involved in the substrate orientation on the GenD2 active site.
Moreover, docking simulations using gentamicin A2 as a ligand corroborated the
hypothetical catalytic mechanism of GenD2. The best-ranked pose of gentamicin A2
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(Figure 6A) indicated that ring I interacts with GenD2 through two hydrogen bonds,
which includes the side chains of Glu239 (not shown) and Tyr293. Ring II is stabilized
by a hydrogen bond with Asp160 and further π-interactions with Trp274, Trp287, and
Phe14 (not shown). Interestingly, the ring III of gentamicin A2 forms three hydrogen
bonds with all amino acids of the proposed catalytic triad including Lys96, Asp177 and
His181, which supports the involvement of these amino acids in the catalysis. The
superposition of all docked poses indicates a well-conserved disposition of both ring I
and III of gentamicin A2 into the narrow cavity of GenD2, while ring II, which is located
in the solvent-exposed region, was able to adopt different conformations due to the
entropic profile of the area (Figure 6B). Noteworthy, the C4 of the nicotinamide ring of
NAD+ and the C3ʹʹ-hydroxyl group of gentamicin A2 are faced each other inside the
cavity (Figure 6C) besides are being surrounded by the proposed catalytic triad (Figure
6D). However, a structure of GenD2 holoenzyme in complex with the substrate or close
analogs is required to further validate the proposed binding mode of gentamicin A2 into
the GenD2 active site.
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Figure 6. GenD2 docking findings. (A) The best-ranked pose of gentamicin A2 (carbons
in green). (B) Superposed poses of the gentamicin A2 at the GenD2 binding cavity. (C)
Merged structures of NAD+ (carbons in yellow) and gentamicin A2 (carbons in green)
through the binding cavity of GenD2. The double-headed red arrow indicates the C3ʹʹ of
ring III of gentamicin and the C4 of the nicotinamide ring of NAD+. (D) Docked ring III
from gentamicin A2 surrounded by the suggested catalytic triad of GenD2 and the
nicotinamide moiety from NAD+. Cartoon protein depicted in white. Carbons of GenD2
in white. Oxygen in red. Nitrogen in blue. Phosphorus in orange. Hydrogen bonds
indicated as black dashed lines. Interatomic distances in angstroms (Å).
CONCLUSION
The substitution of the hydroxyl to amino groups on the sugar molecules is performed by
two enzymatic steps, a dehydrogenase/oxidoreductase that converts the hydroxyl to a
ketone group and this is further acted on by an aminotransferase that replaces the oxygen
to an amino group. Several sets of dehydrogenation/transamination reactions are
identified to act on different hydroxyl groups of aminoglycosides, but the modification at
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position C3ʹʹ of ring III occurs only in gentamicins, sisomicin, tobramycin, and
kanamycin and the biosynthetic gene clusters of these antibiotics all have the specific and
conserved genes that act on this position. To investigate the structural basis of the function
of GenD2, we have obtained its crystal structure in complex with NAD+ at 2.4 Å
resolution. GenD2 belongs to the Gfo/Idh/MocA protein family of oxidoreductases and
has the conserved catalytic residues proposed for deprotonation of the substrate to
facilitate hydride abstraction from the substrate to the cofactor NAD+. ITC analysis has
further confirmed the interaction between NAD+ and GenD2. Interestingly, GenD2 is the
first member of Gfo/Idh/MocA protein family to have a 3D-domain swapping involving
two β-strand of the C-terminal domain. Several features of GenD2 structure have
implication on the substrate recognition including a very negatively charged active site
for the binding of the alkaline substrate and several hydrophobic residues that could
perform π-interaction with the unusual sugar rings to further stabilize the binding. In silico
studies also suggested that Lys96, Asp177 and His181 could be involved in the C3ʹʹ
oxidation of ring III of gentamicin A2 by GenD2. The characterization of the GenD2
structure could provide useful information for increasing the promiscuity of this enzyme
in generating novel valuable aminoglycosides through both in vivo and in vitro
approaches.
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METHODS
Expression and Purification of Recombinant Proteins
The genD2 gene from Micromonospora echinospora ATCC15835 was cloned into the
plasmid pET28a (+) as described by Huang et al.3. Transformation of plasmids was
performed by heat shock in E. coli BL21(DE3) competent cells. E. coli BL21(DE3) cells
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carrying the recombinant plasmids were cultivated in LB (Luria Bertani) medium
supplemented with kanamycin (50 µg/mL) at 37 °C until the cell density reached 0.6 at
600 nm. Overexpression of the protein was induced by β-D-1-thiogalactopyranoside
(IPTG) (0.2 mM) at 18 °C with shaking at 200 rpm overnight. Cells were harvested by
centrifugation (6000 x g, 4 °C, 30 min) and resuspended 1:1 in buffer (50 mM Tris-HCl,
100 mM NaCl, pH 7.7) with the addition of DNAse and lysozyme at a concentration of
50 µg/mL and phenylmethanesulfonyl fluoride (PMSF) (5 µM). Cell lysis was performed
by sonication and the lysate was clarified by centrifugation (17000 x g, 4 °C, 1 h). The
recombinant protein was purified by affinity chromatography using a 5 mL IMAC column
(GE Healthcare), Ni2+ ion-charged His-Bind metal chelating resin (Novagen). GenD2 was
eluted using a linear gradient of imidazole (up to 500 mM) on an Åkta Purifier system
(GE Healthcare). To increase the purity, a gel filtration purification step was further
performed using a HiLoad 16/60 Superdex 200 column (GE Healthcare) equilibrated with
50 mM Tris-HCl, 100 mM NaCl, pH 7.7. Fractions were collected and concentrated up
to 15-20 mg/mL and the enzyme was stored at -80 °C. The purity of GenD2 was
confirmed by SDS-PAGE 12% stained using Coomassie Brilliant Blue.
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Protein crystallization
GenD2 crystallization trials were performed using the sitting-drop method using an Oryx
4 robot (Douglas Instruments) in 96 well plates (MRC2 Swissci). The drops had a total
volume of 0.9 µL, 50% of protein solution at a concentration of 10 mg/mL with 5 mM
cofactor NAD+ and 50% crystallization condition. After obtaining the first crystals hits,
these conditions were optimized manually by hanging drop vapor diffusion method using
24 well VDX plates (Hampton Research). The best and most reproducible crystallization
conditions for GenD2 contained 0.1 M sodium acetate pH 4.6, 2 M sodium formate at a
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protein concentration of 15 mg/mL and the crystals appeared after approximately seven
days in crystallization plates incubated at 30 ºC.
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Diffraction and data collection
Data collection of GenD2 crystals was performed at Deutsche Elektronen-Synchrotron
(DESy), PETRA III, P13 beamline, in Hamburg, Germany. The data analysis of X-ray
diffraction images was performed by XDS25. The data was escalated by AIMLESS26 from
the program CCP4 suite27.
Structure determination
The initial phases for the GenD2 structure were obtained by Molecular Replacement
using the online server MrBUMP 18. The refinement was carried out using Phenix.refine28
from the Phenix crystallographic suite29. The visual inspections were performed using
COOT30 and the stereochemistry quality of the model was accessed using MolProbity31.
All figures were prepared using PyMOL Molecular Graphics System, Version 1.8
(Schrödinger, LLC).
Differential scanning fluorimetry (DSF)
DSF experiments were performed using a Bio-Rad CFX connect Real-Time System with
detection and excitation filters ranging from 450 to 580 nm. For the experiments, 10 μM
of GenD2 in presence of 1 mM of NAD+ diluted in a buffer constituted by 50 mM
HEPES, 100 mM NaCl, pH 7.0 was incubated with different aminoglycoside-like
substrates (kanamycin, geneticin, gentamicin sulfate, apramycin, sisomicin, neomycin,
amikacin, paromamine, and meropenem. This last one was used as negative control) at a
concentration of 125 μM in the presence of the fluorescent dye SYPRO® Orange 5X (Life
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Science). The volumes of 25 μL of protein, each ligand, and fluorescent dye were added
to a well of the 96 well plates (Bio-Rad). The protein solutions were scanned from the
temperature of 25 ºC to 95 ºC with an increment of 0.5º/minute. Tm values were estimated
using Bio-Rad CFX Manager software or using the program Origin and the ΔTm for each
compound was calculated by the difference of protein Tm in the presence of ligand and,
protein Tm in the absence of ligand.
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Isothermal thermodynamic calorimetry (ITC)
ITC experiments were performed using an iT200 microcalorimetry (Malvern) at 25 ºC.
Protein and co-factors (NAD+ and NADP+) were prepared using exactly the same buffer,
which was constituted by 50 mM HEPES, 100 mM NaCl, pH 7.0. The protein
concentration used in the cell was 150 μM and the ligand concentration in the syringe
ranged from 2 to 10 mM. It was performed 18 injections of 10 μL of ligand against to the
protein, spaced of 180 s and a stirring speed of 400 rpm. The ITC curves were fitted using
nonlinear least-squares regression using the noninteraction one-site model from Origen
5.0.
Molecular docking studies
Gentamicin A2 structure was built and energy-minimized with the density functional
theory (DFT), method Becke-3-Lee Yang Parr (B3LYP) with the standard 6-31G* basis
set available in the Spartan'14 program (Wavefunction, Inc., Irvine, CA, USA). The
experimental starting geometry used for the construction of gentamicin A2 was described
previously (PDB entry 5U0N, gentamicin A)13. Molecular docking was performed with
the GOLD 5.2 program (CCDC). The scoring function used was ‘ASP’, which is a default
function of the GOLD 5.2 program. Hydrogen atoms were added to GenD2 according to
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the data inferred by the GOLD 5.2 program on the ionization and tautomeric states. The
docking interaction cavity in the protein was established with a 12 Å radius from the
phenolic hydroxyl group (OH) of Tyr293. The number of genetic operations (crossover,
migration, mutation) in each docking run that was used in the search procedure was set
to 100,000. Docking simulations were performed six times. The figure of the best – and
most frequent – docking pose for gentamicin A2 was generated by the PyMOL Molecular
Graphics System, Version 1.8 (Schrödinger, LLC).
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ASSOCIATED CONTENT
Supporting Information. The supporting information is available free of charge on the
ACS Publication website at DOI:XXXX. GenD2 purification steps (Supporting Figure
1); GenD2 oligomerization interface (Supporting Figure 2); Electrostatic potential surface
of GenD2 tetramer (Supporting Figure 3); Calorimetry parameters for GenD2 against
NAD+ and NADP+ (Supporting Table 1); ITC titration of (A) NAD+ and (B) NADP+
against GenD2 (Supporting Figure 4); Differential Scanning Fluorimetry (DSF) for
GenD2 in presence of different aminoglycoside-like substrates (Supporting Figure 5);
Analysis of DSF for GenD2 in the presence of different aminoglycosides (Supporting
Table 2).
ACKNOWLEDGMENT
This work was supported by FAPESP under grant 2010/15971-3, 2014/50324-0 and
2015/09188-8 to M.V.B.D. and fellowship 2014/07843-6 to P.S.B and a CAPES
fellowship to N.C.A.; and by a project grant from the Medical Research Council, UK
(G1001687) to P.F.L. We also would like to thank R. F. de Souza for helpful discussions.
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AUTHOR CONTRIBUTION
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N.C.A. performed the expression, purification, crystallization and structure refinement,
ITC and DSF assays; P.S.B. determined the structure and performed the refinement. F.H.
performed the cloning and determined the best expression conditions. P.F.L and
M.V.B.D. conceived the work and M.V.B.D. and F.H. wrote the manuscript. M.T.T. and
R.P. performed the docking simulations and reviewed the manuscript.
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AUTHOR INFORMATION
Corresponding author: * 55 (0) 11 3091 7420. E-mail:mvbdias@usp.br; ORCID ID:
0000-0002-5312-0191
Notes
The authors declare no competing financial interest.
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