Identification of residues important for the activity of aldehyde-deformylating oxygenase through investigation into the structure-activity relationship
- Qing Wang†1, 2,
- Luyao Bao†1, 2,
- Chenjun Jia2, 3,
- Mei Li3,
- Jian-Jun Li1, 4Email author and
- Xuefeng Lu1Email author
© The Author(s). 2017
Received: 13 May 2016
Accepted: 8 March 2017
Published: 16 March 2017
Aldehyde-deformylating oxygenase (ADO) is a key enzyme involved in the biosynthetic pathway of fatty alk(a/e)nes in cyanobacteria. However, cADO (cyanobacterial ADO) showed extreme low activity with the k cat value below 1 min−1, which would limit its application in biofuel production. To identify the activity related key residues of cADO is urgently required.
The amino acid residues which might affect cADO activity were identified based on the crystal structures and sequence alignment of cADOs, including the residues close to the di-iron center (Tyr39, Arg62, Gln110, Tyr122, Asp143 of cADO-1593), the protein surface (Trp 178 of cADO-1593), and those involved in two important hydrogen bonds (Gln49, Asn123 of cADO-1593, and Asp49, Asn123 of cADO-sll0208) and in the oligopeptide whose conformation changed in the absence of the di-iron center (Leu146, Asn149, Phe150 of cADO-1593, and Thr146, Leu148, Tyr150 of cADO-sll0208). The variants of cADO-1593 from Synechococcus elongatus PCC7942 and cADO-sll0208 from Synechocystis sp. PCC6803 were constructed, overexpressed, purified and kinetically characterized. The k cat values of L146T, Q49H/N123H/F150Y and W178R of cADO-1593 and L148R of cADO-sll0208 were increased by more than two-fold, whereas that of R62A dropped by 91.1%. N123H, Y39F and D143A of cADO-1593, and Y150F of cADO-sll0208 reduced activities by ≤ 20%.
Some important amino acids, which exerted some effects on cADO activity, were identified. Several enzyme variants exhibited greatly reduced activity, while the k cat values of several mutants are more than two-fold higher than the wild type. This study presents the report on the relationship between amino acid residues and enzyme activity of cADOs, and the information will provide a guide for enhancement of cADO activity through protein engineering.
KeywordsAldehyde-deformylating oxygenase Site-directed mutagenesis Structure-activity relationship Fatty alk(a/e)ne Synechococcus elongatus PCC7942 Synechocystis sp. PCC6803
Fatty alk(a/e)nes, which can be produced by plants, insects, birds, green algae, and cyanobacteria, are the main components of conventional fuels, and have been considered as the ideal replacement for fossil-based fuels [1–5]. It has been accepted that a two-step pathway for fatty alk(a/e)ne biosynthesis exists, involving reduction of fatty acyl-ACP or -CoA into corresponding aldehyde by acyl-ACP reductase and conversion of fatty aldehyde into alk(a/e)ne by aldehyde decarbonylase. In 2010, Schirmer et al. identified two genes involved in fatty alk(a/e)ne biosynthesis in cyanobacteria: acyl-ACP reductase and aldehyde decarbonylase (renamed as aldehyde-deformylating oxygenase, ADO) [1, 6]. Since then cADO (cyanobacterial ADO) has attracted particular interest due to the difficult and unusual reaction it catalyses .
However, cADO showed extreme low activity with the k cat value below 1 min−1, which would present a major barrier to its application in biofuel production [8, 14–17, 22]. In order to address this issue, protein engineering of cADO for improved activity including rational design and/or directed evolution is urgently needed. The knowledge about structure-function relationship of cADOs is the prerequisite for rational protein engineering. Until now, no detailed studies towards structure-function relationship of cADOs have been carried out. Some crystal structures of cADOs from P. marinus strain MIT9313 and Synechococcus elongates strain PCC7942 have been resolved, which have provided a base for identification of the residues important for cADO activity [8, 9].
In the current study, some amino acids which might affect cADO activity were identified through analysis of the cADO crystal structures and sequence alignment of some cADOs. The corresponding enzyme variants were made and characterized. We have found some essential residues for the cADO-catalyzed reaction, which will lay the foundation for improvement of cADO activity through protein engineering.
Identification of the target residues for mutagenesis
The following residues were identified and investigated in the current study. Since the variants including mutations of the residues involved in coordinating the di-iron center negatively affected cADO activity, they were not included in the current study .
Residues whose conformations have changed in the absence of the di-iron center
Residues involved in two hydrogen bonds
Residues close to the di-iron center
The residues around the di-iron center provide conformational constraints that control the geometry of the di-iron center. According to the structure of 1593, residues Tyr39, Arg62, Gln110, Tyr122 and Asp143 are close to the di-iron center, which might interact with the nearby residues including the ligands of di-iron: Tyr39 with Glu60 (ligand), His147 (ligand), Gln110 and Ser111; Arg62 with His147 (ligand) and Asp143; Gln110 with Glu115 (ligand), Asn38 and Tyr39; Tyr122 with Glu32 (ligand), Glu144 (ligand) and Val28 (Fig. 2); Asp143 with Arg62 and His147 (ligand). Residue Tyr39 is highly conserved, and is Phe in 8 cADOs (Additional files 1 and 2). Arg62, Gln110, Tyr122 and Asp143 show high conservativeness among ~150 cADO. Likewise, the residues coordinating the di-iron center like Glu32, Glu44, Glu60, Glu115, Glu144 and His147 were excluded in this study. Residues Tyr39, Arg62, Gln110, Tyr122 and Asp143 of 1593 were selected for investigation.
Residues located on the protein surface
Based on sequence alignment of 150 cADOs, Trp178 of 1593 is highly conserved among ~120 cADOs, and is Arg/Lys in ~30 cADOs. The side-chains of Trp and Arg/Lys exhibited completely different properties: hydrophobicity versus hydrophilicity. Moreover, according to the crystal structure of 1593, Trp178 is positioned at Helix 6 and exposed to the protein surface (Fig. 2). These facts prompted us to presume that hydrophobicity or hydrophilicity of this residue might have some influence on cADO activity. Therefore, this residue was studied.
Site-directed mutagenesis, overexpression and purification
We have observed that 1593 is more active than sll0208 against n-hexadecanal, so Thr146, Leu148 and Tyr150 of sll0208 were respectively mutated into the counterparts of 1593 - Leu, Arg and Phe (Fig. 3). For comparison, Leu146 and Phe150 of 1593 were also mutated into the corresponding ones of sll0208 - Thr and Tyr respectively. Since PMT1231 is more active than sll0208 towards n-hexadecanal under our assay conditions (unpublished results), Asp49 and Asn123 of sll0208 were mutated into the corresponding residues of PMT1231 - His and His respectively (Fig. 3). In order to investigate the effects of the polar side chains of some residues on cADO activity, the single site-directed mutants Y39F, R62A, Q110L, Y122F, D143A and N149A were constructed for 1593. In addition, N123H and W178R of 1593 were made based on the conservativeness.
Enzymatic activities of wild-type cADOs and variants
n-Hexadecanal and n-heptanal were used as the substrates to investigate the effects of mutations on enzymatic activities of 1593 and sll0208. When n-hexadecanal was used as a substrate, the yields of n-pentadecane were quantified. While n-heptanal was used as a substrate, the apparent k cat values were measured.
Apparent k cat values and yields of n-pentadecane of WT 1593, WT sll0208 and variants. The apparent k cat values were determined using 2 mM n-heptanal as the substrate
k cat app (Min−1)
Yield of n-pentadecane (μM)
0.45 ± 0.06
12.1 ± 0.4
0.87 ± 0.1
14.2 ± 1.2
0.31 ± 0.04
8.7 ± 0.7
0.76 ± 0.08
12.2 ± 0.6
0.43 ± 0.05
10.4 ± 0.9
0.72 ± 0.07
14.4 ± 0.6
0.87 ± 0.09
15.0 ± 0.1
0.37 ± 0.05
10.4 ± 0.8
0.23 ± 0.03
5.8 ± 0.2
0.15 ± 0.02
2.1 ± 0.04
0.04 ± 0.001
0.4 ± 0.06
0.36 ± 0.05
9.2 ± 0.2
1.47 ± 0.1
17.4 ± 0.2
0.44 ± 0.05
1.4 ± 0.1
0.17 ± 0.02
1.4 ± 0.1
0.75 ± 0.08
5.1 ± 0.2
0.42 ± 0.04
2.2 ± 0.1
0.22 ± 0.03
1.8 ± 0.1
0.14 ± 0.02
1.0 ± 0.08
0.73 ± 0.08
3.4 ± 0.3
Compared with WT sll0208, the apparent k cat values of sll0208L148R and sll0208D49H/N123H were increased by 70.5 ± 5.3% and 65.9 ± 4.4% respectively, whereas those of sll0208D49H, sll0208N123H and sll0208T146L were significantly reduced (Table 1). The activity of sll0208Y150F was not almost affected. The yields of n-pentadecane of the enzyme variants demonstrated similar trend to the apparent k cat values (Table 1).
Kinetic parameters of WT 1593, WT sll0208 and some variants
K m (mM)
k cat (min−1)
k cat /K m (min−1mM−1)
0.30 ± 0.02
0.48 ± 0.01
1.6 ± 0.2
0.34 ± 0.08
0.94 ± 0.08
2.76 ± 0.3
0.20 ± 0.04
0.75 ± 0.04
3.75 ± 0.4
0.33 ± 0.05
0.87 ± 0.04
2.64 ± 0.2
0.35 ± 0.02
1.04 ± 0.02
2.97 ± 0.3
0.34 ± 0.08
1.81 ± 0.15
5.32 ± 0.4
0.35 ± 0.07
0.59 ± 0.04
1.69 ± 0.2
0.32 ± 0.08
0.91 ± 0.08
2.84 ± 0.3
0.38 ± 0.08
0.83 ± 0.08
2.18 ± 0.2
Interestingly, the k cat values of 1593W178R, 1593Q49H/N123H/F150Y and 1593L146T, close to or above 1 min−1, were obtained. Especially, 1593W178R showed 3.8-fold improved k cat value than WT 1593, and exhibited the highest catalytic efficiency (k cat/K m) among all variants, 3.3-fold higher than WT (Table 2).
Although the conditions of the cADO-catalyzed reaction have been optimized through different attempts, the turnover numbers are still below ~1 min−1 [8, 14–17, 22]. Therefore, the sluggish kinetics of cADO has become a bottle-neck for biofuel production in cyanobacteria, and to identify the activity related key residues for cADO is definitely required. Since it was observed that cADO-1593 showed the highest activity under our assay conditions among cADOs tested (unpublished results), it was selected as the test sequence for BLAST. In the current paper, we have identified some amino acid residues which impact the enzyme activity of cADO through structure and sequence analysis of some cADOs.
Firstly, the results of 1593L146T, 1593F150Y, sll0208T146L and sll0208L148R have clearly demonstrated that the side chains of the residues consisting of the oligopeptide (residues 144 to 150 for 1593) whose conformation changed in the absence of the di-iron center had important effects on cADO activity. Crystal structures reveal that these polar residues form important hydrogen bond interactions with residues from other helices, which may contribute to the local structural stability of the oligopeptide. In addition, the polar side chains of these residues might interact with the nearby residues, for example, in 1593 (Protein code: 4RC5) Arg148 with Asn149 and Glu152, Asn149 with Arg148 and Glu152; in sll0208 (Protein code: 4Z5S) Thr146 with Asp143 and Asn149, Asn149 with Thr146 and Glu152, Tyr150 with Gln49; in PMT1231 (Protein code: 2OC5) Thr159 with Asp156 and Asn162, Asn162 with Thr159 and Glu165, Tyr163 with His62, which are equivalent to the residues 146, 143, 149, 152, 150 and 49 of 1593 and sll0208 respectively. Thr159 of PMT1231 and the equivalent one (Thr146) of sll0208 are also involved in the hydrogen-bonding network close to the di-iron center. Replacement of the residues containing the polar side chains with ones containing the nonpolar side chains led to removal of the possible interaction with nearby residues, which might have severe influence on protein folding, stability, even activity. In contrast, substitution of amino acids with the nonpolar side chains by ones with the polar side chains gave the reverse impact, as observed for 1593L146T, sll0208L148R and 1593F150Y. The enhanced activities of these three variants are presumably due to local stabilization caused by newly established interaction of three residues with the nearby amino acids.
Secondly, based on the structures of sll0208 and PMT1231, a hydrogen bond is possibly established between Tyr150 and Gln49 in 1593 while Phe150 of 1593 was substituted by Tyr. Activities of both F150Y and Q49H/F150Y of 1593 were enhanced, suggesting that the hydrogen bond between these two residues is important for 1593. The triple mutant 1593Q49H/F150Y/N123H showed higher activity than WT 1593, 1593Q49H/F150Y and 1593F150Y, and the double mutant sll0208D49H/N123H exhibited improved activity than WT sll0208, sll0208D49H and sll0208N123H. Thus, some synergistic effects were observed for 1593Q49H/F150Y/N123H and sll0208D49H/N123H, implying that these two hydrogen bonds are beneficial for cADO activity and the co-existence of two His in both hydrogen bonds is more important than the presence of only one His. Unexpectedly, the Y150F substitution of sll0208, in which the hydrogen bond between Tyr150 and Asp49 is removed, had negligible effect on the catalytic activity.
Thirdly, replacement of the residues Arg62, Gln110 and, Tyr122 of 1593 having the polar side chains by those containing the nonpolar ones resulted in significantly decreased activity, which could be possibly due to removal of the interaction with the nearby residues. These results confirmed that the residues close to the di-iron center impacted cADO activity and their polar side chains are important for cADO.
Some amino acids which could affect cADO activity were identified based on the crystal structures and sequence alignment of cADOs. The residues close to the di-iron center, the protein surface, and those involved in the hydrogen-bonding network and the oligopeptide whose conformation changed, exerted great influence on cADO activity. Several mutations led to significantly decreased activity. Some enzyme variants showed improved activity than the wild type, and the k cat values of several of them close to or above 1 min−1 were achieved for the first time in particular. We identified some important residues for the catalytic activity of cADO. The study would be helpful for establishing an efficient cell factory of biofuel production in cyanobacteria.
Chemicals were from Sigma, Merck or Ameresco. Oligonucleotides and the gene encoding ADO (aldehyde-deformylatingoxygenase) Synpcc7942_1593 from Synechococcus elongates PCC7942 with codon optimization were synthesized by Shanghai Sangon Biotech Co. Ltd (China) . Pfu DNA polymerase, restriction endonucleases EcoRI and NotI were from Fermentas or Takara Biotechnology. DpnI was from New England BioLabs. The kits used for molecular cloning were from Omega Bio-tek or Takara Biotechnology. Nickel column and expression vector pET-28a(+) were from Novagen. Amicon YM10 membrane was from Millipore.
Bacterial strains, plasmids, and media
E. coli DH5α was used for routine DNA transformation and plasmid isolation. E. coli BL21(DE3) was utilized for overexpression of cADO. E. coli strains were routinely grown in Luria-Bertani broth at 37 °C with aeration or on LB supplemented with 1.5% (w/v) agar. 100 μg/ml Kanamycin was added when required.
General molecular biology techniques were carried out by standard procedures . DNA fragments were purified from agarose gels using the DNA gel extraction kit. Plasmid DNA was isolated using the plasmid miniprep kit.
The gene sll0208 encoding ADO from Synechocystis sp. PCC6803 (sll0208) was amplified with the forward primer (5′- GCCTTACATATGATGCCCGAGCTTGCTG-3′, the NdeI I restriction site underlined) and the reverse primer (5′- CAACTACTCGAGCTAGACTCCGGCCAAACC-3′, the XhoI restriction site underlined) using genomic DNA as a template. The PCR products were recovered, and digested with restriction enzymes NdeI and XhoI respectively, and re-cloned into the vector pET-28a(+) digested with NdeI and XhoI, respectively.
Construction of site-directed mutants
Site-directed mutants were constructed according to the standard QuikChange Site-Directed Mutagenesis protocol (Stratagene Ltd) using wild-type (WT) 1593 or sll0208 as templates and the primers listed in Table S1 (Additional file 3).
For construction of double and triple mutants, WT 1593 or sll0208 harboring single or double mutation(s) was used as a template respectively following the same protocol as above.
Protein overexpression and purification
WT cADOs and variants were overexpressed in E. coli BL21(DE3) and purified on Nickel column as reported . The purity of protein was checked by SDS-PAGE (Additional file 4). Apo-ADO was prepared according to the published procedure, and the diferrous form of ADO was reconstituted by the addition of the stoichiometric amounts of ferrous ammonium sulfate to the apo-ADO prior to assay [14–16]. Proteins were concentrated with Amicon YM10 membrane (10 kDa cut-off). The protein concentration was determined by the Bradford method using bovine serum albumin as a standard.
Enzyme activity assay
All enzymatic assays were carried out in triplicate. According to the published procedure [16, 17], assays were carried out in HEPES buffer, containing 100 mM KCl and 100 mM HEPES, pH 7.2. The reaction mixtures contain NADH (750 μM), catalase (1 mg/ml), ferrous ammonium sulfate (80 μM), PMS (Phenazine methosulfate) (75 μM), appropriate amount of aldhydes (150 μM for n-hxadecanal, 2 mM for n-heptanal), cADO (20 μM for n-hxadecanal, 5 μM for n-heptanal). When n-hxadecanal was used as the substrate, the yields of n-pentadecane were quantified by GC-MS. While n-heptanal was used as substrate, the apparent k cat values (2 mM of n-heptanal was utilized) were measured. The control using E. coli containing the vector pET-28a only didn’t show any ADO activity (data not shown).
K m and V max values of WT 1593, WT sll0208 and some variants against n-heptanal were determined according to the Michaelis-Menten equation of GraphPad Prism 5 (Additional file 5). The k cat values were calculated from V max on the basis of the molecular weight of enzymes.
Acyl carrier protein
Cyanobacterial aldehyde-deformylating oxygenase
This work was supported by grants from the National Basic Research Program of China (973: 2011CBA00907 and 2011CBA00902), National Science Foundation of China (31170765 and 31370799), CAS Cross and Cooperation Team for Scientific Innovation (Y31102110A), the Excellent Youth Award of the Shandong Natural Science Foundation (JQ201306 to X. Lu) and the Shandong Taishan Scholarship (X. Lu).
Availability of data and materials
The datasets supporting the conclusions of this article are included within the article and its additional files.
JJL and XL designed the experiments. QW and LB performed the experiments, including gene cloning, construction of site-directed mutants, overexpression, purification, characterization, and enzymatic assays. CJ and ML performed structural analysis of cADOs, and assisted in manuscript preparation. JJL, XL, QW, and LB drafted the manuscript. All authors read and approved the final manuscript.
The authors declare that they have no competing interests.
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