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Structural Insights into Phosphite Dehydrogenase Variants Favoring a Non-Natural Redox Cofactor Yuxue Liu, Yanbin Feng, Lei Wang, Xiaojia Guo, Wujun Liu, Qing Li, Xueying Wang, Song Xue, and Zongbao Kent Zhao ACS Catal., Just Accepted Manuscript • DOI: 10.1021/acscatal.8b04822 • Publication Date (Web): 29 Jan 2019 Downloaded from http://pubs.acs.org on January 31, 2019
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ACS Catalysis
Structural Insights into Phosphite Dehydrogenase Variants Favoring a Non-Natural Redox Cofactor Yuxue Liu,†,‡,‖ Yanbin Feng,†,‖ Lei Wang,† Xiaojia Guo,†,‡ Wujun Liu,† Qing Li,†,‡ Xueying Wang,† Song Xue,*,† and Zongbao Kent Zhao*,†,§ †Division
of Biotechnology, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian 116023, China
‡University §State
of Chinese Academy of Sciences, Beijing, 100049, China
Key Laboratory of Catalysis, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian 116023,
China ABSTRACT: Implementation of non-natural cofactors alternative to the ubiquitous redox cofactor nicotinamide adenosine dinucleotide (NAD) is of great scientific and biotechnological interests. Several redox enzymes have been engineered to favor nicotinamide cytosine dinucleotide (NCD), a smaller-sized NAD analogue. However, molecular interactions involving NAD analogues remain elusive, preventing us from devising more enzymes to accept those analogues. Here we took a semi-rational approach to evolve phosphite dehydrogenase (Pdh) and identified variants with substantially improved NCD preference. These mutants are valuable components for regeneration of reduced NCD by using phosphite as the electron donor. We then collected X-ray crystal structures of three Pdh variants and their NCD-complexes to delineate molecular basis for NCD binding. It was found that the incorporation of amino acid residues with large side chains enclosing the NAD-binding pocket led to compacted environment favoring NCD over NAD, and additional interactions between NCD and these side chains. These results guided successful engineering more Pdh mutants with good NCD preference. As many redox enzymes share key structural features, our strategy may be readily adopted to devise NCD-favoring enzymes. We expected that, in the near future, more synthetic systems linked to non-natural cofactors will be created as alternative tools for widespread applications to address challenging problems by chemical and synthetic biologists.
KEYWORDS: phosphite dehydrogenase, non-natural redox cofactor, NAD-binding pocket, nicotinamide cytosine dinucleotide, cofactor preference
The redox cofactor nicotinamide adenosine dinucleotide (NAD, Scheme 1) and its reduced form NADH, are omnipresent in biological systems. NAD(H) involve in enabling catalysis by many redox enzymes, but also have non-redox functions such as providing co-substrates for several important enzymes including poly-ADP-ribose polymerase and some histone deacetylases.1 While enormous efforts have been devoted to intervening the cellular NAD(H) levels,2 it is challenging to predict the consequences of such intervention, largely because redox cofactors are involved in diverse metabolism and processes.3 Novel components such as non-natural nucleotides and non-natural amino acids have been increasingly employed to address challenging biological problems.4 Similarly, there have been continuous interests in implementing nonnatural redox cofactors for chemical and synthetic biology.5 For instance, a biocompatible emissive cofactor has been devised to accomplish real-time visualization of cofactordependent process by fluorescence spectroscopy.5a, 5b In an
early study, we created bioorthogonal redox systems consisted of malic enzyme (ME) mutants that strongly favored the synthetic cofactor nicotinamide cytosine dinucleotide (NCD) and achieved enzymatic regeneration of NCDH.5d We further established a number of functional combinations between enzyme mutants and other NAD analogues.6 We then devised NCD-linked metabolic circuits for reductive carboxylation of pyruvate into malate by using phosphite (Phi) as the electron donor and achieved higher malate yield from glucose by the engineered Escherichia coli cells.5e OH OH N N
N N
O OH
O O
P
OH O
O
NH2
P O
O O
+ N
OH OH
NAD
O
OH OH NH2 O
N
O
N
OH O
OH
P O
P
O
O
O
O
OH OH
NH2
NCD
Scheme 1. Chemical structures of NAD and NCD.
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+ N
NH2
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Cofactor regeneration has major impacts on the application of redox cofactors for biocatalysis and metabolic engineering.7 Phosphite dehydrogenase (Pdh) catalyzes a NAD-dependent oxidation of Phi to phosphate with concurrent formation of NADH in an essentially irreversible way, thus providing a great potential for NADH generation.8 We reported that the wild-type (WT) Pdh from Ralstonia sp. strain 4506 and its I151R mutant were able to mediate NCDH formation, albeit the mutant retained substantial activity with NAD.5e It is critical to engineer redox enzymes with more stringent preference to NAD analogue in order to assemble fully bioorthogonal systems for metabolic control and related applications. However, little is known regarding molecular interactions involving NAD analogues. Herein, we took a semi-rational approach to evolve Pdh, characterized several variants, and revealed molecular bases leading to different NCD-binding capability by X-ray crystal structural analysis. The knowledge guided successful designing additional NCD-preferred mutants. This symbolic study provides valuable knowledge for rational devising non-natural cofactor-dependent systems as unique tools for widespread applications by chemical and synthetic biologists.
Figure 1. Overview of Pdh evolution process. The process is composed of analysis, construction and screening. For analysis, the structure of wild type Pdh is homology modeled, and residues that may affect the cofactor preference are identified. For construction, mutagenesis libraries are generated by primer-based methods. For screening, E. coli colonies are arrayed into 96-deep-well plates, where Pdh variants are expressed and assayed with a colorimetric method against NCD and NAD. Variants with improved activity in the presence of NCD are purified for kinetics assays, and used as parents for next round of evolution if NCD preference is unsatisfactory.
To create NCD-preferred enzyme variants, we conceived that the pocket interacting with the adenine moiety of NAD should be reduced, because the cytosine moiety of NCD is less bulky than the adenine moiety (Scheme 1). Albeit there have been numerous examples shifting cofactor preference between NAD(H) and NADP(H), and some general rules have been identified,9 little is known to reshape the NADbinding pocket for a smaller-sized NAD analogue such as NCD. We applied an iterative saturation mutagenesis (ISM) strategy10 to evolve Pdh_I151R, a Pdh mutant with moderate NCD preference obtained in our previous study.5e The evolution efforts adapted a framework including semi-
rational library construction and high-throughput screening (Figure 1). We first generated a modeled structure of Pdh_I151R using the structure 4E5N11 as the template by the SWISSMODEL homology modeling server12 and identified 9 residues, namely C174, D175, P176, I177, M207, V208, P209, T214 and L217, within 4 Å distance to the adenine moiety of NAD in the complex (Figure S1). We constructed saturated mutagenesis library individually, performed activity assay against NAD and NCD (Figure S2), and identified three mutants, Pdh_I151R/P176E, Pdh_I151R/P176R and Pdh_I151R/M207A, showing significantly reduced activity with NAD but good activity with NCD. Kinetic data revealed that these mutants gained improved NCD preference as indicated by higher (kcat/Km)NCD/(kcat/Km)NAD values (Table 1, Table S1, Figure S3). These results suggested that P176 and M207 are hotspot residues. Interestingly, the improvement was largely contributed by reduced affinity to NAD as over 4fold higher Km,NAD values were observed in comparison with that of Pdh_I151R. Moreover, the fact that Pdh_I151R/M207A showed much higher Km,NAD and Km,NCD values than Pdh_I151R suggested that the M207A mutation had a common role impairing cofactor binding.
Figure 2. Overall structure of Pdh complexed with NCD. (a) The overview structure of Pdh complexed with NCD. NCD is shown as green sticks. The cofactor binding domain and the substrate binding domain are shown in grey and blue, respectively. (b) Close-up view of the NCD cytosine binding pocket. The loops composed the cytosine binding pocket are highlighted in cyan, magentas and green. Loop X, Y and Z represent the residues 206−214, 151−155 and 173−178, respectively. β-Strands and α-helices of the structure are termed in order.
We next took the plasmids encoding Pdh_I151R/P176E and Pdh_I151R/M207A as templates to generate saturated mutagenesis libraries at position 207 and 176, and upon activity screening, Pdh_I151R/P176E/M207A and Pdh_I151R/P176R/M207A, respectively, were identified from the two libraries. Both triple mutants showed high activity with NCD (Figure S4). Kinetic data of purified proteins indicated that the triple mutants gained further improvement in terms of NCD preference compared with their corresponding double mutants (Table 1, Table S1, Figure S3). Pdh_I151R/P176R/M207A showed the highest NCD preference and more than 770- and 14-fold increment in (kcat/Km)NCD/(kcat/Km)NAD value over that of WT and Pdh_I151R, respectively. As noted, the Km,NAD values of Pdh_I151R/P176R/M207A and Pdh_I151R/P176E/M207A were 4.7±1.2 mM and 11.7±2.3 mM, respectively, which were much higher than cellular NAD concentrations in
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ACS Catalysis microbial cells grown under normal conditions.13 Therefore, these two mutants may be barely active in vivo due to incompetent affinity for NAD. To gain further insights into cofactor binding, we collected three sets of X-ray crystal structures of Pdh variants and their NCD-complexes (Table S2). The architecture of Pdh is similar to that of phosphite dehydrogenase from Pseudomonas stutzeri WM88 (Figure S5a).11 The core structure of Pdh is clearly divided into a NAD binding domain and a substrate binding domain (Figure 2a). In the Pdh-NCD complex, the cytosine ring of NCD is surrounded at the pocket consisting of loop X, loop Y and loop Z (Figure 2b). Loop X is recognized as the lid
interacting with NCD, and the distance between P176 of loop Z and T214 of loop X is 8.7 Å (Figure 3a). Superposing the structure of Pdh and that of Pdh-NCD complex showed minute conformational changes with an RMSD of 0.248 Å, indicating a straight-forward binding of NCD by the enzyme (Figure S5b). Thus, major molecular interactions involved in NCD binding include hydrogen bonding with G154, A155, V156, D175, and M207 (Figure 3g). Noting that G154, A155, V156 and D175 located within loop Y forms a typical Rossmann fold (the conserved GXGX2GX17D/E sequence motif) for cofactor binding.14 The cytosine ring makes van der Waals contacts with P176 and V208.
Table 1. Kinetic parameters of Pdh and variants NAD Enzyme
NCD
NCD preference
kcat (s-1)
Km (μM)
kcat/Km (mM-1 s-1)
kcat (s-1)
Km (μM)
kcat/Km (mM-1 s-1)
WT
0.24±0.01
1.1±0.1
218
0.28±0.02
22.2±3.9
12.6
I151R
0.18±0.01
78.8±6.5
2.3
0.11±0.02
15.4±2.5
7.2
3.1
I151R/P176R
0.48±0.02
325.4±14.1
1.46
0.35±0.01
12.1±1.3
28.9
19.8
I151R/P176E
0.31±0.01
684.6±35.5
0.45
0.06±0.00
7.9±0.3
6.96
16.8
I151R/P176R/M207A
0.21±0.07
4.7×103±1.2×103
0.045
0.20±0.02
99.1±14.8
2.04
44.9
0.40±0.05
11.7×103±2.3×103
0.034
0.35±0.02
281.5±54.7
1.24
36.5
I151R/P176E/M207A
0.058
Figure 3. Structural information for NCD binding with Pdh variants. (a), (b) (c) and (d) The closed-up view of the NCD cytosine binding pocket of Pdh, Pdh_I151R, Pdh_I151R/P176E and Pdh_I151R/P176R/M207A, respectively. The residues at 151, 176, 213 and 214 are noted in cyan. The distances between residues at 176 and 214 were shown with a red dotted line. (e) Superposition structures of the complexes of NCD with Pdh (green) and Pdh_I151R/P176E (blue). (f) Superposition structures of the complexes of NCD with Pdh (green) and Pdh_I151R/P176R/M207A (cyan). Residues at 151, 176, 213 and 214 determines the capacity of the cofactor binding pocket. (g), (h) and (i) Overall molecular interactions of NCD within Pdh, Pdh_I151R/P176E and Pdh_I151R/P176R/M207A, respectively.
The structure of Pdh_I151R/P176E also comprises of two molecules in an asymmetric unit, while only one molecule binds NCD (Figure S5c). Comparing with Pdh and Pdh_I151R, Pdh_I151R/P176E apparently presents a more restricted cofactor-binding pocket (Figure 3a-c) and favorable molecular interactions with the cytosine moiety of NCD (Figure 3g, h). Specifically, in Pdh_I151R/P176E, the
side chain of E213 is reoriented away from the cytosine ring (127°) while the side chain of T214 moves inward towards the cytosine ring (Figure 3e). As a result, the distance between the residues situated at 151 and 214 is shortened from 6.0 Å to 5.3 Å (Figure 3c). Additionally, the side chain of R151 prevents the cytosine from flipping. These structural changes induce a favorable environment to
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accommodate a cytosine moiety instead of adenine. Pdh_I151R/P176E uses E176 and R151 to form hydrogenbonds with the cytosine moiety of NCD (Figure 3h), while it shows other electrostatic interactions with NCD similar to those of WT. The structure of Pdh_I151R/P176R/M207A contains four copies of structurally equivalent monomer presented as a closed conformation in an asymmetric unit, however, all four monomers appear as open conformation when complexed with NCD. It seems that the M207A mutation reduces hydrophobic interactions between V156 and I160, leading to a weaker interaction between loop X and α7 of the enzyme. Consequently, loop X flips to the outside of the enzyme and encloses the NCD binding cleft (Figure S5d). Similar to those in Pdh_I151R/P176E, T214 is reoriented and the distance between R151 and T214 is reduced to 4.3 Å (Figure 3d, f). In addition, the side chain of R176 moves inwards, resulting in a further narrowed cleft (Figure 3d) and weaker interaction with the cytosine ring of NCD (Figure 3i). Compared with Pdh and Pdh_I151R/P176E, Pdh_I151R/P176R/M207A lacks hydrogen-bonding interactions between M207 and the hydroxyls of the nicotinamide ribose moiety, which may be a major reason for considerable affinity loss to both NAD and NCD. These structural data reveal that the cofactor-binding pocket of Pdh became more crowded upon introduction of large and polar residues such as Arg or Glu. Noting that these mutations also led to reorienting skeletal amino acid residues inward to narrow the binding cleft. On the other hand, these charged side chains per se provided additional interactions with NCD. Together, these structural changes created a compacted cofactor-binding pocket and molecular interactions beneficial to NCD residence. To further test our speculation that a charged residue at P176 can confer a smaller pocket disfavoring NAD, we used site-directed mutagenesis to replace Pro with Lys and generate two variants, Pdh_I151R/P176K and Pdh_I151R/P176K/M207A. Kinetic analysis of these mutants showed that the corresponding Km,NAD and Km,NCD values are indeed comparable to those of their counterparts where Pro was substituted with either Glu or Arg (Table S1). These data provided additional evidences that mutations can be devised to form a compacted pocket for reduced NAD affinity and improved NCD preference. To examine whether these Pdh variants had substantial differences in terms of phosphite binding, we determined the kinetic constants at saturating concentrations of NAD or NCD (Table S3). Whereas the Km,phosphite value of Pdh was 60.3±6.6 μM in the presence of NCD, Km,phosphite values of other variants were within 3-fold of that of Pdh. Similarly, these Pdh variants had Km,phosphite values within 5-fold of that of Pdh in the presence of NAD. Structural analysis revealed that mutations in the adenine binding pocket of Pdh led to little change in the phosphite-binding pocket (Figure S6). According to previous results,11,15 M53, L75, R237, H293 and R302 in Pdh are active residues that likely involved in positioning phosphite. Compared with Pdh, these residues in Pdh_I151R/P176E and Pdh_I151R/P176R/M207A had modest movement (Figure S6a and b). Also, the NCD molecule was stretched, and the distance between cytosine moiety and nicotinamide was about 18 Å in these co-crystal structures. Such a long distance might provide advantages
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to tune cofactor structure at the site distal to the actual reaction site, i.e., phosphite binding site. Together, our results indicated that these NCD-favoring Pdh variants retained similar substrate binding ability to that of the wild type enzyme. In fact, our previous data also showed that the engineered non-natural cofactor-dependent enzymes retained tight stereoselectivity for both substrate and product,5d further suggesting that these NCD-favoring enzymes may have limited compromise in terms of substrate binding. Furthermore, we did amino acid sequence comparison of Pdh and other NAD-dependent oxidoreductases (Figure S7). It was found that these proteins have a conserved NAD binding domain and highly parallel three-dimensional structures (Figure S8). To generate variants favoring a smaller-sized cofactor, residues located at the loop X, Y and Z may be selected for mutation to introduce amino acids with large side chains. Interestingly, when L310 of ME located at loop Y, the counterpart of I151 in Pdh, was changed to Arg or Lys, the corresponding ME mutants showed significantly reduced affinity to NAD but high affinity to NCD and NFCD,5d which was in perfect agreement with the observations described here. In summary, we revealed structural rules for engineering NAD-dependent enzymes to favor NCD, a smaller-sized redox cofactor. By incorporating amino acid residues with large side chains around the pocket interacting with the adenine moiety of NAD, the engineered enzyme embraces a compacted pocket and molecular interactions favoring NCD over NAD. These mutants are valuable components to establish bioorthogonal redox systems16. We believe that such a paradigmatic study should promote further efforts to engineer other enzymes to favor non-natural redox cofactors, and eventually bring them into life by chemical and synthetic biologists.
ASSOCIATED CONTENT Supporting Information This information is available free of charge on the ACS Publications website. Experimental details for general materials, site-directed mutagenesis and library construction, high-throughput screening, protein expression and purification, enzyme activity and kinetics assays, protein crystallization, data collection and structure determination, sequence alignment, Table S1-S5, Figure S1-S9, and supporting references.
AUTHOR INFORMATION Corresponding Author *
[email protected] *
[email protected] Author Contributions ‖These
authors contributed equally.
Funding Sources This work was supported by National Natural Science Foundation of China (No. 21572227, 21778053, 21721004)
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ACS Catalysis and Dalian Institute of Chemical Physics, DICP&QIBEBT UN201706, DICP ZZBS201605).
CAS
(No.
Notes The authors declare no conflict of interest.
ACKNOWLEDGMENT Dedicated to the 70th anniversary of Dalian Institute of Chemical Physics, CAS.
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