Structural and Kinetic Properties of the Aldehyde Dehydrogenase

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Structural and kinetic properties of the aldehyde dehydrogenase NahF, a broad substrate specificity enzyme for aldehyde oxidation Juliana Barbosa Coitinho, Mozart Silvio Pereira, Debora Maria Abrantes Costa, Samuel Leite Guimaraes, Simara S. de Araujo, Alvan C. Hengge, Tiago A. S. Brandao, and Ronaldo Alves Pinto Nagem Biochemistry, Just Accepted Manuscript • DOI: 10.1021/acs.biochem.6b00614 • Publication Date (Web): 31 Aug 2016 Downloaded from http://pubs.acs.org on September 5, 2016

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Structural and kinetic properties of the aldehyde dehydrogenase NahF, a broad substrate specificity enzyme for aldehyde oxidation Juliana B. Coitinho,a Mozart S. Pereira,b Débora M. A. Costa,a Samuel L. Guimarães,a Simara S. de Araújo,a Alvan C. Hengge,c Tiago A. S. Brandãob,* and Ronaldo A. P. Nagema,* a

Departamento de Bioquímica e Imunologia, Instituto de Ciências Biológicas, Universidade

Federal de Minas Gerais, Belo Horizonte, MG 31270-901, Brazil. b

Departamento de Química, Instituto de Ciências Exatas, Universidade Federal de Minas Gerais,

Belo Horizonte, MG 31270-901, Brazil. c

Department of Chemistry and Biochemistry, Utah State University, Logan, UT 84322-0300,

USA. Corresponding Author * Tiago A. S. Brandão: e-mail: [email protected]; Phone: +55 (31) 3409-5766; Fax: +55 (31) 34095700. *Ronaldo A. P. Nagem: e-mail: [email protected]; Phone: +55 (31) 3409-2626; Fax: +55 (31) 3409-2614

ACS Paragon Plus Environment

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ABBREVIATIONS ALDH, aldehyde dehydrogenase; NahF, salicylaldehyde dehydrogenase; 6xHis-NahF, recombinant NahF with 6xHis N-terminal tag; SA, salicylaldehyde.


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ABSTRACT The salicylaldehyde dehydrogenase (NahF) catalyzes the oxidation of salicylaldehyde to salicylate using NAD+ as a cofactor, the last reaction of the upper degradation pathway of naphthalene in Pseudomonas putida G7. The naphthalene is an abundant and toxic compound in oil and has been used as a model for bioremediation studies. The steady-state kinetic parameters for oxidation of aliphatic or aromatic aldehydes catalyzed by 6xHis-NahF are presented. The 6xHis-NahF catalyzes the oxidation of aromatic aldehydes with large kcat/Km values close to 106 M-1s-1. The active site of NahF is highly hydrophobic and the enzyme shows higher specificity for less polar substrates compared to polar substrates, e.g., acetaldehyde. The enzyme shows α/β folding with three well defined domains: the oligomerization domain, which is responsible for the interlacement between the two monomers; the Rossmann-like fold domain, essential for nucleotide binding; and the catalytic domain. A salicylaldehyde molecule was observed in a deep pocket in the crystal structure of NahF where the catalytic C284 and E250 are present. Moreover, the residues G150, R157, W96, F99, F274, F279 and Y446 were suggested to be important for catalysis and specificity for aromatic aldehydes. Understanding the molecular features responsible for NahF activity allow for comparisons with other aldehyde dehydrogenases (ALDHs), and, together with structural information, provide the information needed for future mutational studies aimed to enhance its stability and specificity, and further its use in biotechnological processes. KEYWORDS Pseudomonas putida G7; naphthalene degradation; salicylaldehyde dehydrogenase; NahF; crystal structure; kinetic; mechanism.


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INTRODUCTION NahF (GenBank: BAE92159.1, 483 amino acid residues and 52,190 kDa) is a salicylaldehyde dehydrogenase from Pseudomonas putida G7 that belongs to the NAD(P)+-dependent aldehyde dehydrogenase (ALDH) superfamily (1). Members of this superfamily catalyze the oxidation of a wide variety of endogenous and exogenous aliphatic and aromatic aldehydes to their corresponding carboxylic acids using NAD+ or NADP+ as a cofactor. They share a similar scaffold with three domains - a NAD(P)+ cofactor-binding domain, a catalytic domain, and a bridging domain - and a number of highly conserved residues necessary for catalysis and cofactor binding, despite their overall low sequence identity (as low as 30%), different modes of oligomerization and substrate specificity (2, 3). Indeed, slight differences in the substrate pockets of ALDH enzymes were suggested to be responsible for their diverse kinetic properties within a variety of aldehyde substrates (4). The catalytic mechanism is proposed to involve cofactor binding, resulting in conformational change and activation of an invariant catalytic cysteine nucleophile. The cysteine and aldehyde substrate form an oxyanion thiohemiacetal intermediate, which undergoes hydride transfer to NAD(P)+ yielding a thioacylenzyme intermediate and NAD(P)H. A highly conserved glutamate residue activates, by general-base catalysis, a nucleophilic water molecule in the deacylation step affording the carboxylic acid product and the free nucleophilic cysteine residue (3-6). NahF catalyzes the oxidation of salicylaldehyde to salicylate using NAD+ as a cofactor, the last reaction of the upper degradation pathway of naphthalene, a typical polycyclic aromatic hydrocarbon (PAH) compound. The upper pathway is composed of enzymes (NahAaAbAcAdBFCED) involved in the conversion of naphthalene to salicylate, while enzymes


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(NahGTHINLOMKJ) in the lower pathway catalyze the conversion of salicylate to pyruvate and acetaldehyde (7, 8). In aerobic degradation of aromatic compounds, peripheral enzymes (upper pathway) catalyze the transformation of structurally diverse pollutants into a few key intermediates, which are further channeled via a few central (or lower) pathways to the central cellular metabolism. While the lower pathway enzymes from various bacteria display significant functional similarity, the peripheral enzymes, which recognize and convert different aromatic pollutants into several central metabolites, play more significant roles in degrading a variety of xenobiotics (9). We focus on Pseudomonas putida G7 for its ability to grow on naphthalene as a sole carbon source. In particular, we are interested in learning to what degree, as a result of these unusually demanding growth conditions, enzymes in the PAH degradation pathway of Pseudomonas putida G7 differ from their previously characterized counterparts in other organisms. There is considerable evidence for the ability of enzymes to evolve more efficient, or even new activities, in a relatively short period. One example is the evolution of phosphotriesterases from lactonases in response to the human introduction of phosphate triesters into the environment in the mid-20th century (10). Previous characterizations of aldehyde dehydrogenases permit us to assess whether the NahF from Pseudomonas putida G7 differs in structural and kinetic characteristics, or in its tolerance of PAH structural variations, from its counterparts in other organisms. Therefore, we report the crystallographic structure of the recombinant NahF from Pseudomonas putida G7, an aldehyde dehydrogenase that particularly oxidizes aromatic aldehydes to their corresponding carboxylic acids. An extensive kinetic characterization of the enzyme is also reported, with kinetic parameters for a wide range of substrates including both


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aromatic and non-aromatic aldehydes. Taken altogether, the structural and kinetic data give evidence for NahF reaction mechanism and substrate specificity and permit comparisons with analogous enzymes from other organisms. These findings are particularly relevant to the prospects for enzyme engineering to develop enzyme-assisted PAH degradation processes. EXPERIMENTAL PROCEDURES Cloning, expression and purification. The nahf gene from Pseudomonas putida G7 was cloned into pET28a(TEV) vector (11) and the recombinant protein was expressed and purified to homogeneity as previously reported (12). Briefly, Escherichia coli BL21 Arctic Express (DE3) cells transformed with the recombinant plasmid pET28a(TEV)-nahf were induced with IPTG (isopropyl β-D-1-thiogalactopyranoside) and the expressed 6xHis-NahF protein (recombinant NahF with 6xHis N-terminal tag) was purified by affinity and gel filtration chromatographies. Several attempts to cleave the 6xHis tag from the recombinant protein were unsuccessful and subsequent experiments were carried out with 6xHis-NahF. Effect of temperature on enzyme activity and stability. The optimal temperature of the reaction catalyzed by 6xHis-NahF was evaluated by using salicylaldehyde (SA) as substrate and the aldehyde dehydrogenase activity was monitored spectrophotometrically following NADH regeneration as an increase in the absorbance at 340 nm. Each assay mixture (total volume 1 mL) containing 200 µM salicylaldehyde, 200 µM NAD+ and 1 to 2 µM 6xHis-NahF in 100 mM sodium phosphate buffer pH 8.5 was assayed over a temperature range of 20 to 70 °C in 10 °C intervals. The enzyme thermostability was analyzed by incubating aliquots of enzyme at different temperatures (40, 50, 60 and 70 °C) for different periods, followed by activity assays using same conditions previously mentioned.


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Steady-state kinetics. The initial rate (v) for the enzyme catalyzed oxidation of the substrates were determined by spectrophotometry at 340 nm considering that v = ∆A/a.b.∆t, where ∆A/∆t, a and b are the respective absorbance variation over time for each reaction, the reaction molar absorptivity taken into consideration the individual contributions (ε) of the substrate (S) and products (P), and cuvette pathlength. The reactions were performed in quartz cuvettes of 1 cm pathlength with the temperature kept at 25.0 ºC by the use of a circulating water bath. The reaction mixture consisted of 50 mM Hepes pH 8.5, 1 mM EDTA, and the ionic strength was kept at 200 mM by addition of NaCl. The substrates were dissolved in acetonitrile before adding to the reaction mixture and the final acetonitrile concentration was 0.5% (v/v) in water. The reactions were initiated by addition of 100 µL of enzyme solution to a final reaction volume of 2 mL using an add-mixer device. The reaction molar absorptivity a is presented as ε(NADH) - ε(S) + ε(P), where ε(NADH) was 6220 M-1 cm-1. For those substrates that absorb appreciably at 340 nm, their molar coefficients (ε) were determined under the same reaction conditions and are presented in the Supporting Information (Table S1). No absorption at 340 nm was considered for reaction products, except for the oxidized form of pyrene-1-carboxaldehyde for which the same molar coefficient of the substrate (S) was considered and the reaction molar absorptivity was 6220 M-1 cm-1. The steady state kinetic parameters were determined by non-linear regression of the initial rate (v) versus [S] or [NAD+] data to the following equations:

v=

Vmax [S] K m + [S]

(1)

v=

Vmax [S] K m + [S] + [S] 2 / K i

(2)


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where Vmax is the maximum velocity, Km is the Michaelis constant, and Ki is the substrate inhibition constant. The kcat value was calculated from kcat = Vmax/[E]o, where [E]o is the total enzyme concentration. pH-rate profile. The effect of pH on the activity of 6xHis-NahF was measured at 25.0 ºC using 200 µM NAD+ and increasing concentrations of salicylaldehyde (SA) from zero to 50 µM. As described above, the extent of reaction was obtained considering both molar coefficients of the substrate (S) and NADH (ε(NADH) = 6220 M-1 cm-1). The molar coefficient for SA varies depending on its protonation state and was measured at different pH values at 25.0 ºC (Supporting Information, Table S2 and Figure S1). The reaction mixture was similar to that described above, except that the following buffers were used: Bis-Tris (pH 5.3-7.3), Hepes (pH 6.7-8.5), Bicine (pH 7.4-9.4), Ches (pH 8.6-9.9) and Caps (pH 9.7-10.3). The steady state kinetic parameters were determined by a fit of the initial rate (v) versus [SA] data to the MichaelisMenten equation. X-ray structure determination and functional analysis. The recombinant protein 6xHis-NahF was crystallized and diffraction data were collected as previously reported (12). The structure was solved using the Molecular Replacement method as implemented in Phaser (13) and a single monomer of the sheep liver cytosolic aldehyde dehydrogenase crystallographic structure (PDB entry 1BXS (14)) was used as the search template. These two enzymes share 37% sequence identity. The 6xHis-NahF model was built using the programs Buccaneer (15) and Coot (16). Restrained refinement of the model was performed using the program REFMAC (17) available in the CCP4 package (18). Final refinement cycles and model adjustments were carried out within the Phenix software suite (19) which allowed better inspection and correction of model parameters


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such as bond lengths, bond angles among others. The refined 6xHis-NahF structure was deposited into the Protein Data Bank (PDB entry 4JZ6). To identify structurally similar proteins to NahF, a 3D search using the DALI-server (http://ekhidna.biocenter.helsinki.fi/dali_server/) (20) was performed. The primary structures of selected ALDHs were aligned and their crystallographic structures (whenever available) were superimposed allowing a comparison of substrate and cofactor binding sites. RESULTS AND DISCUSSION Optimal temperature and thermostability. The enzyme activity for 1 to 2 µM 6xHis-NahF was determined at temperatures between 20 and 70 °C in the presence of 200 µM salicylaldehyde and 200 µM NAD+ in 100 mM sodium phosphate buffer at pH 8.5. As shown in Figure 1a, the enzyme exhibits maximum activity at 60 °C with a sharp decrease at higher temperatures. However, pre-incubation of the enzyme at different temperatures (40, 50 and 60 °C) reveal that the thermostability of the enzyme strongly decreases over time. As observed in Figure 1b, the recombinant NahF retained nearly 60% of its residual activity when incubated at 40 °C for 4 h, but showed marginal activity at 50 °C after 2 h and was fully inactive after 1 h of incubation at 60 °C. Steady-state kinetics and substrate recognition. The steady-state kinetic parameters for oxidation of aliphatic or aromatic aldehydes (RCOH) catalyzed by 6xHis-NahF in the presence of 200 µM NAD+ are presented in Table 1. The Km values range from 0.55 to 5 µM for most aromatic aldehydes, up to 30 µM for 2-formylbenzaldehyde, and from 1.5 µM to 9.6 mM for aliphatic aldehydes with long (C10) to short (C2) carbon chains. These values are uncorrected for the equilibria for aldehyde hydration to the neutral gem-diol and its deprotonation to the


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corresponding anionic species (Scheme 1). A mathematical procedure for correction of Km according to the free aldehyde in solution is presented in the Supporting Information (Scheme S1) along with representative literature data revealing that correction does not substantially change the magnitude of the Km values in Table 1. For example, the highest hydration effect among the series of aldehydes in Table 1 is observed for acetaldehyde, for which the amount of free aldehyde is 52 mol % in water results in an uncorrected Km about twice as high as the actual Km. Most of the aromatic aldehydes in Table 1 are found nearly entirely in their unhydrated form, as for benzaldehyde (over 99 mol % RCOH) at 25 ºC (21). The deprotonation of the neutral gem-diol also has a very small effect on the concentration of the free aldehyde at pH 8.5, which occurs significantly only in very basic conditions. The pKa for the gem-diol of acetaldehyde and 4-nitrobenzaldehyde are 13.6 and 12.1, respectively (22, 23). The kcatap values reported in Table 1 are apparent constants determined in the presence of 200 µM NAD+, which does not represent saturation conditions for the cofactor. The Km value for NAD+ determined in the presence of 70 µM of salicylaldehyde, about 28-fold above its Km value, was 285±6 µM at pH 8.5 (Supporting Information, Figure S2). The kcat value determined under saturation conditions was 32.0±0.2 s-1, which is only 2.5-fold above the kcatap determined in the presence of 200 µM NAD+ and increasing concentrations of salicylaldehyde. In this case, the Km value for salicylaldehyde was 2.48 µM, significantly lower than observed for the His-tagged NahF from P. putida ND6, with a Km value of 145 µM (24). The 6xHis-NahF activity (36.7 µmol min-1 mg-1) is within the range of 1.3 to 85.4 µmol min-1 mg-1 for activities of wild-type NahF’s in P. putida strains G7 (25), ND6 (26), and NCIB 9816 (27). The kcatap/Km and kcatap values in Table 1 are shown in graphical form in Figure 2 to better visualize the catalytic trends. The recombinant NahF catalyzes the oxidation of a broad spectrum


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of aromatic aldehydes with large kcatap/Km values close to 106 M-1 s-1 (Supporting Information, Table S3 and Figure S3). Linear saturated aliphatic aldehydes with short chains exhibit higher Km values and lower kcatap/Km values compared to aromatic substrates. These values show a monotonic change with increase of the aliphatic chain; decanaldehyde has Km and kcatap/Km values comparable to aromatic aldehydes. The Km and kcatap/Km values for ramified aliphatic aldehydes, viz. isovaleraldehyde and citronellal, are comparable to linear saturated aliphatic aldehydes with the same number of carbons in the main chain. Comparisons of kcatap values for different substrates reveal interesting patterns, which are grouped in Figure 2b to illustrate comparisons. The kcatap values for hydroxyl substituted benzaldehydes follow the order 2- > 3- > 4-, and the kcatap value for salicylaldehyde (2hydroxybenzaldehyde) is about 7-fold higher than observed for the unsubstituted benzaldehyde, which shows the importance of the presence and specific position of the hydroxyl group on catalysis. The 5-bromo and 5-methyl salicylaldehydes exhibited similar kcatap values, although 5nitro salicylaldehyde (results not shown) did not seem to be a substrate for this enzyme. The kcatap values for 2-substituted benzaldehydes and 2-pyridinecarboxyaldehyde are similar to salicilaldehyde, but about 6-fold higher in relation to benzaldehyde. The turnover numbers are also not considerably different between salicilaldehyde and the polyaromatic substrates 2naphthaldehyde and pyrene-1-carboxaldehyde. On the other hand, the kcatap values for the oxidation of 3- and 4-substituted benzaldehydes are about 20-fold lower than observed for salicylaldehyde. The kcatap values for aliphatic aldehydes were all lower than observed for the aromatic ones. From hexaldehyde to decanaldehyde the kcat values are quite similar to each other and about 17-


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fold lower than salicylaldehyde. However, the turnover numbers for short-chain aldehydes like acetaldehyde and propionaldehyde are near 45-fold below the observed for salicylaldehyde. Overall, 6xHis-NahF exhibits quite broad substrate specificity for aromatic and long-chain aliphatic aldehydes (Figure 2a), with lower kcatap/Km values for more polar substrates with short alkyl chains, e.g., acetaldehyde. The substrate specificity is not even impaired for polyaromatic substrates as 2-naphthaldehyde and pyrene-1-carboxaldehyde, which are oxidized as fast as salicilaldehyde within the limits of the experimental error in kcatap/Km. Similar observation has been reported for native salicylaldehyde dehydrogenase (SALDH) from carbaryl-degrading Pseudomonas sp. strain C6, which was found to have broad substrate specificity accepting mono- and di-aromatic aldehydes but poor activity on aliphatic aldehydes (28)

. Although SALDH amino acid sequence is partially known, it shares 67% sequence identity

with NahF, which therefore suggests that structural information obtained for NahF can also shed light on the kinetic properties of this SALDH. pH-rate profile. Figure 3 shows the effect of pH on kcatap/Km and kcatap for the oxidation of salicylaldehyde catalyzed by 6xHis-NahF in the presence of 200 µM NAD+. The pH-rate profiles are bell-shaped in both plots with maxima close to pH 8.5 for kcatap/Km and 9.0 for kcatap. The slopes for kcatap/Km are +1 and -2, and about +0.5 and -1 on the acidic and basic limbs for kcatap, respectively. The kinetic data on kcatap/Km and kcatap versus pH were fitted to the logarithmic versions of Equations 3 and 4 using the equilibrium and kinetic parameters in Table 2. The reaction pathway in agreement to these equations is presented in Scheme 2.


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ap

k cat

ap

k cat

ap

(k cat / K m ) max /Km = S E3  K  + K  1 + a  [H ] + 1 + a   [H + ]  K E2 [H + ]    a =

ki ES2 ES2 ES3  [H + ] Ka Ka Ka     K ES1 + 1 + [H + ] + [H + ]2   a 

(3)

+

k max ES3  [H + ]2 Ka  [H + ]    K ES1 K ES2 + K ES2 + 1 + [H + ]  a a  a 

(4)

The observed kcatap/Km in Equation 3 is given by a “maximum” pH-independent term reliant on the protonated substrate with pKaS and two-pKa of the enzyme. The observed kcatap is dependent on a three-pKa system for the enzyme-substrate complex. The ki and kmax represent the pH-independent rate constants for two different enzyme forms. The additive contributions of these forms to the observed rate are given by the first and second term of Equation 4. The first term refers to the “intermediate” form with a deprotonated group of pKaES1 for its conjugate acid and a protonated group with pKaES2. The second term denotes the “maximum” with both groups of the enzyme deprotonated. The acidic form ESAH3–NAD+ is assumed to be catalytically inactive as well as the basic form ESA–NAD+ (Scheme 2).

Overall 6xHis-NahF crystal structure. The 6xHis-NahF was crystallized as previously reported (12) and its crystal structure has been determined and refined at 2.4 Å resolution to a final R-factor of 0.21 (R-free of 0.25). Detailed refinement statistics are shown in Table 3. The protein model comprises all amino acid residues of the native protein (483 residues; NCBI reference sequence YP_534825.1) plus one amino-terminal histidine residue from the expression system. A single enzyme molecule was found in the asymmetric unit of the crystal but a crystallographic two-fold axis is responsible for generating the dimeric biological unit of the enzyme (12). The structure revealed that the enzyme shows a typical α/β aldehyde dehydrogenase


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superfamily fold organized into three domains as shown in Figure 4: the nucleotide-binding, the catalytic and the oligomerization domains. The oligomerization domain formed by three antiparallel β-strands (β6, β7 and β21) is responsible for the interlacement between monomers generating the dimeric biological unit of 6xHis-NahF. Dimer formation involves contacts between α-helices α9 (residues 229 - 243) of both subunits and residues in strands β19 (residues 430 – 433) and β21 (residues 468 – 474) of each subunit. These secondary structure elements form a ten-stranded β-sheet extending through the catalytic and the oligomerization domains. The nucleotide-binding domain is comprised by a Rossmann-like fold with five parallel βstrands (β8 to β12), linked to four α-helices (α6 to α9), two in each side of the β-sheet. No NAD+ cofactor molecule was observed into the nucleotide-binding site but the conformation adopted by the potential residues involved in this interaction (E175, F381, K172, among others) is quite similar to the residues observed in other ALDHs/NAD+ complex structures. Besides, as calculated by the CASTp server (http://sts-fw.bioengr.uic.edu/castp/calculation.php) (29), these residues create a pocket in the dimeric 6xHis-NahF structure for NAD+ binding with 1,469 Å3.

Substrate binding pocket. The substrate binding site is located on the face opposite from where coenzyme binds consisting in a deep funnel formed by an interface between the nucleotide-binding and the catalytic domains. The crystallographic structure of 6xHis-NahF reported here shows the substrate salicylaldehyde inserted into this hydrophobic funnel and surrounded by several hydrophobic residues such as W96, F99, A103, V107, V153, L154, F274, L278, F279, I283, V438 and Y446 (Figure 5a). The volume of this pocket is around 737 Å3 in the absence of the substrate, which is substantially decreased after salicylaldehyde binding.


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Three aromatic residues (W96, F99 and F279) stabilized by π-stacking interactions are observed at the entrance of NahF substrate binding site, which seems to be important for maintaining the catalytic pocket opened. Also, a phenylalanine residue (F274) is observed adjacent to the entrance of the substrate binding pocket, which may play a role in the recognition of aromatic aldehyde substrates together with W96, F99 and F279 (Figure 5a). A set of three or four active-site aromatic residues in other ALDHs has been identified forming a similar arrangement called an “aromatic box”, which seems to promote the recognition and interaction with a variety of aldehyde substrates (30). However, a variation of this arrangement is observed in NahF, wherein only the Y446 is in an equivalent position as other bulky residues found in the aromatic box, in most cases a phenylalanine or a tryptophan. The position of L278 in NahF is commonly occupied by a phenylalanine residue in ALDHs displaying the aromatic box. However, L278 stands between phenylalanine residues F274 and F279, the latter adopting π-stacking interactions with W96 and F99. Curiously, a tryptophan residue often found in the aromatic box of some ALDHs is replaced by an arginine residue (R157) in NahF (Figure 5b); as described in the next section, this residue seems to have a catalytic role. On the other hand, a mutation in the equivalent tryptophan residue of a γ-hydroxymuconic semialdehyde dehydrogenase from Pseudomonas sp. modestly affected enzymatic activity (31), suggesting a different role for these equivalent residues in particularly different ALDHs. Notably, the substrate pocket of NahF is ~12 Å long, suitable for accommodation of relatively elongated substrates, such as decanaldehyde. Taken together, the dimension of this funnel and its hydrophobic environment allow a broad substrate spectrum of NahF, with a clear preference for aromatic and long-chain aliphatic aldehydes.


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Active-site residues and mechanism of catalysis. The formyl group of salicylaldehyde is positioned near the invariant C284 residue (~3.8 Å away from the C284 side chain), and the formyl oxygen atom is hydrogen bonded to the backbone amide N-H atom of C284 (Figure 5b). In addition, the hydroxyl group is stabilized by hydrogen bond with the nitrogen atom of the G150 main chain. Notably, a water molecule is within hydrogen bonding distance of the R157 side chain and is 4.3 Å away from the catalytic E250, which may act as a general base for the hydrolysis of the thioacylenzyme (Figure 5b). The latter observation strongly suggests that R157 is involved in positioning the water molecule required for catalysis. Mutagenesis studies for this residue were made and preliminary kinetic results, to be published elsewhere, indicate a decrease of kcatap/Km up to 280-fold. Analogously, in the succinic semialdehyde dehydrogenase from cyanobacterium Synechococcus, the alanine mutation of residue R139 (equivalent to R157 in NahF) is reported to significantly reduce the catalytic activity by 90% (32). The proposed reaction mechanism of NahF presented in Scheme 3 is in agreement with the kinetic and structural analyses above. The deprotonated sulfhydryl group of C284 (pKaES1 = 6.10 for the conjugate acid) attacks the formyl carbonyl group of the substrate to give the thiohemiacetal intermediate. Following hydride transfer from this intermediate to the nicotinamide group of NAD+, the thioacylenzyme intermediate undergoes hydrolysis by an R157-guided water molecule, assisted by general base catalysis of the carboxylate group of E250 (pKaES2 = 7.98 for the conjugate acid) for the ESAH1-NAD+ species or directly, slower by about 35-fold, for the ESAH2-NAD+ species. This step affords the final product and the deprotonated sulfhydryl group of C284 for a next catalytic round. The pKa for the side chain of E250 is substantially higher than in solution (pKa ~ 4.1) as a result of the hydrophobic environment of


16

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this residue. Similarly, Marchal et al. (33) determined a pKa of 7.6 for the corresponding glutamate residue in the nonphosphorylating glyceraldehyde 3-phosphate dehydrogenase (GAPN) from Streptococcus mutans, which Cobessi et al. (34) observe by x-ray studies to reside into a conserved hydrophobic environment responsible for increasing its pKa. The identity of the residue responsible for pKaES3 was not determined and mutagenesis studies of active site residues are underway to clarify this point. The rate-limiting step for kcatap seems not to depend on the diffusion of the cofactor, NADH, from its binding site. Among the structurally different substrates the kcat varies over two orders of magnitude, which implies that the rate limiting step is the hydride transfer from the thiohemiacetal enzyme to NAD+ or the hydrolysis of the thioester to give the products. The close proximity of G150 with the substrate salicylaldehyde may indicate an important role of this amino acid in binding. The conformation adopted by the region between F277 and T287 places G150 at a distance of approximately 3.0 Å from the substrate. As observed, the oxygen atom of the 2’-hydroxyl group of salicylaldehyde forms a hydrogen bond with the backbone nitrogen of G150 (Figure 5b). Intriguingly, according to an alignment of the consensus sequences of members of the ALDH family, including the aromatic ALDHs (2), the bulky side chain of phenylalanine, tyrosine and leucine residues occupy positions corresponding to that of G150 of NahF, even among those ALDHs that share close structural similarity to NahF (Figure 6). The enzyme NahI, a 2-hydroxymuconate semialdehyde dehydrogenase belonging to the same naphthalene-degradation pathway in P. putida G7, has no activity with salicylaldehyde but is highly specific for its biological substrate, a short-chain aliphatic aldehyde (35). In this regard, our comparative sequence analysis revealed that the residues equivalent to G150 and R157 are, respectively, L156 and W163 in NahI, which suggests that the presence of bulky amino acid


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residues within the catalytic site may present significant interference in accommodating larger substrates. Taking into account the broad range specificity of NahF, it is probable that G150 permits the accommodation of structurally diverse aldehyde substrates.

CONCLUSION The specificity of the recombinant NahF depends on the substrate structure, with less polar substrates favored over polar ones, e.g., acetaldehyde. The oxidation of a variety of aromatic aldehydes with large kcatap/Km values shows a clear preference of NahF for aromatic substrates, even for polyaromatic substrates as 2-naphthaldehyde and pyrene-1-carboxaldehyde, which are oxidized as fast as salicylaldehyde. In turn, although short-chain linear saturated aliphatic aldehydes exhibit higher Km and lower kcatap/Km values compared to aromatic substrates, these values vary as the aliphatic chain increases, and decanaldehyde has Km and kcatap/Km values comparable to aromatic aldehydes. Structural reasons for these observations may lie in the fact that, unlike other ALDHs which have a well-defined “aromatic box”, a variation of this arrangement is observed in NahF, wherein the three aromatic residues W96, F99 and F279 stabilized by π-stacking interactions, together with F274, may play a role in the recognition of aromatic aldehyde substrates. Also, the dimension of the NahF substrate binding pocket and its hydrophobic environment permit catalysis of a broad, and less polar substrate spectrum. While in other ALDHs a tryptophan residue often found in the aromatic box does not seem to show a significant role in catalysis, in NahF this position is occupied by R157, which seems to be involved in positioning a water molecule required for catalysis. Moreover, taking into account the broad range specificity of NahF, it is likely that G150 allows the accommodation of structurally diverse aldehyde substrates


18

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without steric clashes. Lastly, the proposed reaction mechanism of NahF is consistent with the kinetic and structural analyses, which support the involvement of C284 and E250 in catalysis, although the identity of a residue responsible for the third pKa seen in pH-rate profiles remains to be determined. Understanding this particular mechanism and the molecular features responsible for NahF activity and specificity, besides the differences among others ALDHs, provide the necessary information for future structure-based studies to enhance its stability and specificity by systematic mutations and its further use in different biotechnological processes.

Supporting Information Available. Molar absorptivities for NahF substrates at 340 nm. Mathematical procedure for correction of Km. Determination of Km and kcat for NAD+ for the oxidation of salicilaldehyde catalyzed by NahF. Additional kinetic data about the substrate specificity of 6xHis-NahF for aromatic and aliphatic aldehydes. Kinetic data for the activity of 6xHis-NahF in different pH values.

Accession Number. The atomic coordinates and structure factors obtained in this work have been deposited in PDB under the accession code 4JZ6 (NahF in complex with SA).

Author Contributions. RAPN and TASB conceived and designed the experiments; JBC and MSP performed the experiments. JBC, DMAC, SLG, SSA, ACH, TASB and RAPN analyzed the data and wrote the paper.

Funding. This work was supported by FAPEMIG (EDT-0185-0.00-07, Rede-170/08, APQ01006-13 and RED-00010-14), CAPES, CNPq (INCT-Catalysis, 482173/2010-6, 306498/2013-8 and 484232/2013-4) and VALE S.A (RDP-00174-10).


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Page 20 of 35

ACKNOWLEDGMENT We thank Dr. Masataka Tsuda and Dr. Masahiro Sota for kindly providing us the P. putida G7 strain, Laboratório Nacional de Luz Síncrotron (LNLS, Campinas, Brazil), Laboratório Nacional de Biociências (LNBio, Campinas, Brazil), INCT-Catalysis, VALE S.A., and the Brazilian Foundations CNPq, Capes and FAPEMIG for financial support.

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Coitinho, J. B., Costa, D. M., Guimaraes, S. L., de Goes, A. M., and Nagem, R. A. (2012) Expression, purification and preliminary crystallographic studies of NahF, a salicylaldehyde dehydrogenase from Pseudomonas putida G7 involved in naphthalene degradation, Acta Crystallogr Sect F Struct Biol Cryst Commun 68, 93-97. McCoy, A. J., Grosse-Kunstleve, R. W., Adams, P. D., Winn, M. D., Storoni, L. C., and Read, R. J. (2007) Phaser crystallographic software, J Appl Crystallogr 40, 658-674. Moore, S. A., Baker, H. M., Blythe, T. J., Kitson, K. E., Kitson, T. M., and Baker, E. N. (1998) Sheep liver cytosolic aldehyde dehydrogenase: the structure reveals the basis for the retinal specificity of class 1 aldehyde dehydrogenases, Structure 6, 1541-1551. Cowtan, K. (2006) The Buccaneer software for automated model building. 1. Tracing protein chains, Acta Crystallogr D Biol Crystallogr 62, 1002-1011. Emsley, P., Lohkamp, B., Scott, W. G., and Cowtan, K. (2010) Features and development of Coot, Acta Crystallogr D Biol Crystallogr 66, 486-501. Murshudov, G. N., Vagin, A. A., and Dodson, E. J. (1997) Refinement of macromolecular structures by the maximum-likelihood method, Acta Crystallogr D Biol Crystallogr 53, 240-255. Bailey, S. (1994) The CCP4 Suite - Programs for Protein Crystallography, Acta Crystallogr D 50, 760-763. Adams, P. D., Afonine, P. V., Bunkoczi, G., Chen, V. B., Davis, I. W., Echols, N., Headd, J. J., Hung, L. W., Kapral, G. J., Grosse-Kunstleve, R. W., McCoy, A. J., Moriarty, N. W., Oeffner, R., Read, R. J., Richardson, D. C., Richardson, J. S., Terwilliger, T. C., and Zwart, P. H. (2010) PHENIX: a comprehensive Python-based system for macromolecular structure solution, Acta Crystallogr D Biol Crystallogr 66, 213-221. Holm, L., and Rosenstrom, P. (2010) Dali server: conservation mapping in 3D, Nucleic Acids Res 38, W545-549. McClelland, R. A., and Coe, M. (1983) Structure Reactivity Effects in the Hydration of Benzaldehydes, J Am Chem Soc 105, 2718-2725. Bell, R. P. (1966) The Reversible Hydration of Carbonyl Compounds, In Advances in Physical Organic Chemistry (Gold, V., Ed.), pp 1-29, Academic Press. Sayer, J. M. (1975) Hydration of p-nitrobenzaldehyde, J Org Chem 40, 2545–2547. Zhao, H. B., Li, Y. J., Chen, W., and Cai, B. L. (2007) A novel salicylaldehyde dehydrogenase-NahV involved in catabolism of naphthalene from Pseudomonas putida ND6, Chinese Sci Bull 52, 1942-1948. Barnsley, E. A. (1975) The induction of the enzymes of naphthalene metabolism in pseudomonads by salicylate and 2-aminobenzoate, J Gen Microbiol 88, 193-196. Li, S., Li, X., Zhao, H., and Cai, B. (2011) Physiological role of the novel salicylaldehyde dehydrogenase NahV in mineralization of naphthalene by Pseudomonas putida ND6, Microbiol Res 166, 643-653. Shamsuzzaman, K. M., and Barnsley, E. A. (1974) The regulation of naphthalene metabolism in pseudomonads, Biochem Biophys Res Commun 60, 582-589. Singh, R., Trivedi, V. D., and Phale, P. S. (2014) Purification and characterization of NAD+ -dependent salicylaldehyde dehydrogenase from carbaryl-degrading Pseudomonas sp. strain C6, Appl Biochem Biotechnol 172, 806-819. Dundas, J., Ouyang, Z., Tseng, J., Binkowski, A., Turpaz, Y., and Liang, J. (2006) CASTp: computed atlas of surface topography of proteins with structural and


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topographical mapping of functionally annotated residues, Nucleic Acids Res 34, W116118. Riveros-Rosas, H., Gonzalez-Segura, L., Julian-Sanchez, A., Diaz-Sanchez, A. G., and Munoz-Clares, R. A. (2013) Structural determinants of substrate specificity in aldehyde dehydrogenases, Chem Biol Interact 202, 51-61. Su, J., Zhang, C., Zhang, J. J., Wei, T., Zhu, D., Zhou, N. Y., and Gu, L. (2013) Crystal structure of the gamma-hydroxymuconic semialdehyde dehydrogenase from Pseudomonas sp. strainWBC-3, a key enzyme involved in para-Nitrophenol degradation, BMC Struct Biol 13, 30. Yuan, Z. N., Yin, B., Wei, D. Z., and Yuan, Y. R. A. (2013) Structural basis for cofactor and substrate selection by cyanobacterium succinic semialdehyde dehydrogenase, J Struct Biol 182, 125-135. Marchal, S., Rahuel-Clermont, S., and Branlant, G. (2000) Role of glutamate-268 in the catalytic mechanism of nonphosphorylating glyceraldehyde-3-phosphate dehydrogenase from Streptococcus mutans, Biochem 39, 3327-3335. Cobessi, D., Tete-Favier, F., Marchal, S., Branlant, G., and Aubry, A. (2000) Structural and biochemical investigations of the catalytic mechanism of an NADP-dependent aldehyde dehydrogenase from Streptococcus mutans, J Mol Biol 300, 141-152. Araújo, S. S., Neves, C. M., Guimaraes, S. L., Whitman, C. P., Johnson, W. H., Jr., Aparicio, R., and Nagem, R. A. (2015) Structural and kinetic characterization of recombinant 2-hydroxymuconate semialdehyde dehydrogenase from Pseudomonas putida G7, Arch Biochem Biophys 579, 8-17. Gonzalez-Segura, L., Rudino-Pinera, E., Munoz-Clares, R. A., and Horjales, E. (2009) The Crystal Structure of A Ternary Complex of Betaine Aldehyde Dehydrogenase from Pseudomonas aeruginosa Provides New Insight into the Reaction Mechanism and Shows A Novel Binding Mode of the 2 '-Phosphate of NADP(+) and A Novel Cation Binding Site, J Mol Biol 385, 542-557.


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Figure Legends Figure 1. Optimal temperature and thermostability of 6xHis-NahF. (a) Relative initial activity at different temperatures in standard assay conditions. (b) Relative activity in standard assay conditions after pre-incubation of the enzyme for the times shown. Figure 2. Logarithmic plots for kcatap/Km (a) and kcatap (b) for the oxidation of aromatic and aliphatic aldehydes by 6xHis-NahF in the presence of 200 µM NAD+ at 25.0 ºC. Figure 3. Effect of pH on kcatap/Km (a) and kcatap (b) for the oxidation of salicylaldehyde catalyzed by 6xHis-NahF in the presence of 200 µM NAD+ at 25.0 ºC; y-axes are logarithmic scales. Solid lines drawn through the experimental data are fits to Equations 3 and 4. The dot and dash lines in (b) represent the respective contributions of ki and kmax to kcatap. Refer to the Supporting Information (Table S4 and Figure S4) for Michaelis-Menten plots at each pH and additional kinetic data. Figure 4. Different representations of the overall fold of 6xHis-NahF. (a) Biological unit of 6xHis-NahF shown as a dimer. One monomer is shown in cartoon model, whereas the other monomer is represented with surface. The cofactor binding, the catalytic and the oligomerization domains are colored in gray, blue and yellow, respectively. The oligomerization domain contains three antiparallel β-strands protruding across the center of the dimer interface, while the NAD+ binding site is on the opposite side of the dimer interface. Salicylaldehyde oxygen and carbon atoms are depicted as red and yellow spheres, respectively. (b) Topology diagram. Helices are shown as tubes while β-strands are shown as arrows; both are labeled numerically. Color shades from blue to red represent the progress of the chain from N to C-terminus, respectively. The red


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circles identify residues which are located within 5 Å around the substrate: W96, F99, G150, R157, E250, F274, F279, C284 and Y446. Figure 5. Substrate binding pocket and active-site residues. (a) Size and distribution of hydrophobic residues along the substrate binding funnel determine substrate selection. F274 may play a role in the recognition of aromatic aldehyde substrates together with W96, F99 and F279, which are stabilized by π-stacking interactions. (b) Possible interactions established between salicylaldehyde (SA) and residues of the active site, where two water molecules are also found. Electron density maps illustrate the position of water molecules (red spheres) and salicylaldehyde (yellow and red stick-model) at the active site of recombinant NahF. The 2FobsFcalc map (blue) and omit map (green) are contoured at 1.0 and 4.0 σ, respectively. Hydrogen bond interactions are represented by dashed lines. Figure 6. Sequence alignment of NahF with ALDHs. Many ALDHs show high structural similarities to NahF, although the sequence identities are only around 35%. Corynebacterium glutamicum benzaldehyde dehydrogenase (BenzALDH) (PDB entry 3R64, Z-score 48.4, rmsd 2.2 Å, 455 Cα), Bacillus halodurans C-125 glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (PDB entry 3RHH, Z-score 55.1, rmsd 1.5 Å, 470 Cα), Ovis aries liver retinaldehyde dehydrogenase class 1 (ALDH1) (14) (PDB entry 1BXS, Z-score 53.0, rmsd 1.6 Å, 474 Cα), Pseudomonas aeruginosa betaine aldehyde dehydrogenase (BADH) (36) (PDB entry 4CAZ, Zscore 54.0, rmsd 1.5 Å, 477 Cα) and P. putida G7 2-hydroxymuconate semialdehyde dehydrogenase (NahI) (GenBank: BAE92168.1, no crystallographic structure available (35)). Secondary structure elements are drawn on the basis of structure of Pseudomonas putida G7 NahF and shown at the top of the aligned sequences. Residues involved in enzyme catalysis are


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indicated by black circles at the bottom of the aligned sequences. The G150 residue of NahF is indicated by white star.

Scheme 1. Equilibria for aldehyde hydration to the neutral gem-diol and its deprotonation to the corresponding anionic species. Scheme 2. Proposed kinetic model for oxidation of salicylaldehyde catalyzed by 6xHis-NahF in the presence of NAD+. Scheme 3. Proposed reaction mechanism of NahF.


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Table 1. Substrate specificity of 6xHis-NahF for aromatic and aliphatic aldehydes.a

Km

kcatap

kcatap/Km

µM

s-1

103 M-1 s-1

2-Hydroxybenzaldehyde (salicylaldehyde)

2.5±0.3

12.7±0.3b

5100±600

3-Hydroxybenzaldehyde

0.9±0.2

0.74±0.03

800±200

4-Hydroxybenzaldehyde

0.6±0.2

0.186±0.006

300±100

5-Bromosalicylaldehyde

12.1±2.3

4.6±0.5

380±80

5.2±0.4

14.0±0.5

2700±200

0.9±0.2

1.8±0.1

2000±500

4-Nitrobenzaldehyde

4±1

1.00±0.05

220±50

2-Nitrobenzaldehyde

3±1

6±2

2000±1000

2-Formylbenzaldehyde

30±6

11±2

360±90

2-Pyridinecarboxyaldehyde

13±3

14±2

1000±300

Substrate

H3C

5-Methylsalicylaldehyde

CHO OH

Benzaldehyde


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Biochemistry

2-Naphthaldehyde

1.6±0.4

9±1

6000±2000

Pyrene-1carboxaldehyde

0.75±0.09

3.2±0.1

4300±500

Acetaldehyde

9600±500

0.186±0.004

0.020±0.001

Propionaldehyde

1800±200

0.38±0.01

0.21±0.02

Butyraldehyde

2400±200

1.70±0.07

0.70±0.06

52±3

0.75±0.01

14.2±0.9

Octanaldehyde

9.2±0.5

0.66±0.01

72±4

Decanaldehyde

1.5±0.1

0.87±0.01

580±40

Isovaleraldehyde

150±5

0.231±0.003

1.55±0.06

Cinnamaldehyde

7.1±0.6

1.62±0.04

230±20

8±3

0.25±0.06

30±14

Hexaldehyde

Citronellal a

Kinetics were carried out in the presence of 200 µM NAD+ at 25.0 ºC. The steady-state

kinetic parameters were determined from initial rate (v) versus [S] fits using Equations 1 or 2; refer to the Supporting Information (Table S3 and Figure S3) for individual plots and additional kinetic data;

b

The kcat of 32.0±0.2 s-1 was determined under saturation conditions of

salicylaldehyde and NAD+ (see the text).


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Table 2. Calculated equilibrium and kinetic parameters for oxidation of salicylaldehyde catalyzed by 6xHis-NahF in the presence of 200 µM NAD+ at 25.0 ºC.

Parameters (kcatap/Km)max, M-1s-1

Values (9.1±0.3)x106

pKaE2

8.03±0.03

pKaE3

9.44±0.08

pKaS

9.42±0.04

ki, s-1

0.48±0.05

kmax, s-1

16.6±0.3

pKaES1

6.10±0.10

pKaES2

7.98±0.02

pKaES3

9.95±0.06


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Table 3. Detailed refinement data statistics for 6xHis-NahF crystallographic structure. Resolution range (Å)

32.17 – 2.42

No. of reflections

710,305

R-factor† (%)

20.9

R-free† (%)

24.6

No. of protein atoms

3,673

No. of molecules Water

375

Salicylaldehyde

2

Ethylene glycol

5

SO42-

3

Mean B factors (Å2) Protein atoms

31.4

Solvent atoms

33.8

Salicylaldehyde

24.2

Ethylene glycol

57.2

SO4

43.1

r.m.s.d. bond length (Å)

0.005

r.m.s.d. bond angles (o)

0.93

Ramachandran plot (%) Most favored

97.1

Additionally allowed

2.9

PDB code

4JZ6

Rfactor = Σhkl |Fobs| - |Fcalc| / Σhkl |Fobs|; Rfree is the Rfactor value calculated with 5% of the data not included on refinement.

†


29

-1

-1

20 30 40

10 4

10 3

kcat , s

ap

-1

Relative activity (%) 60

40

Relative activity (%)

(a) 100

50 60

(a)

10 0

3H 4- ydr Sa H o l 5- ydr xy- icy B o b la 5- rom xy- enz lde M o be al hy et -s n de d hy al za h e l-s icy ld yd al la eh e ic ld y 4Ni Be yla eh de 2- tro nz lde yde 2- 2- Nit be al hy Py Fo ro n de d rid rm be zal hy e in yl nz de de ec be al hy Py a n d d re 2- rbo zal ehy e ne N x de d -1 ap ya hy e -c ht ld d ar ha eh e bo ld yd x e e Pr Ace ald hyd op ta eh e i ld y Buona eh de ty lde yd He rald hyde Oc xa eh e D tan lde yde Is eca ald hyd ov na eh e a Ci ler ldehyde na ald y m eh de al y Ci deh de tro yd ne e lla l

ap

kcat /Km, M s

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

3H 4- ydr Sa Hy ox li 5- dr y- cy B o b la 5- rom xy- enz lde M o be al hy et -s n de d hy al za h e l-s icy ld yd al la eh e ic ld y 4Ni B yla eh de 2- tro enz lde yde 2- 2- Nit be al hy Py Fo ro n de d rid rm be zal hy e in yl nz de de ec be al hy Py a n d d re 2- rbo zal ehy e ne N x de d -1 ap ya hy e -c ht ld d ar ha eh e bo ld yd x e e Pr Ace ald hyd op ta eh e io lde yd Bu na h e ty lde yd H ral hy e Oc exa deh de D tan lde yde Is eca ald hyd ov na eh e a Ci ler ldehyde na ald y m eh de al y Ci deh de tro yd ne e lla l

Biochemistry

80

20

0 70 0

10 8

5

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Figure 1

(b) 100 80

40 ºC 50 ºC 60 ºC

60

40

20

0 1

Temperature (ºC)

ACS Paragon Plus Environment 2 3 4

Time (h)

Figure 2

(b) 100

10 7

10 6

10 10

2

1

10

10 1

0.1

30

Page 31 of 35

Figure 3

(a)

(b) 8

100

10

7

10

-1

10

kcat , s

-1

-1

kcat /Km, M s

6

ap

10

ap

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

1 5

10

4

0.1

10

5

6

7

8

9

10

11

5

6

pH

7

8

9

10

11

pH


31

Biochemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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Figure 4


32

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Biochemistry

Figure 5

Figure 6


33

Biochemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 34 of 35

Scheme 1

Scheme 2

pK aE2 EAH2

ESAH3

pK aES1

NAD+

H+

ESAH2 NAD+ ki

H+ pK aES2 H+

pK aE3 EAH1

EA H+ S + NAD+

ESAH1

pK aES3

NAD+

H+

ESA NAD+

kmax

E + P + NADH Scheme 3


34

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Biochemistry

FOR TABLE OF CONTENTS USE ONLY. Structural and kinetic properties of the aldehyde dehydrogenase NahF, a broad substrate specificity enzyme for aldehyde oxidation Juliana B. Coitinho, Mozart S. Pereira, Débora M. A. Costa, Samuel L. Guimarães, Simara S. de Araújo, Alvan C. Hengge, Tiago A. S. Brandão, and Ronaldo A. P. Nagem


35

Structural and Kinetic Properties of the Aldehyde Dehydrogenase

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