Structural and biochemical investigations of the catalytic mechanism of an NADP-dependent aldehyde dehydrogenase from Streptococcus mutans

Cobessi, David; Favier, F.; Marchal, Stéphane; Branlant, Guy; Aubry, A.

doi:10.1006/jmbi.2000.3824

Cited by 87 publications

(112 citation statements)

References 22 publications

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“…This interaction would decrease the pKa of the main chain amide of Cys243. While this lysine is strictly conserved only in class 3 ALDHs and in succinic semialdehyde dehydrogenases (25), we note that in the crystal structure of a ternary complex of non-phosphorylating aldehyde dehydrogenase (GAPN) another lysine residue (Lys288, the "distal lysine") in a geometrically analogous position hydrogen bonds instead to the equivalent main chain amide oxygen (51,52). The distal lysine residue is conserved in many classes of ALDH.…”

Section: Discussionmentioning

confidence: 99%

“…The quantitative simulation of a hydride transfer reaction in probable concert with a proton transfer is extremely challenging and will require the development of new methods similar to those reported by Ferrer et al (15). The position of the distal lysine may also have to be placed in a much closer position analogous to that observed in GAPN (51,52) to assess its possible role in facilitating formation of the α-hydroxy sulfide.…”

Section: Discussionmentioning

confidence: 99%

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Mechanistic Implications of the Cysteine−Nicotinamide Adduct in Aldehyde Dehydrogenase Based on Quantum Mechanical/Molecular Mechanical Simulations

2007

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Recent computer simulations of the cysteine nucleophilic attack on propanal in human mitochondrial Aldehyde Dehydrogenase (ALDH2) yielded an unexpected result; the chemically reasonable formation of a dead-end cysteine-cofactor adduct when NAD+ was in the "hydride transfer" position. More recently, this adduct found independent crystallographic support in work on formyltetrahydrofolate dehydrogenase, work which further found evidence of the same adduct on re-examination of deposited electron densities of ALDH2. Although the experimental data showed this adduct was reversible, several mechanistic questions arise from the fact that it forms at all. Here, we present results from further Quantum Mechanical/Molecular Mechanical (QM/MM) simulations toward understanding the mechanistic implications of adduct formation. These simulations revealed that formation of the oxyanion thiohemiacetal intermediate only when the nicotinamide ring of NAD + is oriented away from the active site, contrary to prior arguments. In contrast, and in seeming paradox, when NAD is oriented to receive the hydride, disassociation of the oxyanion intermediate to form the dead-end adduct is more thermodynamically-favored than maintaining the oxyanion intermediate necessary for catalysis to proceed. However, this disassociation to the adduct could be avoided through proton transfer from the enzyme to the intermediate. Our results continue to indicate that the unlikely source of this proton is the Cys302 main chain amide.The overall mechanism of aldehyde dehydrogenase (E.C. 1.2.1.3) shown in Figure 1 involves 1) nucleophilic attack on the aldehyde to form a thiohemiacetal intermediate from which 2) the hydride is transferred to NAD+, to yield NADH and the thioester which 3) hydrolyses to yield the product carboxylic acid followed by 4) release of the cofactor. Specific atomic level details about each of these steps have been obtained from x-ray crystallography and NMR. The side chain of the conserved Asn169 (ALDH2 numbering) and the main chain amide of Cys302 are positioned to form hydrogen bonds to a negatively charged thiohemiacetal intermediate structure. This configuration is often called the "oxyanion hole".In the human mitochondrial ALDH2 structure (1), the Asn169 Nδ-to-crotonaldehyde carbonyl oxygen is 4.1 Å while the main chain amide nitrogen of Cys302 is 3.8 Å away. Three *To whom correspondence should be addressed: Pittsburgh Supercomputing Center 300 S. Craig Street Pittsburgh, PA 15213 e-mail: wymore@psc.edu Phone: 412−268−4960 FAX: 412−268−8200. Supporting Information Available Coordinates of optimized product structures presented in Figure 3 are given in XYZ format. This material is available free of charge via the Internet at http://pubs.acs.org. NIH Public Access Author ManuscriptBiochemistry. Author manuscript; available in PMC 2008 September 5. . The other NAD+ conformation shows the nicotinamide more removed from the active site and is likely the position of the coenzyme during thioester hydrolysis. In this conformation,...

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Mechanistic Implications of the Cysteine−Nicotinamide Adduct in Aldehyde Dehydrogenase Based on Quantum Mechanical/Molecular Mechanical Simulations

2007

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“…The acylation step involves the formation of a hemithioacetal intermediate via the nucleophilic attack of the catalytic Cys-302 (the amino acid numbering used for the biochemical and structural data is that defined by Wang and Weiner (5)) on the aldehydic function followed by hydride transfer that leads to formation of a thioacylenzyme intermediate and NAD(P)H. This intermediate then undergoes a nucleophilic attack by an activated water or CoA molecule. Over the past 15 years both mechanistic and structural aspects of hydrolytic ALDHs have been studied extensively (5)(6)(7)(8)(9)(10)(11)(12)(13). In addition to local conformational reorganizations of the active site induced by ligand binding that provide the required flexibility for an efficient catalysis (14,15), one of the key aspects of the chemical mechanism of this ALDH family is the substantial conformational flexibility of the NMN moiety of the cofactor and in particular of the nicotinamide ring.…”

Section: Structural Dynamics Associated With Cofactor Binding Have Bementioning

confidence: 99%

Adenine Binding Mode Is a Key Factor in Triggering the Early Release of NADH in Coenzyme A-dependent Methylmalonate Semialdehyde Dehydrogenase

Bchini

Dubourg-Gerecke

Rahuel-Clermont

et al. 2012

Journal of Biological Chemistry

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Background: Conformational dynamics of the cofactor are essential for catalysis by hydrolytic ALDHs. Results: Crystallographic and kinetic data reveal the molecular basis for NADH release in MSDH, a CoA-dependent ALDH. Conclusion: Weaker stabilization of the adenine ring triggers early NADH release in MSDH-catalyzed reaction. Significance: First description of the mechanism whereby the cofactor binding mode is partly responsible for the kinetic behavior of CoA-dependent ALDHs.

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“…by Arg 103 , Tyr 155 , and Arg 283 ) and to the carbonyl oxygen at C-1 (Asn 154 and Ser 284 ) (12). However, because Sm-GAPN uses both D-GAP and L-GAP as substrate, the interaction between Arg 437 and the chiral C-2 does not confer any stereospecificity.…”

Section: Fig 2 Structure-based Sequence Alignment Of Tt-gapn Sm-gamentioning

confidence: 99%

“…Subsequently, the resulting thioacyl intermediate is hydrolyzed, and the product and the reduced cosubstrate are released. Despite many biochemical and mutagenesis studies, the mechanism and the role of putatively essential residues of this reaction remain to be elucidated at a molecular level (12). For instance, the invariant Glu 268 (numbering according to class 2 ALDH) has been implied to function as a general base, deprotonating the active-site cysteine in class 2 ALDH (6).…”

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confidence: 99%

The Crystal Structure of the Allosteric Non-phosphorylating Glyceraldehyde-3-phosphate Dehydrogenase from the Hyperthermophilic Archaeum Thermoproteus tenax

Pohl¹,

Brunner²,

Wilmanns³

et al. 2002

Journal of Biological Chemistry

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The NAD؉ -dependent non-phosphorylating glyceraldehyde-3-phosphate dehydrogenase (GAPN) from the hyperthermophilic archaeum Thermoproteus tenax represents an archaeal member of the diverse superfamily of aldehyde dehydrogenases (ALDHs). GAPN catalyzes the irreversible oxidation of D-glyceraldehyde 3-phosphate to 3-phosphoglycerate. In this study, we present the crystal structure of GAPN in complex with its natural inhibitor NADP ؉ determined by multiple anomalous diffraction methods. The structure was refined to a resolution of 2.4 Å with an R-factor of 0.21. The overall fold of GAPN is similar to the structures of ALDHs described previously, consisting of three domains: a nucleotidebinding domain, a catalytic domain, and an oligomerization domain. Local differences in the active site are responsible for substrate specificity. The inhibitor NADP ؉ binds at an equivalent site to the cosubstrate-binding site of other ALDHs and blocks the enzyme in its inactive state, possibly preventing the transition to the active conformation. Structural comparison between GAPN from the hyperthermophilic T. tenax and homologs of mesophilic organisms establishes several characteristics of thermostabilization. These include protection against heat-induced covalent modifications by reducing and stabilizing labile residues, a decrease in number and volume of empty cavities, an increase in ␤-strand content, and a strengthening of subunit contacts by ionic and hydrophobic interactions.

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Structural and biochemical investigations of the catalytic mechanism of an NADP-dependent aldehyde dehydrogenase from Streptococcus mutans

Cited by 87 publications

References 22 publications

Mechanistic Implications of the Cysteine−Nicotinamide Adduct in Aldehyde Dehydrogenase Based on Quantum Mechanical/Molecular Mechanical Simulations

Mechanistic Implications of the Cysteine−Nicotinamide Adduct in Aldehyde Dehydrogenase Based on Quantum Mechanical/Molecular Mechanical Simulations

Adenine Binding Mode Is a Key Factor in Triggering the Early Release of NADH in Coenzyme A-dependent Methylmalonate Semialdehyde Dehydrogenase

The Crystal Structure of the Allosteric Non-phosphorylating Glyceraldehyde-3-phosphate Dehydrogenase from the Hyperthermophilic Archaeum Thermoproteus tenax

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