The First Crystal Structure of a Thioacylenzyme Intermediate in the ALDH Family:  New Coenzyme Conformation and Relevance to Catalysis

Self Cite

Methylmalonate-semialdehyde dehydrogenase (MSDH) belongs to the CoA-dependent aldehyde dehydrogenase subfamily. It catalyzes the NAD-dependent oxidation of methylmalonate semialdehyde (MMSA) to propionyl-CoA via the acylation and deacylation steps. MSDH is the only member of the aldehyde dehydrogenase superfamily that catalyzes a ␤-decarboxylation process in the deacylation step. Recently, we demonstrated that the ␤-decarboxylation is rate-limiting and occurs before CoA attack on the thiopropionyl enzyme intermediate. Thus, this prevented determination of the transthioesterification kinetic parameters. Here, we have addressed two key aspects of the mechanism as follows: 1) the molecular basis for recognition of the carboxylate of MMSA; and 2) how CoA binding modulates its reactivity. We substituted two invariant arginines, Arg-124 and Arg-301, by Leu. The second-order rate constant for the acylation step for both mutants was decreased by at least 50-fold, indicating that both arginines are essential for efficient MMSA binding through interactions with the carboxylate group. To gain insight into the transthioesterification, we substituted MMSA with propionaldehyde, as both substrates lead to the same thiopropionyl enzyme intermediate. This allowed us to show the following: 1) the pK app of CoA decreases by ϳ3 units upon binding to MSDH in the deacylation step; and 2) the catalytic efficiency of the transthioesterification is increased by at least 10 4 -fold relative to a chemical model. Moreover, we observed binding of CoA to the acylation complex, supporting a CoA-binding site distinct from that of NAD(H).Methylmalonate-semialdehyde dehydrogenases (MSDHs) 4 belong to the CoA-dependent aldehyde dehydrogenases (ALDHs), which, together with their hydrolytic counterparts, make up the ALDH superfamily. ALDHs are known to be involved in many essential biological functions such as intermediary metabolism, detoxification, osmotic protection, and cellular differentiation. The enzymes catalyze the NAD(P)-dependent oxidation of a wide variety of aldehydes to their corresponding nonactivated or CoA-activated acids via a common two-step chemical mechanism. The acylation step leads to formation of a thioacyl enzyme intermediate, which then undergoes nucleophilic attack by a water or CoA molecule. Despite mechanistic similarities, previous studies have highlighted major differences in the kinetic mechanism of ALDHs depending on the nature of the deacylation step. In hydrolytic ALDHs, kinetic data support an ordered sequential mechanism in which NAD(P)H dissociates last (1-4). By contrast, CoA-dependent ALDHs exhibit a ping-pong mechanism in which the release of the reduced cofactor occurs before the transthioesterification step (5, 6). Whereas mechanistic and structural aspects have been studied extensively for hydrolytic ALDHs (7-14), considerably less is known about CoA-dependent ALDHs.MSDHs are present in a wide variety of organisms ranging from bacteria and archaea to plants and mammals where they are described to play a d...

Section: Resultsmentioning

confidence: 99%

Methylmalonate-semialdehyde Dehydrogenase from Bacillus subtilis

Talfournier

Stinès-Chaumeil²,

Branlant

2011

Self Cite

“…Of note, replacement of the corresponding conserved glutamate in nonphosphorylating glyceraldehyde-3-phosphate dehydrogenase from Streptococcus mutans did not enhance NAD ϩ binding but trapped the covalent thioacyl-enzyme intermediate (40). Thus, it is not clear at present whether the newly discovered function of the glutamate is a common phenomenon in ALDH enzymes or it is a unique feature of C t -FDH.…”

Section: Discussionmentioning

confidence: 97%

Conserved Catalytic Residues of the ALDH1L1 Aldehyde Dehydrogenase Domain Control Binding and Discharging of the Coenzyme

Tsybovsky¹,

Krupenko

2011

The C-terminal domain (C t -FDH) of 10-formyltetrahydrofolate dehydrogenase (FDH, ALDH1L1) is an NADP ؉ -dependent oxidoreductase and a structural and functional homolog of aldehyde dehydrogenases. Here we report the crystal structures of several C t -FDH mutants in which two essential catalytic residues adjacent to the nicotinamide ring of bound NADP ؉ , Cys-707 and Glu-673, were replaced separately or simultaneously. The replacement of the glutamate with an alanine causes irreversible binding of the coenzyme without any noticeable conformational changes in the vicinity of the nicotinamide ring. Additional replacement of cysteine 707 with an alanine (E673A/ C707A double mutant) did not affect this irreversible binding indicating that the lack of the glutamate is solely responsible for the enhanced interaction between the enzyme and the coenzyme. The substitution of the cysteine with an alanine did not affect binding of NADP ؉ but resulted in the enzyme lacking the ability to differentiate between the oxidized and reduced coenzyme: unlike the wild-type C t -FDH/NADPH complex, in the C707A mutant the position of NADPH is identical to the position of NADP ؉ with the nicotinamide ring well ordered within the catalytic center. Thus, whereas the glutamate restricts the affinity for the coenzyme, the cysteine is the sensor of the coenzyme redox state. These conclusions were confirmed by coenzyme binding experiments. Our study further suggests that the binding of the coenzyme is additionally controlled by a longrange communication between the catalytic center and the coenzyme-binding domain and points toward an ␣-helix involved in the adenine moiety binding as a participant of this communication.Enzymes belonging to the family of aldehyde dehydrogenases (ALDH) 3 are involved in conversions of a broad variety of aliphatic and aromatic aldehydes to their respective carboxylic acids (1-3). These enzymes are either dimers or tetramers of identical subunits (1). Numerous crystal structures showed that subunits of different ALDHs have very similar fold with each composed of catalytic, nucleotide binding, and oligomerization domains (4 -10).Two members of the family, cytosolic ALDH1 and mitochondrial ALDH2, are essential components of the pathway metabolizing ethanol (2, 11). ALDH2 is also involved in mediating the vasodilator activity of nitroglycerine (12). Furthermore, activation of this enzyme correlates with reduced ischemic heart damage in rodent models (13). The polymorphism in the ALDH2 gene found in about 40% of the Asian population causes decreased tolerance to alcohol in heterozygous and homozygous individuals (14). This ALDH2 genetic variant, ALDH2 * 2, has a lysine instead of a glutamate at position 487 within the oligomerization domain of the tetrameric enzyme. This substitution induces conformational changes at the subunit interface, which are reflected by structural alterations within the catalytic center and the coenzyme binding site (5, 15, 16). The final outcome is a strong decrease in the affinity for NAD ...

“…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

Self Cite

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.