Role of Glutamate-268 in the Catalytic Mechanism of Nonphosphorylating Glyceraldehyde-3-phosphate Dehydrogenase from <i>Streptococcus mutans</i>

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.

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

confidence: 99%

“…In class 3 ALDH, however, this residue seems to be involved only in cosubstrate binding. In the case of Sm-GAPN, this residue has been suggested to play an essential role in deacylation by activating a water that hydrolyzes the intermediate (53).…”

mentioning

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

“…It has also been suggested that, in tetrameric class 1 and 2 ALDHs, the proton abstracted by Glu-268 is shuttled to bulk water (10,25). In addition, Glu-268 is also the most likely residue activating a water molecule in the deacylation step of the reaction (5,7,10,23,26).…”

mentioning

confidence: 99%

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 ...

“…This gave us the opportunity to determine the pK app of CoA in the deacylation step, which shifts from ϳ9.6 to 6.8. When compared with a chemical model, the catalytic efficiency of the transthioesterification for the wild-type MSDH was increased at least 10 4 -fold. Therefore, CoA binding to MSDH optimizes its positioning relative to the thioacyl enzyme intermediate and favors a significant decrease of the pK app of its thiol group.…”

mentioning

confidence: 99%

Methylmalonate-semialdehyde Dehydrogenase from Bacillus subtilis

Talfournier

Stinès-Chaumeil²,

Branlant

2011

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...