Chemical mechanism and substrate binding sites of NADP-dependent aldehyde dehydrogenase from Streptococcus mutans

Non-phosphorylating glyceraldehyde 3-phosphate dehydrogenase from Streptoco ccus mutans (GAPN) belongs to the aldehyde dehydrogenase (ALDH) family, whi ch catalyzes the irreversible oxidation of a wide variety of aldehydes into acidic compounds via a two-step mechanism: first, the acylation step invol ves the formation of a covalent ternary complex ALDH-cofactor-substrate, fo llowed by the oxidoreduction process which yields a thioacyl intermediate a nd reduced cofactor and second, the rate-limiting deacylation step. Structu ral and molecular factors involved in the chemical mechanism of GAPN have r ecently been examined. Specifically, evidence was put forward for the chemi cal activation of catalytic Cys-302 upon cofactor binding to the enzyme, th rough a local conformational rearrangement involving the cofactor and Glu-2 68. In addition, the invariant residue Glu-268 was shown to play an essenti al role in the activation of the water molecule in the deacylation step. Fo r E268A/Q mutant GAPNs, nucleophilic compounds like hydrazine and hydroxyla mine were shown to bind and act as substrates in this step. Further studies were focused at understanding the factors responsible for the stabilizatio n and chemical activation of the covalent intermediates, using X-ray crysta llography, site-directed mutagenesis, kinetic and physicochemical approache s. The results support the involvement of an oxyanion site including the si de-chain of Asn-169. Finally, given the strict substrate-specificity of GAP N compared to other ALDHs with wide substrate specificity, one has also ini tiated the characterization of the G3P binding properties of GAPN. These re sults will be presented and discussed fi om the point of view of the evolut ion of the catalytic mechanisms of ALDH. (C) 2001 Elsevier Science Ireland Ltd. All rights reserved.