Orotate phosphoribosyltransferase (OPRTase, EC 2.4.2.10) catalyzes the Mg2+-dependent condensation of orotic acid (OA) with PRPP (5-alpha-d-phosphorylribose 1-diphosphate) to yield diphosphate (PPi) and the nucleotide OMP (orotidine 5'-monophosphate). We have determined the structures of three forms of Saccharomyces cerevisiae OPRTase representing different structural and enzymatic intermediates. The structures include the apoenzyme (2.35 A resolution); a ternary complex of enzyme, Mg2+-PRPP, and OA (1.74 A resolution); and the binary product complex of enzyme with OMP (1.89 A resolution). While the overall structure of the S. cerevisiae OPRTase is similar to that of the Salmonella typhimurium enzyme, as judged by comparison of the two apoenzymes, large conformational transitions occur proceeding from the apoenzyme structure to those of the substrate and product complexes. Comparison of these structures reveals a rotation of the upper hood domain onto the bound ligands by an average of 19.5 degrees in the OMP structure and an average of 24.6 degrees in the OA/Mg2+-PRPP ternary complex. As expected, the conserved loop, composed of residues 104-116, moves extensively and adopts a single stable conformation during the catalytic cycle in order to sequester the substrates from bulk solvent in the ternary complex. The OA and Mg2+-PRPP molecules bound in the ternary complex are oriented for proper attack of the N1 atom of OA onto the C1 atom of the ribose ring. This orientation of substrates, combined with the positioning of the flexible loop, provides a clear picture of a catalytically poised reaction complex for type I phosphoribosyltransferases. The structural asymmetry present in these structures, as well as that found in a recent structure of the S. typhimurium enzyme, combined with the closure of the flexible loop from one subunit into the active site of the opposing subunit in the ternary complex is consistent with the kinetic data [McClard, R. W., et al. (2006) Biochemistry 45, 5330-5342] that demonstrate induced nonequivalence and cooperativity of OPRTase.

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Plant Aldehyde Dehydrogenase10 (ALDH10) enzymes catalyze the oxidation of v-primary or v-quaternary aminoaldehydes, but, intriguingly, only some of them, such as the spinach (Spinacia oleracea) betaine aldehyde dehydrogenase (SoBADH), efficiently oxidize betaine aldehyde (BAL) forming the osmoprotectant glycine betaine (GB), which confers tolerance to osmotic stress. The crystal structure of SoBADH reported here shows tyrosine (Tyr)-160, tryptophan (Trp)-167, Trp-285, and Trp-456 in an arrangement suitable for cation-p interactions with the trimethylammonium group of BAL. Mutation of these residues to alanine (Ala) resulted in significant K m (BAL) increases and V max /K m (BAL) decreases, particularly in the Y160A mutant. Tyr-160 and Trp-456, strictly conserved in plant ALDH10s, form a pocket where the bulky trimethylammonium group binds. This space is reduced in ALDH10s with low BADH activity, because an isoleucine (Ile) pushes the Trp against the Tyr. Those with high BADH activity instead have Ala (Ala-441 in SoBADH) or cysteine, which allow enough room for binding of BAL. Accordingly, the mutation A441I decreased the V max /K m (BAL) of SoBADH approximately 200 times, while the mutation A441C had no effect. The kinetics with other v-aminoaldehydes were not affected in the A441I or A441C mutant, demonstrating that the existence of an Ile in the second sphere of interaction of the aldehyde is critical for discriminating against BAL in some plant ALDH10s. A survey of the known sequences indicates that plants have two ALDH10 isoenzymes: those known to be GB accumulators have a high-BAL-affinity isoenzyme with Ala or cysteine in this critical position, while non GB accumulators have low-BAL-affinity isoenzymes containing Ile. Therefore, BADH activity appears to restrict GB synthesis in non-GB-accumulator plants.

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PaBADH (Pseudomonas aeruginosa betaine aldehyde dehydrogenase) catalyses the irreversible NAD(P)+-dependent oxidation of betaine aldehyde to its corresponding acid, the osmoprotector glycine betaine. This reaction is involved in the catabolism of choline and in the response of this important pathogen to the osmotic and oxidative stresses prevalent in infection sites. The crystal structure of PaBADH in complex with NADPH showed a novel covalent adduct between the C2N of the pyridine ring and the sulfur atom of the catalytic cysteine residue, Cys286. This kind of adduct has not been reported previously either for a cysteine residue or for a low-molecular-mass thiol. The Michael addition of the cysteine thiolate in the 'resting' conformation to the double bond of the α,β-unsaturated nicotinamide is facilitated by the particular conformation of NADPH in the active site of PaBADH (also observed in the crystal structure of the Cys286Ala mutant) and by an ordered water molecule hydrogen bonded to the carboxamide group. Reversible formation of NAD(P)H-Cys286 adducts in solution causes reversible enzyme inactivation as well as the loss of Cys286 reactivity towards thiol-specific reagents. This novel covalent modification may provide a physiologically relevant regulatory mechanism of the irreversible PaBADH-catalysed reaction, preventing deleterious decreases in the intracellular NAD(P)+/NAD(P)H ratios.

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