The crystal structure of lactococcus lactis dihydroorotate dehydrogenase A complexed with the enzyme reaction product throws light on its enzymatic function

Abstract

Dihydroorotate dehydrogenases (DHODs) catalyze the oxidation of (S)-dihydroorotate to orotate, the fourth step and only redox reaction in the de novo biosynthesis of pyrimidine nucleotides. A description is given of the crystal structure of Lacrococcus lacris dihydroorotate dehydrogenase A (DHODA) complexed with the product of the enzyme reaction orotate. The structure of the complex to 2.0 8, resolution has been compared with the structure of the native enzyme.The active site of DHODA is known to contain a water filled cavity buried beneath a highly conserved and flexible loop. In the complex the orotate displaces the water molecules from the active site and stacks above the DHODA flavin isoalloxazine ring, causing only small movements of the surrounding protein residues. The orotate is completely buried beneath the protein surface, and the orotate binding causes a significant reduction in the mobility of the active site loop. The orotate is bound by four conserved asparagine side chains (Asn 67, Asn 127, Asn 132, and Asn 193), the side chains of Lys 43 and Ser 194, and the main chain NH groups of Met 69, Gly 70, and Leu 71. Of these the Lys 43 side chain makes hydrogen bonds to both the flavin isoalloxazine ring and the carboxylate group of the orotate. Potential interactions with bound dihydroorotate are considered using the orotate complex as a basis for molecular modeling. The role of Cys 130 as the active site base is discussed, and the sequence conservation of the active site residues across the different families of DHODs is reviewed, along with implications for differences in substrate binding and in the catalytic mechanisms between these families.

Lactococcus lactis Dihydroorotate Dehydrogenase A Mutants Reveal Important Facets of the Enzymatic Function

“…The crystals of the mutated proteins belong to the same space group (P2 1 ) as the native enzyme. The unit cell parameters are similar and slightly smaller than those obtained earlier for the structures at room temperature (8,9). The average of the cell parameters were a ϭ 53.04 Ϯ 0.22 Å, b ϭ 108.36 Ϯ 0.24 Å, c ϭ 66.01 Ϯ 0.26 Å and ␤ ϭ 103.8 Ϯ 0.11°.…”

“…A few features of the steady state activities are worth noticing. First and as expected, major effects on the activities were produced by alanine mutations of the asparagines (Asn-67, Asn-127, and Asn-193), which form hydrogen bonds to substrate and product in the active site (9). Second, and more surprising, it turned out that a change of the conserved Pro-56 in the cis-proline loop to alanine strongly reduced the steady state activity with all three electron acceptors, while a change of the neighboring and also conserved Arg-57 to alanine increased the steady state activity of DHODA with all three electron acceptors, indicating an important and complex role of the cis-proline loop for optimal function of the active site.…”

“…Cys-130 in the active site loop is only conserved in the class 1 DHODs, but the loop contains three residues that are totally conserved in both classes, Ser-129, Pro-131, and Asn-132. The role of the latter residue was assigned earlier (9), but from our structural investigations studies we were unable to assign any function to the two former residues. Another very highly conserved segment of amino acids, residues 50 -57 in DHODA, found in the DHOD sequences, forms a fixed loop delineating the active site in the DHOD structures (8 -10, 16, 17), which we refer to as the cis-proline loop.…”

“…Two of these (Asn-132 and Lys-43 in DHODA) are important for catalysis (7). Their side chains are hydrogenbonded to the product orotate, and in addition Lys-43 interacts with O4 of FMN (9). Furthermore, in all the known structures Bottom, the A subunit of the native structure in the presence of DTT and absence of orotate and three mutant structures (K213E(Oro), P56A(Oro) and K136E) oriented as the dimer at the top and colorcoded according to the temperature factors of the residues.…”

Estudo do mecanismo e inibição da enzima diidroorotato desidrogenase de Leishmania major

“…A enzima LmDHODH é dimérica, enquanto a enzima humana (classe 2) é monomérica [65]. Como consequência, a interface do dímero é uma região exclusiva na enzima do parasito [56].…”