Structural Studies of a ph-Dependent Tense State of Histidine Decarboxylase from Lactobacillus 30a. Worley, B.S., Monzingo, A.F., Robertus, J.D.

The crystal structure of the D53,54N mutant of Histidine Decarboxylase (HDC) from Lactobacillus 30a has been solved to 3.1Å resolution. HDC is a trimeric, cooperative enzyme whose activity is regulated by pH and histidine levels. The D53,54N mutant is locked in a tense quaternary form, and its structure was solved using molecular replacement methods based upon the known wild-type relaxed state. From the 2.5Å wild-type structure (Gallagher et al., 1993), Asp 53 forms cross-boundary hydrogen bonds with Asp 198 of a neighboring monomer, maintaining structural stability in the inter-subunit active site region and providing an optimal environment for an efficient decarboxylation reaction. Disruption of these interactions through mutation of Asp residues 53 and 54 forces the enzyme into a kinetically locked tense state (Pishko et al., 1995). Similarly, pH-elevation of the wild-type enzyme, also believed to disrupt the Asp 53-Asp 198 interactions, forces it into an initial tense state which converts back to a relaxed form upon excess substrate addition (Recsei and Snell, 1970). Thus, the D53,54N mutant of HDC represents a pH-dependent tense form of a cooperative, multimeric enzyme.

HDC-D53,54N was crystallized into the C222₁ space group with a = 98.23Å, b = 118.92Å, c = 204.39Å, and V_m = 2.88. Systematic OMIT electron density maps of the trimeric asymmetric unit reveal that while the majority of each monomer has remained in a similar position to wild-type HDC, the three active site mutated helices have changed position. Density around two of the helices appears disordered, whereas one, which may be stabilized by a crystallographic symmetry contact, appears more ordered and indicates a positional shift of Asp 53 of greater than 7Å. The Asp 53-Asp 198 bond may thus provide order needed to achieve a globally relaxed enzymatic state. Furthermore, density in similar positions in each active site suggests the possibility of bound reactant or product.