Lactobacillus casei L-lactate dehydrogenase (LCLDH) is activated through the homotropic and heterotropic activation effects of pyruvate and fructose 1,6-bisphosphate (FBP), respectively, and exhibits unusually high pH-dependence in the allosteric effects of these ligands. The active (R) and inactive (T) state structures of unliganded LCLDH were determined at 2.5 and 2.6 A resolution, respectively. In the catalytic site, the structural rearrangements are concerned mostly in switching of the orientation of Arg171 through the flexible intersubunit contact at the Q-axis subunit interface. The distorted orientation of Arg171 in the T state is stabilized by a unique intra-helix salt bridge between Arg171 and Glu178, which is in striking contrast to the multiple intersubunit salt bridges in Lactobacillus pentosus nonallosteric L-lactate dehydrogenase. In the backbone structure, major structural rearrangements of LCLDH are focused in two mobile regions of the catalytic domain. The two regions form an intersubunit linkage through contact at the P-axis subunit interface involving Arg185, replacement of which with Gln severely decreases the homotropic and hetertropic activation effects on the enzyme. These two regions form another intersubunit linkage in the Q-axis related dimer through the rigid NAD-binding domain, and thus constitute a pivotal frame of the intersubunit linkage for the allosteric motion, which is coupled with the concerted structural change of the four subunits in a tetramer, and of the binding sites for pyruvate and FBP. The unique intersubunit salt bridges, which are observed only in the R state structure, are likely involved in the pH-dependent allosteric equilibrium.

Lactobacillus casei allosteric L-lactate dehydrogenase (L-LDH) absolutely requires fructose 1,6-bisphosphate [Fru(1,6)P2] for its catalytic activity under neutral conditions, but exhibits marked catalytic activity in the absence of Fru(1,6)P(2) under acidic conditions through the homotropic activation effect of substrate pyruvate. In this enzyme, a single amino acid replacement, i.e. that of His205 conserved in the Fru(1,6)P(2)-binding site of certain allosteric L-LDHs of lactic acid bacteria with Thr, did not induce a marked loss of the activation effect of Fru(1,6)P(2) or divalent metal ions, which are potent activators that improve the activation function of Fru(1,6)P(2) under neutral conditions. However, this replacement induced a great loss of the Fru(1,6)P(2)-independent activation effect of pyruvate or pyruvate analogs under acidic conditions, consequently indicating an absolute Fru(1,6)P(2) requirement for the enzyme activity. The replacement also induced a significant reduction in the pH-dependent sensitivity of the enzyme to Fru(1,6)P(2), through a slight decrease and increase of the Fru(1,6)P(2) sensitivity under acidic and neutral conditions, respectively, indicating that His205 is also largely involved in the pH-dependent sensitivity of L.casei L-LDH to Fru(1,6)P(2). The role of His205 in the allosteric regulation of the enzyme is discussed on the basis of the known crystal structures of L-LDHs.

L-Lactate dehydrogenase (LDH) from Lactobacillus pentosus is a non-allosteric enzyme, which shows, however, high sequence similarity to allosteric LDHs from certain bacteria. To elucidate the structural basis of the absence of allostery of L. pentosus LDH (LPLDH), we determined the crystal structure of LPLDH at 2.3 A resolution. Bacterial LDHs are tetrameric enzymes composed of identical subunits and exhibit 222 symmetry. The quaternary structure of LPLDH was similar to the active conformation of allosteric LDHs. Structural analysis revealed that the subunit interfaces of LPLDH are optimized mainly through hydrophilic interactions rather than hydrophobic interactions, compared with other LDHs. The subunit interfaces of LPLDH are more specifically stabilized by increased numbers of intersubunit salt bridges and hydrogen bonds, and higher geometrical complementarity. Such high specificity at the subunit interfaces should hinder the rearrangement of the quaternary structure needed for allosteric regulation and thus explain the "non-allostery" of LPLDH.

The nonallosteric and allosteric L-lactate dehydrogenases of Lactobacillus pentosus and L. casei, respectively, exhibited broad substrate specificities, giving virtually the same maximal reaction velocity and substrate K m values for pyruvate and oxaloacetate. Replacement of Pro101 with Asn reduced the activity of the L. pentosus enzyme toward these alternative substrates to a greater extent than the activity toward pyruvate.L-Lactate dehydrogenase (L-LDH; EC 1.1.1.27) and Lmalate dehydrogenase (L-MDH; EC 1.1.1.37) are similar with respect to both protein structure and catalytic machinery, and they catalyze oxidation-reduction of common 2-ketoacids and L-2-hydroxyacids with NAD as the coenzyme (1,19,26). Nevertheless, unless artificially modified, most L-LDHs and LMDHs strictly discriminate their own substrates, pyruvate (L-lactate) and oxaloacetate (L-malate), respectively (15), although a duck ε-crystallin (37) and the Bifidobacterium longum (11) L-LDHs are known to exhibit relatively high L-MDH activities that are only 25-and 44-fold, respectively, lower than their own L-LDH activities.In lactic acid bacteria such as lactobacilli, L-LDHs show a great variety of catalytic properties and play key roles in the fermentation of lactic acid, acting in the last step of the anaerobic glycolysis pathway by converting pyruvate and NADH to L-lactate and NAD ϩ (19). While vertebrate cells possess nonallosteric L-LDH isozymes, depending on the tissue (19), many bacterial cells possess allosteric types of L-LDHs, which usually require fructose 1,6-bisphosphate [Fru(1,6)P 2 ] for activity (13). We are carrying out a comparative study of the nonallosteric and allosteric types of L-LDHs from Lactobacillus pentosus, previously called L. plantarum, and L. casei, respectively, to understand their structure-function relationships (28-30). In the course of this study, we found that these two enzymes exhibit marked activities toward oxaloacetate that are comparable to their activities for pyruvate.The cultivation of Escherichia coli cells harboring expression plasmids for the genes encoding the L-LDHs of L. pentosus JCM1558 (ϭATCC 8041) and L. casei IAM 12473 (ϭATCC 393) and purification of the enzymes were performed essentially as described previously (29,30). Protein concentrations were determined with Bio-Rad protein assay reagent by the Bradford method (4), using bovine serum albumin as the standard. All enzyme assays were performed at 30°C. The 2-ketoacid reduction by L. pentosus L-LDH was assayed in 50 mM sodium MES (morpholineethanesulfonic acid) buffer (pH 6.0) containing 0.1 mM NADH and various concentrations of sodium pyruvate, oxaloacetate, or another 2-ketoacid. The reduction of pyruvate and oxaloacetate by the L. casei enzymes was assayed in 50 mM sodium acetate buffer (pH 5.0) and sodium MOPS (morpholinepropanesulfonic acid) buffer (pH 7.0), respectively. The assay mixtures for oxaloacetate were freshly prepared before each use, using newly purchased oxaloacetic acid (Wako Fine Chemicals, Osaka, Japan). One unit was d...

l-Lactate dehydrogenase (l-LDH) of Lactobacillus casei (LCLDH) is a typical bacterial allosteric l-LDH that requires fructose 1,6-bisphosphate (FBP) for its enzyme activity. A mutant LCLDH was designed to introduce an inter-subunit salt bridge network at the Q-axis subunit interface, mimicking Lactobacillus pentosus non-allosteric l-LDH (LPLDH). The mutant LCLDH exhibited high catalytic activity with hyperbolic pyruvate saturation curves independently of FBP, and virtually the equivalent K(m) and V(m) values at pH 5.0 to those of the fully activated wild-type enzyme with FBP, although the K(m) value was slightly improved with FBP or Mn(2+) at pH 7.0. The mutant enzyme exhibited a markedly higher apparent denaturating temperature (T(1/2)) than the wild-type enzyme in the presence of FBP, but showed an even lower T(1/2) without FBP, where it exhibited higher activation enthalpy of inactivation (ΔH(‡)). This result is consistent with the fact that the active state is more unstable than the inactive state in allosteric equilibrium of LCLDH. The LPLDH-like network appears to be conserved in many bacterial non-allosteric l-LDHs and dimeric l-malate dehydrogenases, and thus to be a key for the functional divergence of bacterial l-LDHs during evolution.