Active and inactive state structures of unliganded Lactobacillus casei allosteric L‐lactate dehydrogenase

Abstract

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.

An alternative allosteric regulation mechanism of an acidophilic l‐lactate dehydrogenase from Enterococcus mundtii 15‐1A

“…The quaternary structure of the ligands‐bound form of LDH‐2 is similar to that of the active state of the other bacterial l ‐LDHs [4,9,14,16]. On the other hand, the crystal structure of the ligands‐unbound LDH‐2 may be an inactive form, although its quaternary structure is very compact compared to that of other l ‐LDHs in the inactive state [5,9,14].…”

“…Another research group has suggested that the acidophilicity of the Lb. casei l ‐LDH is caused by the tendency to adopt an expanded inactive state [14]. They insisted that the inter‐subunit interactions formed between His20 and Asp264 and between His205 and Glu211, which seem necessary in order to form a compact active state, are not generated in the enzyme under a neutral pH condition.…”

“…Structural and mutational analyses of the Lb. casei l ‐LDH indicated that two inter‐subunit salt bridges formed between His20 and Asp264 and between His205 and Glu211 are important to form the active state [14]. The salt bridges seem to be efficiently formed under the acidic condition, since the His residues are sufficiently protonated.…”

Lactate Dehydrogenase A Promotes Communication between Carbohydrate Catabolism and Virulence inBacillus cereus

“…In accordance with the latter hypothesis, we observed that a lack of LdhA led to a greater downregulation of nhe expression under anaerobiosis than that seen under aerobiosis ( Table 4). The idea that LdhA has a regulatory role is not unfounded, because (i) our results indicate that LdhA regulates its own expression, (ii) LdhA is structurally very different from other B. cereus classical fermentative lactate dehydrogenases (2,15), and (iii) lactate dehydrogenases have been identified as single-stranded DNA (ssDNA) binding proteins in eukaryotic cells and speculated to be components of transcriptional coactivator complexes (6,19). Lactate dehydrogenase binding to ssDNA involved a tyrosine residue located near the coenzyme binding site that was regulated by the NADH/NAD ϩ ratio (40).…”

Methyl arachidonyl fluorophosphonate inhibits Mycobacterium tuberculosis thioesterase TesA and globally affects vancomycin susceptibility

“…The best turnover number ( app k cat ) was for palmitoyl-CoA (3.42 AE 0.78) 9 10 À3 Ás À1 . A sigmoidal curve was observed with decanoyl-CoA, suggesting a potential conformational change in the enzyme with this substrate [28]. An oligomeric state of TesA endowed with a homotropic activation mechanism may explain this observation [29].…”