Hiroyuki Uchikoba

Bio: Hiroyuki Uchikoba is an academic researcher from University of Tokyo. The author has contributed to research in topics: Allosteric regulation & Allosteric enzyme. The author has an hindex of 5, co-authored 5 publications receiving 97 citations. Previous affiliations of Hiroyuki Uchikoba include Tokyo University of Science.

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.

Crystal structure of non-allosteric L-lactate dehydrogenase from Lactobacillus pentosus at 2.3 A resolution: specific interactions at subunit interfaces.

Abstract: 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.

An absolute requirement of fructose 1,6-bisphosphate for the Lactobacillus casei L-lactate dehydrogenase activity induced by a single amino acid substitution.

Abstract: Lactobacillus casei allosteric L-lactate dehydrogenase (L-LDH) absolutely requires fructose 1,6-bisphosphate [Fru(1,6)P 2 ] for its catalytic activity under neutral conditions, but exhibits marked catalytic activity in theabsence 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.

Some Lactobacillus l-Lactate Dehydrogenases Exhibit Comparable Catalytic Activities for Pyruvate and Oxaloacetate

Abstract: 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.

A molecular design that stabilizes active state in bacterial allosteric L-lactate dehydrogenases.

Abstract: 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.

Is allostery an intrinsic property of all dynamic proteins

Abstract: Allostery involves coupling of conformational changes between two widely separated binding sites. The common view holds that allosteric proteins are symmetric oligomers, with each subunit existing in "at least" two conformational states with a different affinity for ligands. Recent observations such as the allosteric behavior of myoglobin, a classical example of a nonallosteric protein, call into question the existing allosteric dogma. Here we argue that all (nonfibrous) proteins are potentially allosteric. Allostery is a consequence of re-distributions of protein conformational ensembles. In a nonallosteric protein, the binding site shape may not show a concerted second-site change and enzyme kinetics may not reflect an allosteric transition. Nevertheless, appropriate ligands, point mutations, or external conditions may facilitate a population shift, leading a presumably nonallosteric protein to behave allosterically. In principle, practically any potential drug binding to the protein surface can alter the conformational redistribution. The question is its effectiveness in the redistribution of the ensemble, affecting the protein binding sites and its function. Here, we review experimental observations validating this view of protein allostery.

Recent research on 3-phenyllactic acid, a broad-spectrum antimicrobial compound

Abstract: 3-Phenyllactic acid (PLA), which is an organic acid widely existing in honey and lactic acid bacteria fermented food, can be produced by many microorganisms, especially lactic acid bacteria. It was proved as an ideal antimicrobial compound with broad and effective antimicrobial activity against both bacteria and fungi. In addition, it could be used as feed additives to replace antibiotics in livestock feeds. This article presented a review of recent studies on the existing resource, antimicrobial activity, and measurement of PLA. In addition, microorganism strains and dehydrogenases producing PLA were reviewed in detail, the metabolic pathway and regulation of PLA synthesis in LAB strains were discussed, and high-level bioproduction of PLA by microorganism fermentation was also summarized.

Enantiocomplementary Enzymes: Classification, Molecular Basis for Their Enantiopreference, and Prospects for Mirror-Image Biotransformations

Abstract: One often-cited weakness of biocatalysis is the lack of mirror-image enzymes for the formation of either enantiomer of a product in asymmetric synthesis. Enantiocomplementary enzymes exist as the solution to this problem in nature. These enzyme pairs, which catalyze the same reaction but favor opposite enantiomers, are not mirror-image molecules; however, they contain active sites that are functionally mirror images of one another. To create mirror-image active sites, nature can change the location of the binding site and/or the location of key catalytic groups. In this Minireview, X-ray crystal structures of enantiocomplementary enzymes are surveyed and classified into four groups according to how the mirror-image active sites are formed.

Improvement of multiple-stress tolerance and lactic acid production in Lactococcus lactis NZ9000 under conditions of thermal stress by heterologous expression of Escherichia coli DnaK.

Abstract: The effects of nisin-induced dnaK expression in Lactococcus lactis were examined, and this expression was shown to improve stress tolerance and lactic acid fermentation efficiency. Using a nisin-inducible expression system, DnaK proteins from L. lactis (DnaKLla) and Escherichia coli (DnaKEco) were produced in L. lactis NZ9000. In comparison to a strain harboring the empty vector pNZ8048 (designated NZ-Vector) and one expressing dnaKLla (designated NZ-LDnaK), the dnaKEco-expressing strain, named NZ-EDnaK, exhibited more tolerance to heat stress at 40°C in GM17 liquid medium. The cell viability of NZ-Vector was reduced 4.6-fold after 6 h of heat treatment. However, NZ-EDnaK showed 13.5-fold increased viability under these conditions, with a very low concentration of DnaKEco production. Although the heterologous expression of dnaKEco did not effect DnaKLla production, heat treatment increased the DnaKLla level 3.5- and 3.6-fold in NZ-Vector and NZ-EDnaK, respectively. Moreover, NZ-EDnaK showed tolerance to multiple stresses, including 3% NaCl, 5% ethanol, and 0.5% lactic acid (pH 5.47). In CMG medium, the lactate yield and the maximum lactate productivity of NZ-EDnaK were higher than the corresponding values for NZ-Vector at 30°C. Interestingly, at 40°C, these values of NZ-EDnaK were not significantly different from the corresponding values for the control strain at 30°C. Lactate dehydrogenase (LDH) activity was also found to be stable at 40°C in the presence of DnaKEco. These findings suggest that the heterologous expression of dnaKEco enhances the quality control of proteins and enzymes, resulting in improved growth and lactic acid fermentation at high temperature.