Recognition Site for the Side Chain of 2-Ketoacid Substrate in D-Lactate Dehydrogenase

Abstract

Replacement of Tyr52 with Val or Ala in Lactobacillus pentosus d-lactate dehydrogenase induced high activity and preference for large aliphatic 2-ketoacids and phenylpyruvate. On the other hand, replacements with Arg, Thr or Asp severely reduced the enzyme activity, and the Tyr52Arg enzyme, the only one that exhibited significant enzyme activity, showed a similar substrate preference to the Tyr52Val and Tyr52Ala enzymes. Replacement of Phe299 with Gly or Ser greatly reduced the enzyme activity with less marked change in the substrate preference. Except for the Phe299Ser enzyme, these mutant enzymes with low catalytic activity consistently stimulated NADH oxidation in the absence of 2-ketoacid substrates. However, the double mutant enzymes, Tyr52Arg/Phe299Gly and Tyr52Thr/Phe299Ser, did not exhibit synergically decreased enzyme activity or the substrate-independent NADH oxidation, but rather increased activities toward certain 2-ketoacid substrates. These results indicate that the coordinative combination of amino acid residues at two positions is pivotal in both the functional recognition of the 2-ketoacid side chain and the protection of the bound NADH molecule from the solvent. Multiplicity in such combinations appears to provide d-LDH-related 2-hydroxyacid dehydrogenases with a great variety of catalytic and physiological functions.

A New Family ofD-2-Hydroxyacid Dehydrogenases That ComprisesD-Mandelate Dehydrogenases and 2-Ketopantoate Reductases

“…It has been reported that only minor structural changes, such as amino acid replacements, cause drastic change, in the enzyme function in this family, as for example, conversions from a D-lactate dehydrogenase (D-LDH) to a D-hydroxyisocaproate dehydrogenase (D-HicDH), 17,18) and even from a formate dehydrogenase to a D-2-HydDH. [19][20][21] Although the enzymes in this family generally exhibit strict specificity toward NAD, it was reported recently that Holoferax mediterranei D-2-HydDH, which belongs to the D-2-HydDH family, utilizes both NAD and NADP as coenzymes, acting on bulky 2-ketoacid substrates.…”

“…[21][22][23][24] In spite of the great variety of their catalytic functions, however, there is no reported enzyme in the D-2-HydDH family that exhibits high catalytic activity toward C3-branched substrates. 17,18,22,25) Enzymes purified from Lactobacillus curvatus, 26) Enterococcus faecalis, 27,28) and also the yeast Rhodotorula graminis 29) are known to catalyze efficiently the conversion between benzoylformate (C 6 H 5 -CO-COO À ) and D-mandelate (C 6 H 5 -CHOH-COO À ), which possess a branched side chain at the C3 position, and are called D-mandelate dehydrogenases (D-ManDHs). Nevertheless, their structural relation to the D-2-HydDH family remains uncertain, because little is known about the protein structure of D-ManDHs.…”

Highly stereoselective biosynthesis of (R)-α-hydroxy carboxylic acids through rationally re-designed mutation of d-lactate dehydrogenase

“…In order to improve the reduction activity of d -nLDH toward α-hydroxy carboxylic acids with larger groups at C-3, certain residues of d -nLDH, which are around the C-3 group of α-hydroxy carboxylic acids, should be re-designed. Previous studies of d -nLDH stereo structure revealed that Tyr52 and Phe299 are mainly responsible for hindering larger substrates because of their short distance from the side chain of α-keto carboxylic acids and their steric orientation28303132. Moreover, Tokuda et al have succeeded in converting the d -nLDH of Lactobacillus pentosus into d -α-hydroxyisocaproate dehydrogenase by replacing of Tyr52 with an aliphatic amino residue, Leu33.…”

Construction of a Chimeric Biosynthetic Pathway for the De Novo Biosynthesis of Rosmarinic Acid in Escherichia coli

“…[62] The key mutations they used had been shown previously to convert the enzyme from a D-lactate dehydrogenase to a D-hydroxyisocaproate dehydrogenase [63] and increase activity against phenylpyruvic acid. [64] While Yao et al do not report on the level of de novo production of 4-HPL 2 or 3,4-DHPL 3 without precursor feeding or engineering of upstream pathways for tyrosine overproduction, they were able to show up to 7.1 g/L 3,4-DHPL 3 with their tyrosine-overproducing strain in a batch-fed process. [62] The implementation of our 3,4-DHPL 3 biosynthetic pathway in a tyrosine overproducing strain could therefore lead to similarly high titers of 3,4-DHPL 3 .…”