D- -Lactate

Figure A8.18 A racemic mixture of a-hydroxyacids (like L, D-lactate) can be transformed via the corresponding a-ketoacid (pyruvate) to the desired L-amino acid (L-alanine) with cofactor recycling.

In a related approach, Adam ef al. used glycolate oxidase with D-lactate dehydrogenase for the deracemization of a wide range of racemic a-hydroxy acids (20) (Figure 5.13) [23]. 

Adam, W., Lazarus, M., Saha-Moller, C.R. and Schreier, P. (1998) Quantitative transformation of racemic 2-hydroxy acids into (R)-2-hydroxy acids by enantioselective oxidation with glycolate oxidase and subsequent reduction of 2-keto acids with D-lactate dehydrogenase. Tetrahedron Asymmetry, 9 (2), 351-355. 

L, loading module DH, dehydratase KS, p-ketosynthase KR, ketoreductase MT methyltransferase PS, pyran synthase DHh and KRh are DH and KR-like sequences, together with the FkbH domain, they are involved in the formation of D-lactate starter unit HMG-CS, hydroxy-methyl-glutaryl CoA synthase. Acyl-carrier-protein domains are shown as small filled balls with chain attached by the thiol group. The box shows the HMG-CS pathway for the formation of exocyclic enoate. 

Fig. 5. Coupled electrochemical process for the formation of D-lactate from pyruvate (l-LDH = L-lactate dehydrogenase)...

The successful synthetic application of this electroenzymatic system has first been shown for the in-situ electroenzymatic reduction of pyruvate to D-lactate using the NADH-dependent D-lactate dehydrogenase. Electrolysis at — 0.6 V vs a Ag/AgCl-reference electrode of 50 mL of a 0.1 M tris-HCL buffer of pH 7.5 containing pentamethylcyclopentadienyl-2,2 -bipyridinechloro-rhodium(III) (1 x 10 3 M), NAD+ (2 x 10 3 M), pyruvate (2 x 10 2 M), 1300 units D-lactate dehydrogenase (divided cell, carbon foil electrode) after 3 h resulted in the formation of D-lactate (1.4 x 10 2 M) with an enantiomeric excess of 93.5%. This means that the reaction occurred at a rate of 5 turnovers per hour with respect to the mediator with a 70% turnover of the starting material. The current efficiency was 67% [67],... 

Alizade, M. A., Simon, H. Studies on mechanism and compartmentation of the L- and D-lactate formation from L-malate and D-glucose by Leuconostoc mesenteroides. Hoppe-Seylers Z. Physiol. Chem. 354, 163—168 (1973). 

Structural variations on the Ac2-L-Lys-D-Ala-D-Ala ligand also have a sizeable impact on the energetics of binding with vancomycin thus, replacing the o-Ala terminal with D-lactate [197] or D-Ser [198] reduces the binding affinity by 4.1 and 1.8 kcal/mol, respectively, while changing the terminal amide NH with a methylene (CH2) costs 1.5 kcal/mol [197]. 

StoU VS, Manohar AV, Gillon W, Mac Farlane EL, Hynes RC, et al. 1998. A thioredoxin fusion protein of VanH, a d-lactate dehydrogenase from Enterococcus Faecium cloning, expression, purification, kinetic analysis, and crystallization. Protein Sci 7 1147-1155. 

IMIDAZOLEACETATE HYDROXYLASE ISOVALERYL-CoA DEHYDROGENASE KYNURENINE 3-HYDROXYLASE d-LACTATE DEHYDROGENASE (CYTOCHROME)... 

ISOTOPE EXCHANGE AT EQUILIBRIUM d-LACTATE DEHYDROGENASE (CYTO-... 

The advantage of this reaction is that lactate is mnch cheaper than pyruvate, so the reaction is not only useful for the regeneration of NADH but also for the production of pyruvate. The disadvantage is that lactate dehydrogenases are enantiospecific, so a D-lactate as well as an L-lactate dehydrogenase are reqnired for a full conversion of racemic lactate (Figure 10.7). 

Similarly to the case of amino acids, hydroxy acids can also be deracemized by combining an enantioselective oxidation with a non-enantioselective reduction with sodium borohydride. For example, the group of Soda has reported the transformation of DL-lactate into D-lactate in >99% (Scheme 5.38) [78]. 

Figure 10.15 Metabolism of lactose, glucose, galactose, d- and L-lactic acid in Emmental cheese. Cheese transferred to hot room (22-24°C) at 14 days. , D-lactate O, acetate , galactose , L-lactate , glucose 0 lactose , propionate.

The lactic streptococci used in cheese manufacture produce only the l( + ) isomer of lactic acid (Lawrence et al. 1976). However, ripened cheeses contain both d(-) and l( + ) lactate isomers (T irner and Thomas 1980). Nonstarter bacteria (pediococci and lactobacilli) form d( —) lactate from residual lactose or by conversion of l( + ) lactate (Thomas and Crow 1983). 

Hydrolytic cleavage, as indicated in the diagram, yields the product D-lactate.7 Thus, we have a direct displacement with inversion followed by an additional hydrolytic step. This is an example of covalent catalysis, the enzyme providing a well-oriented reactive group instead of generating a hydroxyl group from a bound water molecule. 

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