Lactobacilli brevis

For reduction of acetylenic ketones, two oxidoreductases were used [25]. Lactobacillus brevis alcohol dehydrogenase (LBADH) gave the (R)-alcohols and Candida parapsilosis carbonyl reductase (CPCR) afforded the (S)-isomer, both in good yield and excellent enantioselectivity. By changing the steric demand of the substituents, the enantiomeric excess values can be adjusted and even the configurations of the products can be altered (Figure 8.34). 

Considerable interest has developed during the last ten years in the potential utility of condensed 1,2,3-triazine derivatives as medicinals, although the first indication that these compounds might be useful appears to be the report by Woolley and Shaw in 1951 that the 2-azadenine derivative (183) inhibited hypoxanthine activity in Lactobacillus brevis. l,2,3-Benzotriazin-4-one (10, R = H) is reported... 

Lactobacillus brevis also produces an (R)-spedfic ADH (LB-ADH) [4]. These two enzymes have similar properties. An alignment of the protein sequences of both short-chain dehydrogenases revealed an identity of 88% (Fig. 2.2.4.5). The nucleotide sequences of LK-ADH and LB-ADH show an identity of 78%. 

An NADP(H)-dependent ADH of Lactobacillus brevis (LBADH) was identified as a suitable catalyst accepting a broad range of diketo esters A as substrate [8]. This stable enzyme is easily available in the form of a crude cell extract (recLBADH) from a recombinant E. coli strain [9]. The reaction with diketo esters la-lc was performed on a preparative scale, using substrate-coupled regeneration of NADPH (Scheme 2.2.7.2). 

Lactobacillus brevis whole-cell biotransformation When the reduction of diketo ester la was performed with whole cells of Lactobacillus brevis or L. kefir, formation of the 3,5-dihydroxy ester (3R,5S)-5a was observed [10, 22]. This was surprising since it is known that the prevailing alcohol dehydrogenase in I. brevis is the one described as LBADH [23] and since, moreover, this enzyme does not reduce P-keto 5-hydroxy ester 2a to the corresponding dihydroxy ester (Scheme 2.2.7.6). Under the conditions tested, further alcohol dehydrogenase activity is clearly present in I. brevis and I. kefir. Pfruender et al. optimized the production of L. kefir cells and used this biocatalyst for the one-pot synthesis of dihydroxy ester syn-(3R,5S)-5a using diketo ester la as starting material [24]. 

Scheme 3.1.1 Enantio- and diastereoselective enzymatic synthesis of l-phenylpropane-1, 2-diol stereoisomers in a reaction sequence. recLbADH, recombinant ADH from Lactobacillus brevis Th.sp.-ADH, ADH from Thermoanaerobium species. After crystallization ee > 99%.

Lactobacillus brevis - [ANTIBIOTICS-ELFAMYCINS] (Vol 2) -inhibited by sorbates [SORBIC ACID] (Vol 22)... 

The ADH from Lactobacillus brevis (Riebel, 1997) has a broad substrate specificity and converts even bulky aromatic ketones with high activity (Hummel, 1999 Wolberg, 2001). In addition, the enzyme is the best characterized completely (R)-specific ADH. The enzyme belongs to the class of short-chain dehydrogenases and its 3D structure has recently been solved (Niefind, 2003). The recombinant form of L. brevis ADH in E. coli accepts a variety of /j.d-dikelo esters as was determined in the synthesis of potential building blocks for HMG CoA reductase inhibitors (see also Chapter 13, Section 13.3.2) (Wolberg, 2001). tert-Butyl 3,5-dioxohexanoate and tert-butyl 3,5-dioxoheptanoate were reduced on a preparative scale to afford the corresponding (R)-<5-hydroxy-/3-keto esters with 99.4% e.e. and 98.1% e.e., respectively. 

ADH from Lactobacillus brevis afforded a 72% yield of enantiopure tert-butyl (S)-6-chloro-5-hydroxy-3-oxohexanoate ... 

The crystal structure of R-spedfic alcohol dehydrogenase from Lactobacillus brevis suggests the structural basis of its metal dependency, J. Mol. Biol. 2003, 327, 317-328. 

W. Hummel, and D. Schomburg, Crystallization and preliminary characterization of crystals of R-alcohol dehydrogenase from Lactobacillus brevis, Acta Crystallogr D Biol Crystallogr. 2000, 56, 1696-1698. 

Another way to statin side chains, via the intermediate syn-(i K,5,S )-6-chloro-hexanoate, employs regioselective and (K)-specific reduction with alcohol dehydrogenase (ADH) from Lactobacillus brevis to yield the intermediate (5S)-6-chloro-3-ketohexanoate from the 3,5-diketo acid (Wolberg, 2001) (Figure 13.16). Further reduction of (5S)-6-chloro-3-ketohexanoate to syn-(3R,5S)-6-chlorohexanoate is afforded chemically with NaBH4/B(OMe)Et2. 

L. brevis Lactobacillus brevis L. lactis Lactococcus lactis Candida magnoliae ... 

Purification of Alcohol Dehydrogenase from Lactobacillus brevis.168... 

LBADH [90] (.Lactobacillus brevis)a) ADH (Isoenzymes I-III) [91] (Saccharomyces cerevisiae) ... 

The ADH from Lactobacillus brevis can be purified by exactly the same chromatographic procedures as applied for the purification of L. kefir ADH. 

Table 12. Purification of alcohol dehydrogenase from Lactobacillus brevis. (Phenyl-and octylsepharoses are materials for hydrophobic interaction chromatography Mono Q is an anionic exchanger.)...

Table 13. Substrate specificity of alcohol dehydrogenase from Lactobacillus brevis ...

Fig. 3. Alignment of the JV-terminal sequence of the ADH from Lactobacillus brevis and homo-logues proteins.

The primary structure of the ADH from L. brevis contains several structures which are typical for short-chain ADHs. The N-terminus, with a length of approximately 30 amino acids, is widely regarded as the coenzyme binding site with the conserved motif G-X-X-X-G-X-G, which is G-G-T-L-G-I-G for Lactobacillus brevis. A second conserved domain found in the L. brems-ADH sequence is a hydrophobic region comprising 10 or 11 residues, respectively. It contains two highly conserved glycines (G82 and G92), separated by nine amino acids. Such structures seem to be located inside the protein and determine the conformation of the enzyme. 

Fig. 4. DNA and protein sequence of the recombinant (R)-alcohol dehydrogenase from Lactobacillus brevis in E. coli (upper line DNA sequence lower line corresponding amino acid in one letter code). The sequences of the primers are given in bold-type, sequences obtained by amino acid sequencing are underlined...

Hydroxyhept-6-enoates have been key intermediates in the synthesis of a variety of natural products, especially of the arachidonic acid metabolic pathway, including prostaglandins, leukotrienes, and isoprostanes [105, 113-118]. The usage of two enantiocomplementary enzymes allowed convenient access to both enantiomers via an ADH-catalyzed reduction of 5-oxo-hept-6-enoate. Alcohol dehydrogenase from Lactobacillus brevis (ADH-LB) furnished the (S )-enantiomer, Thermoanaerobacter sp. ADH (ADH-T) the (7 )-enantiomer in excellent enantiomeric access respectively [119]. A cross-metathesis reaction followed by cyclopropanation led to the formal synthesis of constanolactones C and D (Fig. 16) [86, 120, 121]. 

---