L-aspartate-a-decarboxylase

In contrast to mammals, /i-alanine 3 is generated in Escherichia coli [12] mainly by decarboxylation of L-aspartate 4 [13] (Scheme 1.6.3). The tetrameric enzyme, l-aspartate-a-decarboxylase (EC 4.1.1.15), was isolated from E. coli [13], cloned [14], and its crystal structure [15] was determined. In bacteria, pantothenate synthase converts /(-alanine to pantothenate, a constituent of coenzyme A [16],... 

Glide docking into L-aspartate a-decarboxylase 333,761 compounds including Maybridge, ZINC, NCI, and FDA drugs were docked in the crystal structure and then narrowed to 703 hits and further limited to 28 and then eight compounds. No experimental validation was reported Sharma et al. (90)... 

L-aspartate-a-decarboxylase, which leads to the formation of p-alanine, and further nitrogenous products are formed, as shown in Fig. 15.2. 

Konst, P.M., Franssen, M.C.R., Scott, E.L., Sanders, J.P.M., 2009. A study on the applicability of L-aspartate a-decarboxylase in the biobased production of nitrogen containing chemicals. Green Chemistry 11, 1646-1652. 

P. Strop, H. Gehring, J. N. Jansonius, and P. Christen, Conversion of aspartate aminotransferase into an L-aspartate /3-decarboxylase by a triple active-site mutation, J. Biol. Chem. 1999, 274, 31203-31208. 

A number of decarboxylase enzymes have been described as catalysts for the preparation of chiral synthons, which are difficult to access chemically (see Chapter 2).264 The amino acid decarboxylases catalyze the pyridoxal phosphate (PLP)-dependent removal of C02 from their respective substrates. This reaction has found great industrial utility with one specific enzyme in particular, L-aspartate-P-decarboxylase (E.C. 4.1.1.12) from Pseudomonas dacunhae. This biocatalyst, most often used in immobilized whole cells, has been utilized by Tanabe to synthesize L-alanine on an industrial scale (multi-tons) since the mid-1960s (Scheme 19.33).242-265 Another use for this biocatalyst has been the resolution of racemic aspartic acid to produce L-alanine and D-aspartic acid (Scheme 19.34). The cloning of the L-aspartate-P-decarboxylase from Alcaligenes faecalis into E. coli offers additional potential to produce both of these amino acids.266... 

Fig. 8.22 The biosynthesis of [R]-pantothenate in E. coli [112]. Enzymatic activities ADC, L-aspartate-1 -decarboxylase KPHM, a-ketopantoate hydroxymethyltransferase KPR, a-ketopantoate reductase PS, pantothenate synthase.

A continuous production system using immobilized Pseudomonas dacunhae cells with high L-aspartate 3-decarboxylase activity is currently under investigation [12]. The reaction proceeds as shown below. 

Production of L-alanine by decarboxylation of L-aspartic acid under the action of the intracellular L-aspartate-P-decarboxylase in Pseudomonas dacunhae (Tanabe Seiyaku Co., Ltd). A 1000-liter pressurized column bioreactor can typically yield 5 tons of L-alanine per month. 

I)-Aspartases from Escherichia coli and Brevibacterium flavum catalyse the stereospecific addition of ammonia to fumaric acid. Nanning Only-Time in China, Kyowa Hakko Kogyo and Tanabe Seiyaku in Japan produce aspartic acid accordingly. Using an (L)-aspartate-jS-decarboxylase, alanine can be prepared in a subsequent step as well. [62]... 

L-Aspartate- 8-decarboxylase, a pyridoxal 5 -phosphate enzyme, catalyzes the y8-decarboxylation of L-aspartate to L-alanine [Eq. (1)] as... 

Amino Acid Systems Glutamine binding sites, 46, 414 labeling of the active site of r-aspartate /3-decarboxylase with yS-chloro-r-ala-nine, 46, 427 active site of r-asparaginase reaction with diazo-4-oxonorvaline, 46, 432 labeling of serum prealbumin with N-bro-moacetyl-L-thyroxine, 46, 435 a pyridoxamine phosphate derivative, 46, 441. 

Also Tanabe have extended the use of that aspartic acid producing process by using the L-aspartic acid as the substrate for L-alanine production using P. dacunae cells with L-aspartate decarboxylase activity. This process has been operating since 1982 using sequential colunms of iimnobihsed E. coli and P. dacunae cells (Chibata, Tosa and Takamatsu, 1987). Also, DL-aspartic acid can be used as the feed in this process. Then, D-aspartic acid is obtained as an additional product, for which there is a modest demand. 

As very high degrees of conversion are achieved in a PFR with much smaller reactor volumes than in a CSTR (Lilly, 1976 Vieth, 1976) a PFR with immobilized enzymes is most suitable if complete conversion has to be achieved, as in the case of isomerization of glucose or the decarboxylation of D,L-aspartate with L-aspar-tate-/l-decarboxylase. 

Because of the difficulties of stabilizing the pH at the pH optimum of 6.0 owing to liberation of C02, a loop reactor was developed which keeps the C02 dissolved at lObar and thus helps to stabilize the pH. Co-immobilization of E. coli and Ps. dacunhae cells for direct production of L-alanine from fumaric acid was not successful because E. coli cells work best at a pH of 8.5, in comparison with a pH of 6.0 for Ps. dacunhae cells and the decarboxylase. The sequential process has been run since 1982. The high enantioselectivity of L-aspartate-/kdecarboxylase (ADC) led to a process, in 1989, in which inexpensive DL-aspartate was converted to L-alanine and D-aspartate [Eq. (7.1)] the latter commands interest for synthetic penicillins ... 

In addition to resolution approaches, there are three main methods to prepare amino acids by biological methods addition of ammonia to an unsaturated carboxylic acid the conversion of an a-keto acid to an amino acid by transamination from another amino acid, and the reductive animation of an a-keto acid. These approaches are discussed in Chapter 19 and will not be discussed here to avoid duplication. The use of a lyase to prepare L-aspartic acid is included in this chapter as is the use of decarboxylases to access D-glutamic acid. 

A similar process is also used for the production of L-malic acid from fumarate, in this case using a hydratase enzyme derived from Brevibacterium ammoniagenes. Another variation of the Tanabe technology involves the synthesis of L-alanine from L-aspartic acid through the use of immobilized whole cells (P dacunae) containing aspartate-decarboxylase. 

For two transaminases the remaining unknown stereochemical parameter was determined by demonstrating an internal transfer of tritium (dialkyl amino acid transaminase) [28] or deuterium (pyridoxamine-pyruvate transaminase) [27] from the a-position of the substrate L-alanine to C-4 of the cofactor. Internal hydrogen transfer from the a-position of the substrate amino acid to C-4 of PLP has also been demonstrated for two of the abortive transamination reactions, those catalyzed by tryptophan synthase fi2 protein [32] and by aspartate-/8-decarboxylase [31]. In addition, the same phenomenon must occur in alanine transaminase, as deduced from the observation that the enzyme catalyzes exchange of the /8-hydrogens of... 

The biocatalytic synthesis can be extended towards the production of L-alanine when using additionally an L-aspartate decarboxylase for a subsequent decarboxylation step. Such a process has been reported by Tanabe Seiyaku, and gave the desired L-alanine in 86% yield and with 99% ee [37]. A key feature of the decarboxylase process is the high substrate concentration of 2.5 M, and a space-time yield of 170 g/(L d) of L-alanine. Based on this two-step approach, L-alanine has been produced in annual amounts of ca. 60 tons [32b, 37]. 

The combined utilization in a single reactor of both aspartase from Brevibacterium flavum and aspartate-P-decarboxylase from Pseudomonas dacunhae, thereby catalyzing the reaction from fumaric acid via L-aspartic acid to L-alanine (5), has also been developed by Mitsubishi 5. ... 

L-Alanine is produced from L-aspartate by a one-step enzymatic method using aspartate -decarboxylase ... 

The decarboxylation of L-aspartic acid to L-alanine is catalysed by a pyridoxal-P-dependent j8-decarboxylase whose reaction mechanism is clearly different from that of the a-decarboxylases since the initial step probably involves C -H bond cleavage. The steric course at during the normal decarboxylation reaction has recently been shown [23b] to be inversion. In addition to this, however, the enzyme will also catalyse the decarboxylation of amino-malonic acid to glycine and Meister and coworkers [24,25] have shown that this process involves loss of the Si carboxyl group with overall retention at C . 

Aminomalonic acid is a substrate for aspartate /5-decarboxylase (EC 4.1.1.12). When (3R)- and (3S)-[3- C]aminomalonates were prepared from [3- C]-and [l- C]serines, respectively, and incubated with this enzyme, the 3-pro-R carboxyl group was lost (346). Since the decarboxylation had been shown to incorporate label into the 2-pro-S hydrogen of glycine (78), the decarboxylation was deemed to have occurred with retention of configuration. 

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