Branching enzymes structure-function

MMP inhibitor development constitutes an important branch of research in both academic and industrial settings and advances our knowledge on the structure-function relationship of these enzymes. Targeting... 

In this section, enzymes in the EC 2.4. class are presented that catalyze valuable and interesting reactions in the field of polymer chemistry. The Enzyme Commission (EC) classification scheme organizes enzymes according to their biochemical function in living systems. Enzymes can, however, also catalyze the reverse reaction, which is very often used in biocatalytic synthesis. Therefore, newer classification systems were developed based on the three-dimensional structure and function of the enzyme, the property of the enzyme, the biotransformation the enzyme catalyzes etc. [88-93]. The Carbohydrate-Active enZYmes Database (CAZy), which is currently the best database/classification system for carbohydrate-active enzymes uses an amino-acid-sequence-based classification and would classify some of the enzymes presented in the following as hydrolases rather than transferases (e.g. branching enzyme, sucrases, and amylomaltase) [91]. Nevertheless, we present these enzymes here because they are transferases according to the EC classification. 

The repair of stalled replication forks entails a coordinated transition from replication to recombination and back to replication. The recombination steps function to fill the DNA gap or rejoin the broken DNA branch to recreate the branched DNA structure at the replication fork. Lesions left behind in what is now duplex DNA are repaired by pathways such as base-excision or nucleotide-excision repair. Thus a wide range of enzymes encompassing every aspect of DNA metabolism ultimately take part in the repair of a stalled replication fork. This type of repair process is clearly a primary function of the homologous recombination system of every cell, and defects in recombinational DNA repair play an important role in human disease (Box 25-1). 

As indicated in Table 4.12, four regions which constitute the catalytic regions of amylolytic enzymes are conserved in the starch-branching isoenzymes of maize endosperm, rice seed and potato tuber, and the glycogen-branching enzymes of E. coli.286,281 It would be of interest to know whether the seven highly conserved amino acid residues of the a-amylase family listed in bold letters in Table 4.12 are also functional in branching enzyme catalysis. Further experiments, such as chemical modification and analysis of the three-dimensional structure of the BEs, would be needed to determine the nature of its catalytic residues and mechanism. 

Primary, secondary and tertiary structures of amylolytic enzymes from a wide variety of sources and functions (the a-amylases, bacterial cyclomaltodextrin glucanosyltrans-ferases, isoamylases and starch-branching enzymes) have been found to be closely related, and have been placed into the so-called structural a-amylase family.176,177 These enzymes have been studied with regard to the number, structure, organization and function of domains.178... 

It is worth noting that the understanding of the starch synthases lags behind that of the ADPGlc PPase and the branching enzymes. To cover that ground, it will be necessary to achieve expression of the plant enzymes in E. coli so that studies of structure-function relationships can be facilitated. 

The monomer-up approach opens up the possibility of enzymatically grafting branched polysaccharides from functionalized substrates in order to make hybrid materials bearing a highly branched amylose part. Following the same route we are currently synthesizing hybrid materials bearing (hyper)branched polysaccharide structures as shown in Figure 9.11 with the described tandem reaction of two enzymes. 

Indeed, studies by Kuriki et with respect to the different N- and C-termini indicate that these amino acid sequence regions are important with respect to BE specificity with respect to substrate preference (amylose or amylopectin) as well as in size of chain transferred and extent of branching. The C-terminal was functional with respect to substrate preference while the N-terminal was functional with respect to the size of chain transferred in the BE catalysis. Furthermore, truncation of 113 amino acids of the N-terminal of the E. coli BE, causes it to branch longer branch chains than the wild-type enzyme " further indicating that the N-terminal region was involved in specifying the size of chain transferred. Recently, the crystal structure of a truncated form of the E. coli branching enzyme has been elucidated. 

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