Amino acids, cellobiose

Since the primary structure of a peptide determines the global fold of any protein, the amino acid sequence of a heme protein not only provides the ligands, but also establishes the heme environmental factors such as solvent and ion accessibility and local dielectric. The prevalent secondary structure element found in heme protein architectures is the a-helix however, it should be noted that p-sheet heme proteins are also known, such as the nitrophorin from Rhodnius prolixus (71) and flavocytochrome cellobiose dehydrogenase from Phanerochaete chrys-osporium (72). However, for the purpose of this review, we focus on the structures of cytochromes 6562 (73) and c (74) shown in Fig. 2, which are four-a-helix bundle protein architectures and lend themselves as resource structures for the development of de novo designs. 

The CBH I (D) is identical in composition and activity to the CBH I (D) previously described (2) from T. reesei QM 9123. The close correspondence of their amino acid contents (Table VI), the nearly identical content of neutral carbohydrate 6.8% by weight for the CBH I (D) produced in the presence of sophorose and 6.7% for T. reesei QM 9123 CBH I (D) grown on cellulose (2), and identical electrophoretic properties clearly argue for a common molecular structure for these CBH s I (D). The CBH II is clearly different from all other CBH s in electrophoretic mobility (Figure 12) and amino acid composition (41), but is devoid of endoglucanase activity and produces predominantly cellobiose (>90% by weight of soluble products) from cellulose. It has a sedimentation coefficient of 3.71 in comparison to CBH I (D), for which a value of 3.66 was obtained. 

Oxidation of two out of 13 tryptophan residues in a cellulase from Penicillium notatum resulted in a complete loss of enzymic activity (59). There was an interaction between cellobiose and tryptophan residues in the enzyme. Participation of histidine residues is also suspected in the catalytic mechanism since diazonium-l-H-tetrazole inactivated the enzyme. A xylanase from Trametes hirsuta was inactivated by N-bromosuc-cinimide and partially inactivated by N-acetylimidazole (60), indicating the possible involvement of tryptophan and tyrosine residues in the active site. As with many chemical modification experiments, it is not possible to state definitively that certain residues are involved in the active site since inactivation might be caused by conformational changes in the enzyme molecule produced by the change in properties of residues distant from the active site. However, from a summary of the available evidence it appears that, for many / -(l- 4) glycoside hydrolases, acidic and aromatic amino acid residues are involved in the catalytic site, probably at the active and binding sites, respectively. 

Physical or chemical modification of a substrate may additionally selectively affect transformation or uptake Keil and Kirchman (1992) compared the degradation of Rubisco uniformly labeled with 3H amino acids produced via in vitro translation to Rubisco that was reductively methylated with 3H-methane. Although both Rubisco preparations were hydrolyzed to lower molecular weights at approximately the same rate, little of the methylated protein was assimilated or respired. The presence of one substrate may also inhibit uptake of another, as has been demonstrated for anaerobic rumen bacteria. Transport and metabolism of the monosaccharides xylose and arabinose were strongly reduced in Ruminococcus albus in the presence of cellobiose (a disaccharide of glucose), likely because of repression of pentose utilization in the presence of the disaccharide. Glucose, in contrast, competitively inhibited xylose transport and showed noncompetitive inhibition of arabinose transport, likely because of inactivation of arabinose permease (Thurston et al., 1994). 

One of the cellobiose oxidoreductases present in S. pulverulentum has been characterised and named cellobiose oxidase (Ander and Eriksson, 1978). The enzyme contains both haem and flavin co factors and binds irreversibly to concanavalin A-Sepharose, suggesting that it is a glycoprotein. Cellobiose oxidase from S. pulverulentum has now been purified to homogeneity by Morpeth (1985). The carbohydrate and amino acid compositions of the enzyme have been determined. The enzyme contains FAD and cytochrome b prosthetic groups and is a monomer with an Mr of 74400 determined by sedimentation equilibrium. 

Thus far, EMRs have been successfully used with macromolecular substrates, as for the saccharification of cellulose11-13 15 26 and protein hydrolysis,19 or with low molecular weight substrates, as for cellobiose hydrolysis9 and L-malic acid production from fumaric acid.20 Bench and large scale plants are already in operation for the preparation of N-acetyl-D,L derivatives from L-amino acids by means of acylase and the production of L-malic acid from fumaric acid by means of fumarase.20 In the case of fumarase, conversions of up to 86% with resolution rates of up to 85% have been attained. 

Figure 2 Unrooted phylogenetic tree for eight protein sequences of cellobiose dehydrogenases. The numbers in nodes represent bootstrap values for 100 replicates. The scale bar indicates the branch length corresponding to 0.1 amino acid substitutions per site.

Cellulases enzymes which hydrolyse cellulose to cellobiose, and which are found in plants, bacteria and fungi, TTie C. of Penicillium notatum is well-studied it consists of 324 amino acid residues (M, 35,000) with a disulfide bridge and no free SH groups. C. are used in digestion tablets, to remove undesired cellulose in foods, and for the preparation of cellobiose from cellulose. 

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