Catalytic residues, conserved

A prior distribution for sequence profiles can be derived from mixtures of Dirichlet distributions [16,51-54]. The idea is simple Each position in a multiple alignment represents one of a limited number of possible distributions that reflect the important physical forces that determine protein structure and function. In certain core positions, we expect to get a distribution restricted to Val, He, Met, and Leu. Other core positions may include these amino acids plus the large hydrophobic aromatic amino acids Phe and Trp. There will also be positions that are completely conserved, including catalytic residues (often Lys, GIu, Asp, Arg, Ser, and other polar amino acids) and Gly and Pro residues that are important in achieving certain backbone conformations in coil regions. Cys residues that form disulfide bonds or coordinate metal ions are also usually well conserved. 

Each CHS monomer consists of two structural domains (Fig. 12.5, left). The upper domain exhibits the a-p-a-p-a pseudo-symmetric motif observed in fatty acid P-ketoacyl synthases (KASs) (Fig. 12.5, right).20 Both CHS and KAS use a cysteine as a nucleophile in the condensation reaction, and shuttle reaction intermediates via CoA thioester-linked molecules or ACPs, respectively. The conserved architecture of the upper domain maintains the three-dimensional position of the catalytic residues of each enzyme Cysl64, His303, and Asn336 in CHS correspond to a Cys, His, and His in KAS I and II. 

Figure 12.5 A. Comparison of the CHS monomer (left) and P-ketoacyl synthase monomer (right). The structurally conserved secondary structure of each monomer s upper domain is colored in blue (a-helix) and gold (P-strand). Portions of each protein monomer forming the dimer interface are colored purple. The side-chains of the catalytic residues of CHS (Cysl64, His303, Asn336) and P-ketoacyl synthase (Cysl63, His303, His340) are shown. B. Sequence conservation of the catalytic residues of CHS, 2-PS, p-ketoacyl synthase (FAS II), and the ketosynthase modules of 6-deoxyerythronolide B synthase (DEBS), actinorhodin synthase (ActI) and tetracenomycin synthase (TcmK). The catalytic residues are in red.

Wu, P. Y. et al. A conserved catalytic residue in the ubiquitin-conjugating enzyme family. Embo J 2003, 22, 5241-50. 

The core-enzymes, prepared in our laboratory, and containing the active centers, were successfully crystallized (Dr. Jones, Uppsala, communicated) and tertiary structures will be described in the near future. Chemical modification studies on these enzymes are currently being undertaken in our laboratory identification of important catalytic residues and location of the active centers will lead to more functional information on these enzymes. Other cellulases such as some endoglucanases from Clostridium thermocel-lum (EG A, EG B, EG D) (10) and EngA and Exg from Cellulomonas fimi (19) also contain sequences of conserved, terminally located and sometimes reiterated, amino acids. Some of these sequences are preceded by proline-serine rich domains. Thus, a bistructural-bifunctional organization seems to be a rather common feature among cellulases, at least for EngA and Exg from C. fimi and the enzymes from Trichoderma reesei. 

Further analysis was done on the multiple sequence alignment of the H. pylori protein with members of the AstE AspA family. Amino acid residues corresponding to bovine carboxypep-tidase A responsible for Zn binding (Glu-72, His-69), carboxy-late binding (Arg-145), and catalytic residues (Glu-270) (58) are conserved in the H. pylori sequence. However, Zn-binding residues corresponding to His-69 are substituted by Gin, suggesting the possibility that this H. pylori protein is related to this enzymatic family (Fig. 8). 

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. 

---