Histidine decarboxylase (HDC) from Lactobacillus catalyzes the reaction histidine --- greater than histamine + CO2. This particular enzyme is synthesized in an inactive form and activates itself by cleavage of the peptide bond between Ser 81 and Ser 82. As part of this process, Ser 82 is converted to a pyr which serves as the enzymatic cofactor for the decarboxylation. Chemical studies have shown that several general acids and bases must be involved in the auto-activation and catalytic processes; X-ray studies have suggested several candidates, based on their proximity to the active site. We have cloned and sequenced the genes for HDC and one activation mutant. The proteins have been expressed in E. coli from plasmids and we propose to carry our a program of oligonucleotide directed site specific mutagenesis of HDC.
We aim to analyze the contribution of various residues to both the auto-activation scheme and catalytic mechanism of the enzyme. Among the mutations to be made are the conversion of Ser 82 to both Cys and Thr. Ser 81, a conserved residue in pyruvate requiring decarboxylases which may stabilize the auto-activation intermediate, will be converted to an Ala. Lys 155, which may bind the substrate carboxyl, will be converted to an Gln. In addition, three acidic groups, Glu 197, Glu 66 and Asp 63, which are near the active site will be altered, initially to amides, while two Tyr groups, 62 and 262, which may act in auto-activation, will initially be converted to Phe.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM035989-03
Application #
3289549
Study Section
Biochemistry Study Section (BIO)
Project Start
1987-08-01
Project End
1991-07-31
Budget Start
1989-08-01
Budget End
1991-07-31
Support Year
3
Fiscal Year
1989
Total Cost
Indirect Cost
Name
University of Texas Austin
Department
Type
Schools of Arts and Sciences
DUNS #
City
Austin
State
TX
Country
United States
Zip Code
78713
Robertus, J D; Monzingo, A F; Marcotte, E M et al. (1998) Structural analysis shows five glycohydrolase families diverged from a common ancestor. J Exp Zool 282:127-32
Hollis, T; Honda, Y; Fukamizo, T et al. (1997) Kinetic analysis of barley chitinase. Arch Biochem Biophys 344:335-42
Chaddock, J A; Monzingo, A F; Robertus, J D et al. (1996) Major structural differences between pokeweed antiviral protein and ricin A-chain do not account for their differing ribosome specificity. Eur J Biochem 235:159-66
Day, P J; Ernst, S R; Frankel, A E et al. (1996) Structure and activity of an active site substitution of ricin A chain. Biochemistry 35:11098-103
Pishko, E J; Potter, K A; Robertus, J D (1995) Site-directed mutagenesis of intersubunit boundary residues in histidine decarboxylase, a pH-dependent allosteric enzyme. Biochemistry 34:6069-73
Hart, P J; Pfluger, H D; Monzingo, A F et al. (1995) The refined crystal structure of an endochitinase from Hordeum vulgare L. seeds at 1.8 A resolution. J Mol Biol 248:402-13
Pishko, E J; Robertus, J D (1993) Site-directed alteration of three active-site residues of a pyruvoyl-dependent histidine decarboxylase. Biochemistry 32:4943-8
Robertus, J D (1992) The structure of plant toxins as a guide to rational design. Targeted Diagn Ther 7:133-49
Robertus, J (1991) The structure and action of ricin, a cytotoxic N-glycosidase. Semin Cell Biol 2:23-30
Katzin, B J; Collins, E J; Robertus, J D (1991) Structure of ricin A-chain at 2.5 A. Proteins 10:251-9

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