Diaminopropionate Ammonia-lyase from Sulmonellu typhimurium PURIFICATION AND CHARACTERIZATION OF THE CRYSTALLINE ENZYME, AND SEQUENCE DETERMINATION OF THE PYRIDOXAL 5’-PHOSPHATE BINDING

We have found a wide occurrence of a,@-diaminopro- pionate ammonia-lyase in bacteria and actinomycetes. Considerable amounts of this enzyme were found in Salmonella typhirnurium. The enzyme was purified and crystallized from S. typhimurium (IF0 12529). The relative molecular mass of the native enzyme, estimated by the ultracentrifugal equilibrium method, is 89,000 Da, and the enzyme consists of two subunits identical in molecular mass. The enzyme exhibits ab- sorption maxima at 278 and 413 nm and contains 2 mol of pyridoxal 5’-phosphate(pyridoxal-P)/mol of en- zyme. The enzyme catalyzes the a,@-elimination reaction of both L- and D-a,@-diaminopropionate, the most suitable substrates, to form pyruvate and ammonia. The L- and D-isomers of serine were also degraded, though slowly. After the internal Schiff base with pyr- idoxal-P had been reduced with sodium borohydride, followed by trypsin or lysyl endopeptidase digestion of the enzyme, we determined the sequence of about 20 amino acid residues around the lysine residue which binds pyridoxal-P. No homology was found in either the amino acid sequence of the pyridoxal-P binding peptide or the amino-terminal amino acid sequence between the enzyme

' The abbreviation used is: DAP, a$-diaminopropionate. out. Little information is available on the enzyme, and many of its physicochemical and catalytic properties remain unclear. It is noteworthy that the enzyme is the first reported pyridoxal-P-dependent enzyme that catalyzes elimination of the amino group at the @position.
In the present study, we purified and crystallized the DAP ammonia-lyase from Salmonella typhimurium. We characterized the crystalline enzyme in detail and determined the amino acid sequence of the pyridoxal-P binding peptide of the enzyme.

DISCUSSION
The study described here dealt with the enzymological and physicochemical characterization of the Salmonella DAP ammonia-lyase. The enzyme was purified and crystallized from S. typhimurium, as DAP ammonia-lyase occurs most abundantly in this species. Previously, DAP ammonia-lyase was shown to be present in a pseudomonad for the first time and purified by Vijayalakshmi et al. (10). However, the homogeneity of the enzyme purified from the pseudomonad seems to be doubtful in view of its catalytic and structural properties. Our crystalline enzyme catalyzes the a,p-elimination of L-DAP to pyruvate at the rate of 46.9 pmol/min/mg of protein at 30 "C. However, the highest specific activity of the Pseudomonas enzyme was only 4.0 pmollminlmg of protein at 37 "C (10). Although the molecular mass (89,000) and subunit structure (two subunits) of the Salmonella DAP ammonialyase resembled those of the Pseudomonas enzyme (lo), the pyridoxal-P contents and absorption spectra are different (Salmonella enzyme, 2 mol of pyridoxal-P/mol of the enzyme, Xmax = 413 nm; Pseudomonas enzyme, 1 mol of pyridoxal-P/ mol of the enzyme, X , , , = 388 nm). The Pseudomonas enzyme was highly specific for L-DAP, D-DAP being about 10% as effective as L-DAP as a substrate, although the optical purity of the D-compound was unclear (10). None of the structurally related compounds tested were affected by the enzyme (10). As to the Salmonella enzyme, the D-isomer of DAP was about 66.3% as effective as L-DAP as a substrate. D-Serine acted as a more preferred substrate than the L-isomer, though their activities were much lower than those of L-and D-DAP. Other physicochemical and catalytic properties of the Pseudomonas enzyme remain unknown.
Portions of this paper (including "Experimental Procedures," "Results," Figs. 1-8, and Tables I-VI) are presented in miniprint at the end of this paper. Miniprint is easily read with the aid of a standard magnifying glass. Full size photocopies are included in the microfilm edition of the Journal that is available from Waverly Press.
The Salmonella enzyme is very unique in that it acts on not only L-isomers but also D-isomers of DAP and serine. In addition, both L-and D-alanine competitively inhibited the enzyme. Thus, the Salmonella DAP ammonia-lyase has almost no optical specificity for substrates, although its substrate specificity, itself, is very high. It is known that tyrosine phenol-lyase also catalyzes the a,@-elimination reaction of both L-and D-serine (47), which is likely to be related to the enzyme ability to catalyze the slow racemization of alanine (48). By contrast, the racemization of alanine and methionine was not catalyzed by DAP ammonia-lyase, even though a large amount of the enzyme was added and the incubation was carried out for a long time. The D-and L-isomers of 3chloroalanine which are known as suicide substrates for some pyridoxal-P-dependent enzymes were also attacked by the enzyme at very low rates. The removal of the a-proton of 3chloroalanine results in the simultaneous elimination of C1from the @-carbon position. Thus, the Salmonella enzyme can remove the a-proton from both the L-and D-isomers without racemization. Detailed molecular mechanisms of the nonstereospecific proton subtraction by the enzyme are unknown.
Multiple pyridoxal-P enzyme reactions such as the @-replacement reaction and the reverse reaction of a,@-elimination were not detected with the DAP ammonia-lyase. The enzyme thus belongs to a group of pyridoxal-P dependent enzymes including D-serine dehydratase (EC 4.2.1.4) and Lserine dehydratase (EC 4.2.1.13) and is the only enzyme that can eliminate an amino group at the &carbon position.
The amino acid sequence of the DAP ammonia-lyase including the active site lysyl residue constitutes additional information as to the active site structures of more than 20 pyridoxal-dependent enzymes thus far studied (44). A computer search for sequence homology has been carried out with the NBRF protein sequence data bank (National Biomedical Research Foundation) using a program for local homology alignment with the Integrated Database and Extended Analysis System (IDEAS) (46) executed on a FACOM M-380Q computer at the Institute for Chemical Research, Kyoto University. However, no significant homology of the active site sequence of DAP ammonia-lyase with those of pyridoxal enzymes and other proteins with known primary structures has been found. The partial sequence in the N-terminal region (22 residues) also showed no homology with the sequences of about 3500 proteins in the NBRF data base. . soiutron was centrlfuged to remove them before the enzyme began to crystallize. The enzyme began to crystallize raprdly at 5-C. The presence of crystalllzed materials was evldent from t h e silky sheen whlch appeared whan the m i x t u r e w a s strrred. The crystalllzed enzyme was collected by centrlfuqatlon and then dlssalved ~n a m l n l m u m volume of t h e same buffer. The enavme was recrvstalllzed by the addrtlon of solld ammonium sulfate to about 3 5 % aaturatlon.
This recrystalllzatlon procedure was repeated t w l c e . A summary of the purlflcation procedures is presented in TableP.
Winter and Xarlson 1161. The denslty gradient used, wlth a pH range of 3 - The lsoelectrlc polnt of the enzyme w a s deterrnlned a s descrlbed by mgl, which had been exhaustively dlalyzed aqarnst 1.3 M glyclne, was applred . .  Histidine l i r y r n l n e