HEADER RACEMASE 06-JUL-93 2MNR COMPND MANDELATE RACEMASE (E.C.5.1.2.2) SOURCE (PSEUDOMONAS PUTIDA) (ATCC 12633) STRUCTURAL GENE EXPRESSED SOURCE 2 IN (ESCHERICHIA COLI) AUTHOR D.J.NEIDHART,G.A.PETSKO REMARK 1 REMARK 1 REFERENCE 1 REMARK 1 AUTH D.J.NEIDHART,P.L.HOWELL,G.A.PETSKO,V.M.POWERS,R.LI, REMARK 1 AUTH 2 G.L.KENYON,J.A.GERLT REMARK 1 TITL MECHANISM OF THE REACTION CATALYZED BY MANDELATE REMARK 1 TITL 2 RACEMASE. 2. CRYSTAL STRUCTURE OF MANDELATE REMARK 1 TITL 3 RACEMASE AT 2.5 ANGSTROMS RESOLUTION: REMARK 1 TITL 4 IDENTIFICATION OF THE ACTIVE SITE AND POSSIBLE REMARK 1 TITL 5 CATALYTIC RESIDUES REMARK 1 REF BIOCHEMISTRY V. 30 9264 1991 REMARK 1 REFN ASTM BICHAW US ISSN 0006-2960 033 REMARK 1 REFERENCE 2 REMARK 1 AUTH D.J.NEIDHART,G.L.KENYON,J.A.GERLT,G.A.PETSKO REMARK 1 TITL MANDELATE RACEMASE AND MUCONATE LACTONIZING REMARK 1 TITL 2 ENZYME ARE MECHANISTICALLY DISTINCT AND REMARK 1 TITL 3 STRUCTURALLY HOMOLOGOUS REMARK 1 REF NATURE V. 347 692 1990 REMARK 1 REFN ASTM NATUAS UK ISSN 0028-0836 006 REMARK 2 REMARK 2 RESOLUTION. 1.9 ANGSTROMS. REMARK 3 REMARK 3 REFINEMENT. REMARK 3 PROGRAM XPLOR; PROLSQ REMARK 3 AUTHORS A.BRUNGER; W.HENDRICKSON REMARK 3 R VALUE 0.162 REMARK 3 RMSD BOND DISTANCES 0.014 ANGSTROMS REMARK 3 RMSD BOND ANGLES 2.7 DEGREES REMARK 3 REMARK 3 NUMBER OF REFLECTIONS 25592 REMARK 3 RESOLUTION RANGE 6.0 - 1.9 ANGSTROMS REMARK 3 DATA CUTOFF 1.0 SIGMA(F) REMARK 4 REMARK 4 MANDELATE RACEMASE IS TIGHTLY PACKED AS AN OCTAMER IN THE REMARK 4 CRYSTALLINE STATE AND IS PROBABLY OCTAMERIC IN SOLUTION. REMARK 4 ONE OF THE TWO-FOLD INTERFACES IS ESPECIALLY INTIMATE AND REMARK 4 PROVIDES INTERPENETRATION OF THE ACTIVE SITE OF THE REMARK 4 TWO-FOLD RELATED SUBUNIT. THUS, MANDELATE RACEMASE IS REMARK 4 PROBABLY BEST DESCRIBED AS A TETRAMER OF DIMERS. ONE OF REMARK 4 THESE FUNCTIONAL DIMERS MAY BE GENERATED BY APPLYING THE REMARK 4 SYMMETRY OPERATION (X, -Y, -Z) TO THE SUPPLIED MONOMERIC REMARK 4 COORDINATES. THE ORIGIN OF THE SUPPLIED COORDINATES LIES REMARK 4 ON THE 422 POINT OF SYMMETRY AT THE CENTER OF THE OCTAMER. REMARK 5 REMARK 5 MANDELATE RACEMASE ABSOLUTELY REQUIRES A DIVALENT METAL REMARK 5 ION FOR ACTIVITY AND IS MOST ACTIVE WITH A MAGNESIUM (II) REMARK 5 ION BOUND IN THE ACTIVE SITE. IN THIS CRYSTAL STRUCTURE, REMARK 5 MANGANESE (II) WAS EXCHANGED INTO THE ACTIVE SITE IN ORDER REMARK 5 TO FACILITATE X-RAY REFINEMENT OF THE POSITION OF THE BOUND REMARK 5 METAL ION. MANGANESE (II) SUBSTITUTED MANDELATE RACEMASE REMARK 5 IS ABOUT 48% AS ACTIVE AS HOLOENZYME CONTAINING MAGNESIUM REMARK 5 (II); HOWEVER, VERY LITTLE STRUCTURAL CHANGE OF THE ENZYME REMARK 5 IS APPARENT BETWEEN THESE FORMS. GIVEN THE MUCH HIGHER REMARK 5 NATURAL ABUNDANCE OF MAGNESIUM (II) VS. MANGANESE (II), IT REMARK 5 IS LIKELY THAT MAGNESIUM (II) IS THE PREDOMINANT COFACTOR REMARK 5 FOR MANDELATE RACEMASE IN VIVO. REMARK 6 REMARK 6 SITE MTL CONSTITUTES THE DIRECT METAL ION LIGANDS. REMARK 7 REMARK 7 SITE ACT CONSTITUTES THE ACID/BASE CATALYSTS RESPONSIBLE REMARK 7 FOR PROTON ABSTRACTION AND REDELIVERY IN THE RACEMIZATION REMARK 7 REACTION. REMARK 8 REMARK 8 SITE CAR CONSTITUTES THE BINDING SITE FOR THE CARBOXYL REMARK 8 GROUP OF SUBSTRATE. THESE RESIDUES FUNCTION, ALONG WITH REMARK 8 THE METAL ION, AS ELECTROPHILIC CATALYSTS. REMARK 9 REMARK 9 BETA SHEETS ARE NAMED FOR THE MAJOR DOMAINS IN WHICH THEY REMARK 9 OCCUR: "N" FOR N-TERMINAL DOMAIN, "B" FOR BETA-BARREL REMARK 9 DOMAIN, AND "C" FOR C-TERMINAL DOMAIN. "F" REFERS TO THE REMARK 9 ACTIVE SITE FLAP. LIKEWISE, ALPHA HELICES ARE NAMED WITH REMARK 9 TWO CHARACTERS, THE FIRST REFERRING TO THE DOMAIN IN WHICH REMARK 9 THEY OCCUR. REMARK 10 REMARK 10 THE SHEET PRESENTED AS *B* ON SHEET RECORDS BELOW IS REMARK 10 ACTUALLY AN EIGHT-STRANDED BETA-BARREL. THIS IS REMARK 10 REPRESENTED BY A NINE-STRANDED SHEET IN WHICH THE FIRST REMARK 10 AND LAST STRANDS ARE IDENTICAL. REMARK 11 REMARK 11 WATER MOLECULES HOH 597, HOH 598, HOH 612, AND HOH 614 LIE REMARK 11 ON TWO-FOLD AXES OF SYMMETRY WITHIN THE OCTAMERIC STRUCTURE REMARK 11 OF MANDELATE RACEMASE. REMARK 12 REMARK 12 THE E. COLI EXPRESSION VECTOR FOR MANDELATE RACEMASE REMARK 12 ENCODES AN ENZYME IN WHICH MET 1 AND SER 2 ARE ABSENT. IN REMARK 12 PSEUDOMONAS, MET 1 MAY BE REMOVED CONTRANSLATIONALLY. REMARK 12 PRELIMINARY CRYSTALLOGRAPHIC ANALYSIS OF THE PRODUCT ENZYME REMARK 12 FROM E. COLI CONSTRUCTS IN WHICH MET 1 AND SER 2 WERE REMARK 12 ENCODED INDICATED THAT MET 1 AND SER 2 ARE COMPLETELY REMARK 12 DISORDERED. THEREFORE, IN THE INTEREST OF EXPEDITING REMARK 12 CONSTRUCTION OF MUTAGENESIS VECTORS, MET 1 AND SER 2 WERE REMARK 12 DELETED FROM THE CONSTRUCTS FOR "WILD TYPE" AND MUTANT REMARK 12 ENZYMES. NO FUNCTIONAL DIFFERENCE IS NOTED BETWEEN THE REMARK 12 RESULTANT N-TERMINALLY TRUNCATED ENZYME AND ENZYME REMARK 12 EXPRESSED IN E. COLI VECTORS THAT MIGHT BE EXPECTED TO REMARK 12 EXPRESS MET 1 AND/OR SER 2 IN THE MATURE ENZYME. SEQRES 1 357 GLU VAL LEU ILE THR GLY LEU ARG THR ARG ALA VAL ASN SEQRES 2 357 VAL PRO LEU ALA TYR PRO VAL HIS THR ALA VAL GLY THR SEQRES 3 357 VAL GLY THR ALA PRO LEU VAL LEU ILE ASP LEU ALA THR SEQRES 4 357 SER ALA GLY VAL VAL GLY HIS SER TYR LEU PHE ALA TYR SEQRES 5 357 THR PRO VAL ALA LEU LYS SER LEU LYS GLN LEU LEU ASP SEQRES 6 357 ASP MET ALA ALA MET ILE VAL ASN GLU PRO LEU ALA PRO SEQRES 7 357 VAL SER LEU GLU ALA MET LEU ALA LYS ARG PHE CYS LEU SEQRES 8 357 ALA GLY TYR THR GLY LEU ILE ARG MET ALA ALA ALA GLY SEQRES 9 357 ILE ASP MET ALA ALA TRP ASP ALA LEU GLY LYS VAL HIS SEQRES 10 357 GLU THR PRO LEU VAL LYS LEU LEU GLY ALA ASN ALA ARG SEQRES 11 357 PRO VAL GLN ALA TYR ASP SER HIS SER LEU ASP GLY VAL SEQRES 12 357 LYS LEU ALA THR GLU ARG ALA VAL THR ALA ALA GLU LEU SEQRES 13 357 GLY PHE ARG ALA VAL LYS THR LYS ILE GLY TYR PRO ALA SEQRES 14 357 LEU ASP GLN ASP LEU ALA VAL VAL ARG SER ILE ARG GLN SEQRES 15 357 ALA VAL GLY ASP ASP PHE GLY ILE MET VAL ASP TYR ASN SEQRES 16 357 GLN SER LEU ASP VAL PRO ALA ALA ILE LYS ARG SER GLN SEQRES 17 357 ALA LEU GLN GLN GLU GLY VAL THR TRP ILE GLU GLU PRO SEQRES 18 357 THR LEU GLN HIS ASP TYR GLU GLY HIS GLN ARG ILE GLN SEQRES 19 357 SER LYS LEU ASN VAL PRO VAL GLN MET GLY GLU ASN TRP SEQRES 20 357 LEU GLY PRO GLU GLU MET PHE LYS ALA LEU SER ILE GLY SEQRES 21 357 ALA CYS ARG LEU ALA MET PRO ASP ALA MET LYS ILE GLY SEQRES 22 357 GLY VAL THR GLY TRP ILE ARG ALA SER ALA LEU ALA GLN SEQRES 23 357 GLN PHE GLY ILE PRO MET SER SER HIS LEU PHE GLN GLU SEQRES 24 357 ILE SER ALA HIS LEU LEU ALA ALA THR PRO THR ALA HIS SEQRES 25 357 TRP LEU GLU ARG LEU ASP LEU ALA GLY SER VAL ILE GLU SEQRES 26 357 PRO THR LEU THR PHE GLU GLY GLY ASN ALA VAL ILE PRO SEQRES 27 357 ASP LEU PRO GLY VAL GLY ILE ILE TRP ARG GLU LYS GLU SEQRES 28 357 ILE GLY LYS TYR LEU VAL HET MN 360 1 MANGANESE ++ HET SO4 361 5 SULFATE FORMUL 2 MN MN1 ++ FORMUL 3 SO4 O4 S1 FORMUL 4 HOH *211(H2 O1) HELIX 1 NA THR 55 ILE 73 1 KINKS AT ALA 58, ALA 70 HELIX 2 NB PRO 80 ALA 94 1 KINK AT ARG 90 HELIX 3 NC GLY 98 VAL 118 1 HELIX 4 ND LEU 123 LEU 127 1 HELIX 5 BA GLY 144 GLU 157 1 HELIX 6 BB LEU 172 VAL 186 1 HELIX 7 BC VAL 202 GLU 215 1 HELIX 8 BD TYR 229 SER 237 1 HELIX 9 BE PRO 252 ILE 261 1 HELIX 10 BH ALA 271 ILE 274 1 HELIX 11 BF VAL 277 PHE 290 1 HELIX 12 BG GLN 300 ALA 308 1 HELIX 13 CA ALA 322 ILE 326 1 HELIX 14 CB GLU 351 LYS 356 1 SHEET 1 N 3 LEU 5 VAL 16 0 SHEET 2 N 3 ALA 32 THR 41 -1 O ALA 40 N GLY 8 SHEET 3 N 3 VAL 45 PHE 52 -1 O VAL 45 N THR 41 SHEET 1 B 9 VAL 134 HIS 140 0 SHEET 2 B 9 ALA 162 LYS 166 1 O ALA 162 N ASP 138 SHEET 3 B 9 GLY 191 ASP 195 1 O GLY 191 N VAL 163 SHEET 4 B 9 TRP 219 GLU 221 1 O TRP 219 N VAL 194 SHEET 5 B 9 PRO 242 MET 245 1 O PRO 242 N ILE 220 SHEET 6 B 9 ARG 265 MET 268 1 N MET 268 O MET 245 SHEET 7 B 9 PRO 293 HIS 297 1 N SER 295 O ALA 267 SHEET 8 B 9 HIS 314 ARG 318 1 O TRP 315 N SER 296 SHEET 9 B 9 VAL 134 HIS 140 1 O GLN 135 N LEU 316 SHEET 1 C 3 THR 331 GLU 333 0 SHEET 2 C 3 ASN 336 VAL 338 -1 N VAL 338 O THR 331 SHEET 3 C 3 VAL 134 ALA 136 -1 O VAL 134 N ALA 337 SHEET 1 F 2 VAL 22 THR 24 0 SHEET 2 F 2 GLY 27 VAL 29 -1 O VAL 29 N VAL 22 TURN 1 T1 THR 24 GLY 27 TURN 2 T2 THR 41 GLY 44 TURN 3 T3 ILE 73 GLU 76 TURN 4 T4 GLY 187 PHE 190 TURN 5 T5 LEU 225 ASP 228 TURN 6 T6 GLN 236 LEU 239 TURN 7 T7 GLY 246 TRP 249 TURN 8 T8 THR 310 ALA 313 TURN 9 T9 GLU 333 ASN 336 SITE 1 MTL 3 ASP 195 GLU 221 GLU 247 SITE 1 ACT 2 LYS 166 HIS 297 SITE 1 CAR 2 LYS 164 GLU 317 CRYST1 125.000 125.000 106.200 90.00 90.00 90.00 I 4 2 2 16 ORIGX1 1.000000 0.000000 0.000000 0.00000 ORIGX2 0.000000 1.000000 0.000000 0.00000 ORIGX3 0.000000 0.000000 1.000000 0.00000 SCALE1 0.008000 0.000000 0.000000 0.00000 SCALE2 0.000000 0.008000 0.000000 0.00000 SCALE3 0.000000 0.000000 0.009416 0.00000