Proc. Natl. Acad. Sci. USA Vol. 77, No. 3, pp. 1260-1264, March 1980 Biochemistry Molecular structure and intermolecular interactions of N1'-methoxycarbonylbiotin methyl ester: A model for carboxybiotin (ureido electronic configuration/evolution of a coenzyme/enzymatic carboxyl transfer/x-ray diffraction) WILLIAM C. STALLINGS*, C. T. MONTI*, M. DANIEL LANEt, AND GEORGE T. DETITTAt *Institute for Cancer Research, 7701 Burholme Avenue, Fox Chase, Philadelphia, Pennsylvania 19111; tDepartment of Physiological Chemistry, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205; and tMedical Foundation of Buffalo, Inc., 73 High Street, Buffalo, New York 14230 Communicated by David Harker, October 24, 1979 ABSTRACT The crystal structure of N1'-methoxycarbon- In the first half-reaction, enzyme-bound biotin is carboxylated ylbiotin methyl ester, a model for M'-carboxybiotin, has been at Ni', generating N1'-carboxybiotin (II). In the second half- determined. The ureido carbonyl bond has more double bond reaction, C02 is transferred, as a carboxyl group, from car- (keto) character than does.the corresponding bond in free biotin, which has single bond (enolate) character. In addition, there is boxybiotin to another molecule. Biotin enzymes are, in general, an interesting intermolecular interaction between the ureido high molecular weight, multisubunit structures; the half-re- carbonyl oxygen and a methyl group. Comrparison of the mo- actions generally occur at separate active sites which are located lecular structure and crystal packing with those of free biotin on different subunits of the enzymes. suggests. that the coenzyme may have evolved with the incor- Here, we report the molecular structure of N1'-methoxy- poration of the ureido moiety because the electronic configu- carbonylbiotin methyl ester (III), which is a model for Ni'- ration of this region of the molecule is sensitive to MN' carbox- carboxybiotin (II). Structural results were obtained by using ylation. On decarboxylation, the ureido carbonyl bond becomes more polarized (C-Os° thereby-facilitating the deprotonation the technique of single-crystal x-ray diffraction. Previous of NI' and increasing its nucleophilicity. As a result, carboxy- crystallographic studies have established the molecular struc- lation can occur readily. On carboxylation, the carbonyl bond tures of free biotin (I) (2), dethiobiotin (3), azabiotin (4), oxy- is depolarized (C==O), allowing the carboxylated coenzyme to biotin, selenobiotin, biotin methyl ester, biotin sulfone, and interact with nonpolar groups and carboxy ate them. Thus, the biotin-d-sulfoxide, and carbobiotin (5). Differences between carboxylation and decarboxylation of biotin appear to act as a the molecular geometries and therefore the electronic config- mechanistic switch, turning off and'on the polarization of the ureido ca'rbonyl bond as well as modulating the nucleophilicity urations of the ureido moieties of I and III are of great interest of NI'. because the ureido ring is the catalytically active portion of the biotin molecule. The previous studies have stressed the notion (+)-Biotin (I) is a coenzyme involved in carbon dioxide me- that, in free biotin and its'derivatives, the polarized forms of tabolism and carboxyl transfer. Structurally, the molecule the biotin resomers (those with 'formal charge separation) consists of a bicyclic ring system whose mirror symmetry is 16110 02'11 02 02' all /OCH3 >HN' HN3 1.NH HN3 1.N-COO HN3 1N-C 0 I S S iCH2A (CH2)4- COON COOH 0 OCH3 II III coax a b C broken by a valeric acid side chain; the top ring of the bicyclic system is a ureido ring and the bottom ring, containing sulfur, contribute significantly to their observed electronic structures. is a tetrahydrothiophene ring. Biotin is covalently attached to As will be pointed out, this is less the case for the carboxylated an enzyme by means of an amide linkage between the side derivative (III). In addition, we shall also detail some aspects chain carboxyl group of biotin and the E amino group of a lysine of the intermolecular interactions which occur in the crystal residue of the enzyme. structure and which have possible biochemical relevance. Biotin enzymes (1) (carboxylases, transcarboxylases, and decarboxylases) function by means of a two-step reaction se- quence: EXPERIMENTAL Nl'-Methoxycarbonylbiotin methyl'ester, C13H20N205S (III), Enz-biotin + donor-COy - Enz-biotin-CO- + donor was synthesized according to the procedure described by Enz-biotin-CO- + acceptor - Enz-biotin + acceptor- Knappe et al. (6); the crystals are clear, colorless needles. The dimensions of the crystal used were 0.35 X 0.07 X 0.08 mm. The CO2. space group is P212121 with a = 24.737(4), b = 26.997(4), and c = A with Z = there are two molecules in the The publication costs of this article were defrayed in part by page 4.6469(4) 8; charge payment. This article must therefore be hereby marked "ad- asymmetric unit. The calculated density is 1.35 g-cm-3. vertisement" in accordance with 18 U. S. C. §1734 solely to indicate Three-dimensional x-ray intensity data were collected on an this fact. automated diffractometer with the 6-20 scan technique and 1260 Downloaded by guest on September 25, 2021 Biochemistry: Stallings et al. Proc. Natl. Acad. Sci. USA 77 (1980) 1261 Table 1. Positional parameters* for N1'-methoxycarbonylbiotin methyl ester Molecule A Molecule B Atom x y z x y z S 0.3661 0.0458 0.3300 0.2656 0.6212 0.4716 02' 0.5014 0.1660 0.183 0.4349 0.5519 0.675 Ni' 0.4206 0.1570 0.438 0.3620 0.5344 0.371 N1'-Methoxycarbonylbiotifn N3' 0.4846 0.1008 0.478 0.3977 0.6098 0.377 methyl ester C2' 0.4722 0.1437 0.346 0.4023 0.5647 0.497 C 0.3929 0.2007 0.385 0.3564 0.4837 0.391 0 0.3511 0.2094 0.509 0.3237 0.4620 0.247 O lOb 4 O' 0.4154 0.2289 0.194 0.3882 0.4633 0.587 C' 0.3893 0.2751 0.118 0.3813 0.4117 0.632 C2 0.4213 0.0315 0.576 0.3123 0.6520 0.230 C3 0.4443 0.0824 0.674 0.3553 0.6142 0.158 C4 0.3991 0.1225 0.648 0.3293 0.5616 0.161 1404 o IO& C5 0.3465 0.0977 0.559 0.2710 0.5661 0.252 C6 0.4622 -0.0043 0.444 0.3337 0.7010 0.351 C7 0.4365 -0.0558 0.388 0.2895 0.7394 0.391 C8 0.4741 -0.0916 0.234 0.3108 0.7878 0.500 C9 0.4460 -0.1398 0.185 0.2689 0.8264 0.545 Cdo 0.4748 -0.1755 -0.015 0.2914 0.8740 0.681 OlOa 0.5171 -0.1688 -0.127 0.3337 0.8735 0.804 OlOb 0.4470 -0.2174 -0.048 0.2651 0.9101 0.660 ClH 0.4694 -0.2546 -0.232 0.2859 0.9536 0.825 HN3' 0.521 0.088 0.46 0.421 0.634 0.36 HiC' 0.398 0.299 -0.11 0.412 0.400 0.71 H2C' 0.347 0.267 0.06 0.350 0.400 0.73 H3C' 0.377 0.289 0.25 0.390 0.395 0.48 HC2t 0.403 0.014 0.76 0.290 0.660 0.04 HC3t 0.459 0.080 0.89 0.373 0.622 -0.05 O l0b HC4t 0.394 0.140 0.86 0.332 0.545 -0.05 1207 HlC5t 0.325 0.084 0.75 0.246 0.571 0.06 o0 H2C5t 0.321 0.123 0.44 0.259 0.534 0.37 HlC6t 0.495 -0.009 0.60 0.364 0.715 0.21 Biotin H2C6t 0.478 0.010 0.25 0.352 0.693 0.56 FIG. 1. (Upper) Bond lengths determined for N1'-methoxycar- HlC7t 0.401 -0.050 0.26 0.260 0.725 0.55 bonylbiotin methyl eter. Values for molecule A are above those for H2C7t 0.425 -0.072 0.59 0.269 0.746 0.19 molecule B. The average estimated standard deviation is 0.009 A. HlC8t 0.510 -0.097 0.36 0.341 0.801 0.35 (Lower) Bond lengths determined for free biotin in a previous study H2C8t 0.484 -0.076 0.02 0.332 0.781 0.71 (2). HlC9t 0.405 -0.133 0.10 0.237 0.812 0.68 H2C9t 0.442 -0.158 0.40 0.252 0.836 0.34 at calculated positions and their positional and thermal pa- HlCll 0.446 -0.289 -0.19 0.249 0.969 0.84 rameters were not refined. The quantity minimized in the H2Cll 0.469 -0.240 -0.42 0.306 0.932 1.05 least-squares calculations was wf IIFoI - IFcI J2. The weights, H3Cll 0.501 -0.259 -0.21 0.322 0.961 0.77 w, were o-2(F), with zero weight for those reflections below the threshold value. Published atomic scattering factors (7, 8) * The estimated standard deviations for carbon, nitrogen, and oxygen were used in all calculations. The starting material in the syn- atoms average 3 X 10-4, 2 X 10-4, and 2 X 10-3 for x, y, and z, re- spectively. Equivalent values for the sulfur atoms are 1 X 10-4, 1 thesis of Nl'-methoxycarbonylbiotin methyl ester was X 10-4, and 4 X 10-4; for refined hydrogen atoms, these values av- biotin, whose absolute configuration has been established (9); erage 4 X 10-3, 3 X 10-3, and 2 X 10-2.