1. The 2.0 Å Crystal Structure of Cephalosporin Acylase
Youngsoo Kim, Ki-Hong Yoon, Yongho Khang, Stewart Turley, Wim G.J. Hol 
Structure 
Volume 8, Issue 10, Pages (October 2000)
DOI: /S (00)

2. Figure 1 Electron Density and Overall Structure of CAD
(a) Stereoview of part of the MAD experimental electron density map, calculated after solvent flipping and contoured at 1.3σ. The electron density is superimposed upon the refined coordinates of several active-site residues (see also Figure 3).
(b) Stereoview of the heterodimeric structure of CAD. The enzyme is a heterodimer consisting of one α subunit, in green, and one β-subunit, in purple. The view is looking down the deep active-site cleft. The N-terminal active-site residue of β-subunit (Ser1β) represented by a ball-and stick model is shown in the center. The first six and last 11 residues of the α-subunit are disordered, hence only residues Gln7α to Gly158α are depicted. All residues of the β-subunit (Ser1β to Phe520β) are shown. The label “C” for the β-subunit indicates the location of the C-terminus. The labels “N” and “C” in the α-subunit indicate the locations of Gln7α and Gly158α, respectively.
(c) An orthogonal view of Figure 1b representing four different topological regions. The central purple region represents a distinctive structural motif of the N-terminal hydrolase family. The motif consists of four layers of α-helices and β-sheets. The critical active-site residue, Ser1β, is shown in a ball-and stick representation. The part of the α-subunit consisting of seven α-helices and three α-helix turns is shown in green. The β-strand rich “B-knob” region, shown in red, consists of seven β-strands that form a distorted β-barrel-like domain. The helix-rich “A-knob” region is depicted in blue. The label “C” for the β-subunit in purple indicates the location of the C-terminus, and the labels “N” and “C” for the α-subunit in green indicate the locations of Gln7α and Gly158α, respectively
Structure 2000 8, DOI: ( /S (00) )

3. Figure 4
Sequence Alignment of the Class I Cephalosporin Acylases with Known Amino Acid Sequences
Fourteen residues in the active-site cleft of CAD are completely conserved in this class of enzymes, as shown in colored boxes. The green boxes represent the residues involved in chemical catalysis, and the yellow boxes indicate the ones forming the pocket for binding side chains of substrates. Secondary structural elements are represented as a cylinder or an arrow for an α-helix and a β-strand at the top of each sequence, respectively. The colors of secondary structural elements represent the same color code of the four structural regions as shown in Figure 1c. Where the three matching sequences are all identical, they are shown in black letters; otherwise they are shown in pink. The residue numbers are shown at the front and end of each line. Thirteen out of the 14 residues in the active-site cleft are identical among the three class I cephalosporin acylases with known sequences, and only one residue, Asn244β in CAD, is not identical. However, a side-chain amide NH of Asn244β is possibly conserved in the other two class I CAs as a side-chain NH of an equivalent arginine residue. If there are no corresponding residues in the sequences, they are marked by a hyphen. The labels “P130” and “C427” refer to CAs from Pseudomonas sp.130 [9] and Pseudomonas C427 [22], respectively.
The amino acid sequence converted from the DNA sequence [17] has been corrected in the region 193β – 283β (Y. K.– H., unpublished results) on the basis of the amino acid sequence that was determined by our high quality experimental electron density map without any ambiguity except for asparagine/aspartic acid and glutamine/glutamic acid pairs. Interestingly, as a consequence of that correction, the sequence identity of CAD with PGA increased from 14% to 26% in this variable region, and this result further supports the correction. The sequence identities of PGA with P130 and C427 remain 13% and 14%, respectively. Whether the sequences in the other class I CAs need to be corrected is subject to further investigations
Structure 2000 8, DOI: ( /S (00) )

4. Figure 2
Surface Presentation of CAD Structure Showing the Active-Site Pocket
The active-site cleft is shown in the depressed center of the αβ heterodimer that has the shape of a baseball glove. This is a 30°-rotated view with respect to Figure 1c. Blue and red correspond to positively and negatively charged areas, respectively. The side-chain binding pocket is shown by an arrow, and the Oγ of the N-terminal active site Ser1β is indicated by the asterisk. There are no negative charges in the side-chain binding pocket
Structure 2000 8, DOI: ( /S (00) )

5. Figure 3 Stereoview of Active-Site Residues of the CAD Structure
The residues located in the active-site pocket are shown in ball-and-stick. Four residues involved in chemical catalysis are shown in green, and ten residues forming a pocket for binding side chains of substrates, CPC or GL-7ACA, are represented in yellow. The Cα backbones of the α-subunit and the β-subunit are in green and purple, respectively
Structure 2000 8, DOI: ( /S (00) )

6. Figure 5
Structural Comparison of a Cephalosporin AcyIase and a Penicillin AcyIase
(a) Overall comparison of CAD and PGA [19]. The α- and β-subunits of CAD are colored in green and purple, respectively, and the α- and β-subunits of PGA are shown in blue and red, respectively. The label “C” for β-subunits of PGA and CAD indicate the location of C-termini. The label “N” of the α-subunit of PGA indicates the location of the N-terminus. The C-terminus of the α-subunit of PGA is not labeled in order to obtain a clear view. The labels “N” and “C” for the α-subunit of CAD indicate the locations of Gln7α and Gly158α, respectively. The N-terminal active-site residues of β-subunits in both structures, Ser1β, are shown in ball-and stick. The cores of the Ntn hydrolase region, the “B-knob” regions and the helix-regions of the α-subunit are highly homologous whereas the “A-knob” regions differ most in the two structures.
(b) Active-site superposition between CAD and PGA [19]. The Cα atoms of the 61 residues located in close proximity of active-site clefts were used for the superposition. The 14 residues forming the active-site pocket for chemical catalysis and side-chain binding are shown. The Cα traces of α- and β-subunits for CAD are colored in green and purple, respectively, and the α- and β-subunits of PGA are shown in light-green and light-purple. The residues involved in chemical catalysis of CAD and PGA are colored in green and blue, respectively, and the ones for the side-chain binding pocket of CAD and PGA are colored in yellow and orange. Arg57β of CAD, corresponding to Val56β of PGA, may be most critically involved in altering substrate specificities between two enzymes. All 14 residues of CAD are labeled by a normal, bigger font, and the four residues of PGA that are not correctly aligned by PIMA [23] (also see Figure 6) are labeled in an smaller, italic font
Structure 2000 8, DOI: ( /S (00) )

7. Figure 6
Partial Structural Alignment of CAD and PGA and Sequence-Based Alignment of the Cephalosporin Acylases CAD, A14 [25], N176 [3], acyI [5] and the Penicillin Acylase, PGA [19]
The amino acid sequences of CAD (a Class I CA), A14 from Pseudomonas sp. A14 (a class II CA), N176 from Pseudomonas diminuta N176 (a Class III CA), acyI from Pseudomonas sp. SE83, and PGA are used for alignments. First, CAD is structurally aligned to PGA as shown in the first and sixth rows. The names of the two sequences are represented by purple letters. Second, CAD is sequence-based aligned individually to A14, N176, acyI, and PGA by PIMA [23] and those four individual sequence-based alignments are manually put together in the second, third, fourth, and fifth rows with respect to the CAD sequence. In this way, those four sequence-based alignments can be compared to the structure-based alignment by using the CAD and PGA of which alignments are shown in the first and sixth rows. The names of the four sequences used for the sequence-based alignments are in black letters. The 14 active-site residues from the structural alignment between CAD and PGA in Figure 5b are boxed yellow in the first and sixth rows (“CADα” or “CADβ” in purple letters and “PGAα” or “PGAβ” also in purple letters). Ten out of these 14 residues of CAD and PGA are aligned correctly by PIMA (compared to the structural alignment). Those ten corresponding residues in A14, N176, acyI, and PGA are boxed red. The four residues of PGA incorrectly aligned by PIMA (“PGAα” or “PGAβ” in the fifth row) are boxed grey and numbered 1, 2, 3, and 4 underneath the sequence alignment. The corresponding amino acids of CAD are labeled likewise, but above the sequence alignment. The residue numbers of each sequence are shown at the beginning and end of the lines. The residues that were mutated to improve the activity of N176 with respect to CPC (Met164α, Met31β, Tyr32β, and Ala33β) are boxed turquoise [7, 8]
Structure 2000 8, DOI: ( /S (00) )