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Exploring the Structurally Conserved Regions and Functional Significance in Bacterial N-Terminal Nucleophile (Ntn) Amide-Hydrolases - PubMed Skip to main page content
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. 2024 Jun 21;25(13):6850.
doi: 10.3390/ijms25136850.

Exploring the Structurally Conserved Regions and Functional Significance in Bacterial N-Terminal Nucleophile (Ntn) Amide-Hydrolases

Israel Quiroga  1 , Juan Andrés Hernández-González  1 , Elizabeth Bautista-Rodríguez  1   2 , Alfredo C Benítez-Rojas  1
Affiliations

Exploring the Structurally Conserved Regions and Functional Significance in Bacterial N-Terminal Nucleophile (Ntn) Amide-Hydrolases

Israel Quiroga et al. Int J Mol Sci. .

Abstract

The initial adoption of penicillin as an antibiotic marked the start of exploring other compounds essential for pharmaceuticals, yet resistance to penicillins and their side effects has compromised their efficacy. The N-terminal nucleophile (Ntn) amide-hydrolases S45 family plays a key role in catalyzing amide bond hydrolysis in various compounds, including antibiotics like penicillin and cephalosporin. This study comprehensively analyzes the structural and functional traits of the bacterial N-terminal nucleophile (Ntn) amide-hydrolases S45 family, covering penicillin G acylases, cephalosporin acylases, and D-succinylase. Utilizing structural bioinformatics tools and sequence analysis, the investigation delineates structurally conserved regions (SCRs) and substrate binding site variations among these enzymes. Notably, sixteen SCRs crucial for substrate interaction are identified solely through sequence analysis, emphasizing the significance of sequence data in characterizing functionally relevant regions. These findings introduce a novel approach for identifying targets to enhance the biocatalytic properties of N-terminal nucleophile (Ntn) amide-hydrolases, while facilitating the development of more accurate three-dimensional models, particularly for enzymes lacking structural data. Overall, this research advances our understanding of structure-function relationships in bacterial N-terminal nucleophile (Ntn) amide-hydrolases, providing insights into strategies for optimizing their enzymatic capabilities.

Keywords: N-terminal nucleophile (Ntn) amide-hydrolase; protein design; protein modeling; structure–function relationship.

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Conflict of interest statement

The authors certify that they have NO affiliations with or involvement in any organization or entity with any financial interest (such as honoraria; educational grants; participation in speakers’ bureaus; membership, employment, consultancies, stock ownership, or other equity interest; and expert testimony or patent-licensing arrangements), or non-financial interest (such as personal or professional relationships, affiliations, and knowledge or beliefs) in the subject matter or materials discussed in this manuscript.

Figures

Figure 1
Figure 1
Structural alignment of N-terminal nucleophile (Ntn) amide-hydrolases S45 family. (A) The structural alignment of two proteins of each group, specifically referencing entries 1AI4, 1CP9, 7REO, 7REP, 3K3W, 3ML0, 6NVW, 8BRR, 7EA4, 7EBY, 4HSR, 4HST, 4YF9, 5C9I, 3SRA, 5UBK, 1GK1, and 1OR0. (B) The structural alignment of SCRs of two proteins of each group, specifically referencing entries 1AI4, 1CP9, 7REO, 7REP, 3K3W, 3ML0, 6NVW, 8BRR, 7EA4, 7EBY, 4HSR, 4HST, 4YF9, 5C9I, 3SRA, 5UBK, 1GK1, and 1OR0. α-helices are marked in red; β-sheets are marked in cyan; loops are marked in white; turns are marked in green; and penicillin is depicted in stick representation for reference [PDB code: 1FXV].
Figure 2
Figure 2
SCRs of the chain A of N-terminal nucleophile (Ntn) amide-hydrolases S45 family are depicted both in a structural overview and in a topological illustration. (A) Structurally conserved regions (SCRs) of chain A in penicillin acylases, cephalosporin acylases, and D-succinylase using a topological representation. These conserved regions were identified through STAMP alignment, with their positions highlighted in distinct colors. α-helices are depicted as bold lines, while β-sheets are represented by arrows. (B) This provides a structural overview of the structurally conserved regions (SCRs) of chain A in penicillin acylases, cephalosporin acylases, and D-succinylase. These conserved regions, identified through STAMP alignment, are superimposed on the reference structure of penicillin acylase from Escherichia coli [PDB code: 1FXV]. The SCRs are depicted in various colors, while the variable regions are represented in grey.
Figure 3
Figure 3
SCRs of the chain B of N-terminal nucleophile (Ntn) amide-hydrolases S45 family are depicted both in a structural overview and in a topological illustration. (A) Structurally conserved regions (SCRs) of chain B in penicillin acylases, cephalosporin acylases, and D-succinylase using a topological representation. These conserved regions were identified through STAMP alignment, with their positions highlighted in distinct colors. α-helices are depicted as bold lines, while arrows represent β-sheets. (B) This provides a structural overview and structural alignment of penicillin acylases, cephalosporin acylases, and D-succinylase. Structurally conserved regions are depicted in colors, while the variable regions are represented in grey. The most extensive variable region is depicted at the top of the image.
Figure 4
Figure 4
Active site of N-terminal nucleophile (Ntn) amide-hydrolases S45 family. (A) The active site overlay of PDB structures of N-terminal nucleophile (Ntn) amide-hydrolases S45 family from various sources is depicted as follows: E. coli penicillin G acylase [PDB code: 1FXH] is represented in black sticks for reference, Pseudomonas aeruginosa acyl-homoserine lactone acylase [PDB code: 2WYB] in blue lines, Bacillus megaterium penicillin G acylase [PDB code: 6NVW] in orange lines, Kluyvera cryocrescens penicillin G acylase [PDB code: 7REO] in yellow lines, Brevundimonas diminuta cephalosporin C acylase [PDB code: 1KEH] in pink lines, Acidovorax sp. MR-S7 acyl-homoserine lactone acylase [PDB code: 4YF9] in green lines, Cupriavidus sp. P4-10-C D-succinylase [PDB code: 7EA4] in red lines, Brevundimonas diminuta cephalosporin C acylase [PDB code: 1FM2] in cyan lines, and Pseudomonas cephalosporin acylase [PDB code: 4HSR] in violet lines. Penicillin is represented in green stick configuration as a point of reference [PDB code: 1FXV]. The annotated amino acids correspond to those in E. coli penicillin G acylase [PDB code: 1FXH]. Amino acids corresponding to spatial positions in other crystallographic structures are detailed in Table 1. (B) The amino acids identified as constituents of the active site in the N-terminal nucleophile (Ntn) amide-hydrolases S45 family in this study are depicted in black sticks. The annotated amino acids correspond to those present in E. coli penicillin G acylase [PDB code: 1FXH]. Panels A and B are shown from the same perspective. (C) The first line corresponds to the consensus numbering of 83 crystal structure sequences. Residue and chain IDs of the crystallographic structures are indicated on the sides of the alignment. Active site residues are denoted by an asterisk (*). Structurally conserved regions (SCRs) are marked by a hyphen (-) and a vertical bar (|) at the start and at the end. The "#" in "SCR#A" and "SCR#B" is used as a placeholder to indicate that any number can be applied, generalizing the reference to structurally conserved regions in chain A and chain B, respectively.
Figure 4
Figure 4
Active site of N-terminal nucleophile (Ntn) amide-hydrolases S45 family. (A) The active site overlay of PDB structures of N-terminal nucleophile (Ntn) amide-hydrolases S45 family from various sources is depicted as follows: E. coli penicillin G acylase [PDB code: 1FXH] is represented in black sticks for reference, Pseudomonas aeruginosa acyl-homoserine lactone acylase [PDB code: 2WYB] in blue lines, Bacillus megaterium penicillin G acylase [PDB code: 6NVW] in orange lines, Kluyvera cryocrescens penicillin G acylase [PDB code: 7REO] in yellow lines, Brevundimonas diminuta cephalosporin C acylase [PDB code: 1KEH] in pink lines, Acidovorax sp. MR-S7 acyl-homoserine lactone acylase [PDB code: 4YF9] in green lines, Cupriavidus sp. P4-10-C D-succinylase [PDB code: 7EA4] in red lines, Brevundimonas diminuta cephalosporin C acylase [PDB code: 1FM2] in cyan lines, and Pseudomonas cephalosporin acylase [PDB code: 4HSR] in violet lines. Penicillin is represented in green stick configuration as a point of reference [PDB code: 1FXV]. The annotated amino acids correspond to those in E. coli penicillin G acylase [PDB code: 1FXH]. Amino acids corresponding to spatial positions in other crystallographic structures are detailed in Table 1. (B) The amino acids identified as constituents of the active site in the N-terminal nucleophile (Ntn) amide-hydrolases S45 family in this study are depicted in black sticks. The annotated amino acids correspond to those present in E. coli penicillin G acylase [PDB code: 1FXH]. Panels A and B are shown from the same perspective. (C) The first line corresponds to the consensus numbering of 83 crystal structure sequences. Residue and chain IDs of the crystallographic structures are indicated on the sides of the alignment. Active site residues are denoted by an asterisk (*). Structurally conserved regions (SCRs) are marked by a hyphen (-) and a vertical bar (|) at the start and at the end. The "#" in "SCR#A" and "SCR#B" is used as a placeholder to indicate that any number can be applied, generalizing the reference to structurally conserved regions in chain A and chain B, respectively.
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