Conserved structural modules and bonding networks in isopenicillin N synthase related non-haem iron-dependent oxygenases and oxidases☆
Introduction
In the multi-step β-lactam biosynthetic pathway carried out by various bacteria and fungi [1], three enzymes, isopenicillin N synthase (IPNS), deacetoxycephalosporin C synthase (DAOCS) and deacetylcephalosporin C synthase (DACS), are known to bind iron with a non-haem coordinated ligand environment and also concomitantly use oxygen and ascorbate for their catalysis [2], [3]. Based on these properties and their amino acid sequence relatedness, IPNS, DAOCS and DACS have been classified under a subfamily of mononuclear non-haem iron-binding oxygenases and oxidases [2]. Most enzyme members within this subfamily are involved in catalyzing the biosyntheses of plant secondary metabolites. For example, flavanone 3β-hydroxylase (FL3OH), flavonol synthase (FS), leucoanthocyanidin dioxygenase (LDOX) and anthocyanidin synthase (ANS) are involved in the biosynthesis of flavanoids [4]; 1-aminocyclopropane-1-carboxylic acid oxidase (ACCO) catalyses the last step in the biosynthesis of ethylene [5]; hyoscyamine 6β-hydroxylase (H6H) is involved in synthesizing the plant alkanoid scopolamine [6]; gibberellin 20-oxidase (G20O) and desacetoxyvindoline-4-hydroxylase (DVH) are involved in the biosynthesis of gibberellins [7] and vindolines [8], respectively. In this report, these enzymes are collectively named as IPNS related non-haem iron-dependent oxygenases and oxidases or designated as the NHIDOX family in this study.
Iron, oxygen and ascorbate are commonly used for catalysis although the NHIDOX enzymes carry out different oxidative reactions and exhibit different substrate selectivities. For instance, IPNS is instrumental in catalyzing a desaturative ring closure reaction whereby a tripeptide substrate l-α-aminoadipyl-l-cysteinyl-d-valine (LLD-ACV) is cyclized to form the four-membered β-lactam ring and the five-membered thiazolidine ring of isopenicillin N [1]. DAOCS, also known as expandase, catalyses the oxidative ring expansion of the five-membered thiazolidine ring of penicillin N to form the six-membered dihydrothiazine ring of deacetoxycephalosporin C (DAOC) and DACS is responsible for hydrolyzing the methyl group at the C3 position of DAOC.
Another enzyme of interest, H6H, is involved in catalyzing an epoxidation reaction in which the substrate hyoscyamine is converted to form the plant alkaloid scopolamine [6]. With the exception of IPNS and ACCO, the other members of NHIDOX family also require 2-oxoglutarate as the co-substrate for catalysis.
To date, extensive biochemical and functional studies of various enzyme members have shown conclusively the conserved residues responsible for binding iron and substrate/co-substrate [9], [10], [11], [12], [13], [14], [15], [16], [17], [18]. Separate reports [2], [19], [20] on the high-resolution crystal structure complexes of Aspergillus nidulans IPNS (IPNS_AN) and Streptomyces clavuligerus DAOCS (DAOCS_SC) has been reported. Such information will provide clues on how an enzyme’s function can be modulated by certain amino acids or strings of sequences. In this study, we have described the conserved architectural scaffolds as well as the key factors that are responsible for modulating the functional differences and substrate selectivities of enzymes in the NHIDOX family.
Section snippets
Data retrieving
The analysis is based on comparison of 142 protein sequences and 2 protein structures available in the databank. All sequences were retrieved from the National Center of Biotechnology Information (NCBI)-supported sequence search program and the structure coordinates were downloaded from the Protein Data Bank (PDB). The 142 sequences include 11 IPNS, 4 DAOCS, 2 DACS, 20 FL3OH, 11 LDOX, 5 ANS, 7 FS, 19 GO, 2 H6H, 52 ACCO and 1 DVH homologous enzymes as well as proteins isolated from Campylobacter
Comparative primary sequence analysis
An extensive sequence analysis of NHIDOX enzymes was carried out 5 years ago [9]. This analysis included 52 non-redundant enzyme members, consisting only of those involved in making β-lactams in certain prokaryotic and eukaryotic lineages and those involved in synthesizing secondary signaling products in various plant species. Unexpectedly, our sequence searches and analyses revealed that several proteins isolated from more diverse origins, such as the fruit fly, fission yeast, C. jejuni,
Conclusions
Comparison of the protein sequences of NHIDOX enzymes essentially reveals their linear relationships of having seven highly conserved residues. However, analysis of their two- and three-dimensional structure relationships showed that these enzymes have evolved common topological scaffolds, comprising of highly conserved antiparallel running β-strands and bondings with certain amino acid residues, to form the jelly-roll motif structure for their active centers. The remarkable conservation of
Acknowledgements
The assistance of Francis Ng Hoong Kee in the computational calculations is greatly appreciated. This research was supported by National University of Singapore (Singapore) Research Grant Number R-182-000-019-112.
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Supplementary data associated with this article can be found at doi: 10.1016/S1381-1177(03)00037-7.