Functional Diversity and Structural Analysis of SAM‐dependent Aminobutanoyl Transferases

Abstract

Aminobutanoyl transferases (ABT) catalyze the transfer of 2‐aminobutanoate from S‐adenosyl‐L‐methionine (SAM) in the production of a variety of natural products. Nicotianamine (NA), a metal‐chelator found in plants, is a classic example and is biosynthesized by nicotianamine synthase through the condensation of three aminobutanoyl moieties from three successive SAM molecules with the first moiety formed into an azetidine ring. Although NA is common in plants, fungal, archaeal and bacterial species have also been shown to produce NA‐like molecules using ABTs. Examples have been characterized from the bacterium S. aureus and the archaeal species M. thermautotrophicus. Structural and functional data remains limited, but existing data suggests a diversity of products using one or more SAM moieties and/or additional amino acid substrates. We used Enzyme Function Initiative (EFI) tools to create sequence similarity and genome neighborhood networks to explore the diversity of ABT‐containing operons and to guide our predictions for the functional roles of distinct ABTs. We also used multi‐sequence alignments of our EFI dataset and structural alignments of the two existing ABT crystal structures to identify structural motifs that may be important to function. We have identified five functional ABT classes producing distinct product types. Example hypotheses from our analysis include the identification of consecutive asparagines deep in the active site pocket that appear to predict processivity and the presence of a serine/alanine substitution that correlates with stereoselectivity. To begin testing hypotheses developed from our bioinformatic analysis, we have selected four ABTs for heterologous expression and purification. We have successfully purified a carboxyazetidine‐forming ABT from P. aeruginosa and have begun crystallization trials. Purification of additional ABTs and the development of an HPLC‐based activity assay is ongoing. Through the characterization of ABTs selected from our bioinformatic analysis we expect to establish rules for predicting processivity, carboxyazetidine ring formation and the use of amino acid substrates by uncharacterized ABTs and to expand the limited available structural data using X‐ray crystallography.