Crystal Structure and Mechanistic Implications of N2-(2-Carboxyethyl)arginine Synthase, the First Enzyme in the Clavulanic Acid Biosynthesis Pathway

Abstract

The initial step in the biosynthesis of the clinically important ␤-lactamase inhibitor clavulanic acid involves condensation of two primary metabolites, D-glyceraldehyde 3-phosphate and L-arginine, to give N 2 -(2-carboxyethyl)arginine, a ␤-amino acid. This unusual N-C bond forming reaction is catalyzed by the thiamin diphosphate (ThP 2 )-dependent enzyme N 2 -(2-carboxyethyl)arginine synthase. Here we report the crystal structure of N 2 -(2-carboxyethyl)arginine synthase, complexed with ThP 2 and Mg 2؉ , to 2.35-Å resolution. The structure was solved in two space groups, P2 1 2 1 2 1 and P2 1 2 1 2. In both, the enzyme is observed in a tetrameric form, composed of a dimer of two more tightly associated dimers, consistent with both mass spectrometric and gel filtration chromatography studies. Both ThP 2 and Mg 2؉ cofactors are present at the active site, with ThP 2 in a "V" conformation as in related enzymes. A sulfate anion is observed in the active site of the enzyme in a location proposed as a binding site for the phosphate group of the D-glyceraldehyde 3-phosphate substrate. The mechanistic implications of the active site arrangement are discussed, including the potential role of the aminopyrimidine ring of the ThP 2 . The structure will form a basis for future mechanistic and structural studies, as well as engineering aimed at production of alternative ␤-amino acids.

Purified recombinant hARD1 does not catalyse acetylation of Lys532 of HIF‐1α fragments in vitro

“…Controls were run with several proteins that contained an N-terminus consisting of a glycyl, serinyl or threoninyl residue, under the same conditions as those used for hARD1 and HIF substrates. The control proteins were FIH [9], HIF , open reading frames 2 [23], 15 and 16 from the clavulanic acid biosynthesis pathway (unpublished data), and phytanoyl coenzyme A hydroxylase [24], and were donated by D. Lancaster, A. Hardy, Dr. M. Caines and Dr. T. Searls, respectively (Chemistry Research Laboratory, Oxford University).…”

“…The resulting GSH-hARD1 was incubated with [1-14 C]-AcCoA and controls were run with several proteins that contained a glycine residue at the N-terminus, under the same conditions as those used for hARD1 and HIF-1a substrates. The control proteins were FIH [9], HIF , open reading frames 2 [23], 15 and 16 from the clavulanic acid biosynthesis pathway, (unpublished data) and phytanoyl coenzyme A hydroxylase [24]. Autoradiographic analysis revealed that the band corresponding to GSH-hARD1 was [ 14 C]-labeled, suggesting that the acetylation was indeed self-mediated; a faint band corresponding to acetylated residual (His) 6 -tagged hARD1 was also visible in assays containing enzyme with both [1-14 C]-AcCoA and ORFs 2, 15 and 16 (Fig.…”

ORF17 from the Clavulanic Acid Biosynthesis Gene Cluster Catalyzes the ATP-dependent Formation of N-Glycyl-clavaminic Acid

“…Three of these steps are catalyzed by a single 2-oxoglutarate (2-OG)-dependent oxygenase, clavaminic acid synthase (CAS2) (10, 18 -20), and one is catalyzed by proclavaminate amidinohydrolase (PAH) (21), to give the bicyclic clavam ring system (22)(23)(24). Crystal structures have been reported for the first four enzymes in the pathway (25)(26)(27)(28) as well as for the gene product of orf6 (OAT2), an ornithine acetyltransferase (29,30) proposed to be involved in the biosynthesis of the L-arginine feedstock for the pathway. The final step in the pathway has also been identified and shown to involve the NADPH-dependent reduction of the labile (3R,5R)-clavaldehyde to give (3R,5R)-clavulanic acid (31).…”

Carboxyethylarginine Synthase Genes Show Complex Cross-Regulation in Streptomyces clavuligerus

“…To gain further insights, a fifth type of ceaS2 mutant was created in which the ceaS2 gene was modified by a site-directed mutation of 2 bp, with GA changed to TG, such that the glutamate codon at amino acid 57 became a tryptophan codon. Structural analyses of CeaS2 have suggested that the glutamate residue at position 57 (E57) is intimately involved in the catalytic function of the protein (27), and so the E57W mutant CeaS2 protein was expected to be defective in CeaS2 activity but otherwise unchanged from the wild type. A cloned copy of the E57W mutant version of ceaS2 was conjugated into a ceaS2::apr mutant, whereupon the apr-disrupted ceaS2 was replaced by E57WceaS2.…”

“…Furthermore, both the ceaS2::apr and ceaS2-FS mutants could potentially produce truncated or C-terminally mistranslated CeaS2 proteins. Since the CeaS protein is known to exist as a tetramer (a dimer of dimers) (27), the defective Ceas2 subunits might interact with active CeaS1 subunits to affect activity.…”