Crystal structure of anN^ω-hydroxy-L-arginine hydrolase found in theD-cycloserine biosynthetic pathway

Abstract

DcsB, one of the enzymes encoded in the d-cycloserine (d-CS) biosynthetic gene cluster, displays a high sequence homology to arginase, which contains two manganese ions in the active site. However, DcsB hydrolyzes N ! -hydroxy-larginine, but not l-arginine, to supply hydroxyurea for the biosynthesis of d-CS.Here, the crystal structure of DcsB was determined at a resolution of 1.5 Å using anomalous scattering from the manganese ions. In the crystal structure, DscB generates an artificial dimer created by the open and closed forms. Gel-filtration analysis demonstrated that DcsB is a monomeric protein, unlike arginase, which forms a trimeric structure. The active center containing the binuclear manganese cluster differs between DcsB and arginase. In DcsB, one of the ligands of the Mn A ion is a cysteine, while the corresponding residue in arginase is a histidine. In addition, DcsB has no counterpart to the histidine residue that acts as a general acid/base during the catalytic reaction of arginase. The present study demonstrates that DcsB has a unique active site that differs from that of arginase.

Catalytic mechanism of DcsB: Arginase framework used for hydrolyzing its inhibitor

“…The crystallization of wild‐type and mutated DcsB was conducted by the method described previously 11 . The typical formula of reservoir solution to obtain the crystals was 0.1 M Tris–HCl buffer (pH 8.5) containing 32% PEG4,000 and 0.2 M MgCl 2 .…”

“…In the crystal of DcsB, an artificial dimer is formed between a closed form, in which a flexible loop connecting the α8 and α9 helices covers the active site, and an open form, in which the loop is protruded into the solvent region. 11 In the crystal, the α1 helix in the closed form is inserted into the active site in the open form. Although the loop structure is different between the open and closed forms, dimanganese cluster consisting of Mn A and Mn B is commonly found at the active center in both forms.…”

“…The plasmid for the expression of wild-type DcsB with Cterminal His 6 -tag was constructed by using pET-21a(+), as described previously. 11 Mutants of DcsB were made using a partially codon-optimized gene fragment, because dcsB gene cloned from genome of S. lavendulae ATCC11924 displays a high GC content (72.9%). All oligonucleotides and synthetic gene sequences (gblocks) used for making mutants of DcsB were synthesized by Integrated DNA Technologies (IDT) (Table S3).…”

“…9,10 HU is supplied by the hydrolysis of N ω -hydroxy-Larginine (L-NOHA) by DcsB (Figure 2). 11 L-NOHA is produced by the hydroxylation of L-arginine (L-Arg) by the heme-containing enzyme, DcsA. 12 The generated Oureido-L-serine is racemized by DcsC 9,13,14 and subsequently cyclized with the help of DcsG, which is a member of the ATP-grasp fold family enzymes.…”

Crystal structure of O‐ureidoserine racemase found in the d‐cycloserine biosynthetic pathway

“…8,9 Its acetyl group is replaced with an O-ureido group by the pyridoxal phosphatedependent enzyme DcsD using hydroxyurea. 10,11 Hydroxyurea is supplied from N ω -hydroxy-L-arginine via hydrolysis by DcsB, 12 while N ω -hydroxy-L-arginine is supplied via hydroxylation of L-arginine by the heme-containing enzyme DcsA. 13 The generated O-ureido-L-serine (L-OUS) is racemized by DcsC, 10,14 and its enantiomer O-ureido-D-serine (D-OUS) is subsequently cyclized by DcsG, which is a member of the ATP-grasp fold family.…”