Structural and biochemical characterization of the biuret hydrolase (BiuH) from the cyanuric acid catabolism pathway of Rhizobium leguminasorum bv. viciae 3841

Abstract

Biuret deamination is an essential step in cyanuric acid mineralization. In the well-studied atrazine degrading bacterium Pseudomonas sp. strain ADP, the amidase AtzE catalyzes this step. However, Rhizobium leguminosarum bv. viciae 3841 uses an unrelated cysteine hydrolase, BiuH, instead. Herein, structures of BiuH, BiuH with bound inhibitor and variants of BiuH are reported. The substrate is bound in the active site by a hydrogen bonding network that imparts high substrate specificity. The structure of the inactive Cys175Ser BiuH variant with substrate bound in the active site revealed that an active site cysteine (Cys175), aspartic acid (Asp36) and lysine (Lys142) form a catalytic triad, which is consistent with biochemical studies of BiuH variants. Finally, molecular dynamics simulations highlighted the presence of three channels from the active site to the enzyme surface: a persistent tunnel gated by residues Val218 and Gln215 forming a potential substrate channel and two smaller channels formed by Val28 and a mobile loop (including residues Phe41, Tyr47 and Met51) that may serve as channels for co-product (ammonia) or co-substrate (water).

An unexpected vestigial protein complex reveals the evolutionary origins of an s-triazine catabolic enzyme

“…Previously, AtzE had been reported to be a biuret hydrolase (1,5,14,19), largely through comparison with the non-homologous biuret hydrolase of Rhizobium sp. (Table 1).…”

“…viciae 3841 (BiuH) (AM236084.1) and barbituric acid hydrolase from Nocardioides sp. JS614 coding for the protein ABL81019 (ABL81019.1) were expressed in E. coli as described elsewhere (12,19).…”

“…There was no detectable biuret amidohydrolase activity with AtzE ( Fig. 2A), but it was detected using the biuret amidohydrolase from a Rhizobium species, BiuH (19) (Fig. 2B).…”

“…AtzF is sometimes substituted by the homologous TrzF found in Enterobacter cloacae strain 99 (70 % identity to AtzF; (14)) and AtzE has been replaced with the non-homologous biuret amidohydrolase (BiuH, a (/)8 TIM barrel cysteine hydrolase) in Rhizobium leguminasorum bv. viciae 3841 (Table 1) (1,19).…”

Microbial biodegradation of biuret: defining biuret hydrolases within the isochorismatase superfamily

“…The crystal structure for the R. leguminosarum bv. viciae 3841 BH was recently solved (Esquirol e t al ., ). Here, we took a comparative approach using the R. leguminosarum structure to concretely identify structure–function signatures of BH subfamily.…”