Biochemical and structural characterization ofKlebsiella pneumoniaeoxamate amidohydrolase in the uric acid degradation pathway

Abstract

HpxW from the ubiquitous pathogen Klebsiella pneumoniae is involved in a novel uric acid degradation pathway downstream from the formation of oxalurate. Specifically, HpxW is an oxamate amidohydrolase which catalyzes the conversion of oxamate to oxalate and is a member of the Ntn-hydrolase superfamily. HpxW is autoprocessed from an inactive precursor to form a heterodimer, resulting in a 35.5 kDa subunit and a 20 kDa subunit. Here, the structure of HpxW is presented and the substrate complex is modeled. In addition, the steady-state kinetics of this enzyme and two active-site variants were characterized. These structural and biochemical studies provide further insight into this class of enzymes and allow a mechanism for catalysis consistent with other members of the Ntn-hydrolase superfamily to be proposed.

Coping with inevitable accidents in metabolism

“…Yet it might generate oxamate in a S ‐ureidoglycine degradation pathway: ureidoglycine could not be tested in the assays if the protein precluding identification of this activity if it was indeed present (Li et al ., ). Subsequently, oxamate would release oxalate and ammonium, as observed for oxamate amidohydrolase HpxW in Klebsiella michiganensis (Hicks and Ealick, ). To substantiate this view, I note that, using the phyloprofile software in B. subtilis shows that protein YwrD is very similar to the Klebsiella HpxW counterpart and that it co‐evolves with most of the enzymes involved in the xanthine degradation pathway (Table S2).…”

Ureide metabolism in plant-associated bacteria: purine plant-bacteria interactive scenarios under nitrogen deficiency

“…The further degradation of oxalurate to carbamoyl phosphate and oxamate is catalyzed by the enzyme oxamate carbamoyl transferase in E. coli (Hasegawa et al 2008;Li et al 2011) and in S. allantoicus (Bojanowski et al 1964); in contrast to the direct conversion of oxalurate into oxamate and NH 3 described in K. pneumoniae (Hicks and Ealick 2016). Oxamate is finally converted to oxalate by the enzyme oxamate amidohydrolase as shown in K. pneumoniae (Hicks and Ealick 2016;Danchin 2017 for growth (Vander Wauven et al 1986) suggests the possible existence of interconnections between routes 1 and 2. As shown in E. coli, the enzyme NAD(P) + -dependent ureidoglycolate dehydrogenase, a member of Lsulfolactate dehydrogenase-like family, may oxidize ureidoglycolate to oxalurate, allowing urease-negative E. coli to preserve N and energy resources more efficiently (Werner et al 2010).…”

“…The route 2 commences with the conversion of (S)-allantoate to (S)-ureidoglycine by the enzyme allantoate amidohydrolase and into oxalurate via the reaction catalyzed by the enzyme Mn 2+ -, Co 2+ -, and Ni 2+ -dependent (S)-ureidoglycine aminotransferase, concomitantly with the synthesis of α-amino acids from α-keto acids (French and Ealick 2010). The further degradation of oxalurate to carbamoyl phosphate and oxamate is catalyzed by the enzyme oxamate carbamoyl transferase in E. coli (Hasegawa et al 2008;Li et al 2011) and in S. allantoicus (Bojanowski et al 1964); in contrast to the direct conversion of oxalurate into oxamate and NH 3 described in K. pneumoniae (Hicks and Ealick 2016). Oxamate is finally converted to oxalate by the enzyme oxamate amidohydrolase as shown in K. pneumoniae (Hicks and Ealick 2016;Danchin 2017 for growth (Vander Wauven et al 1986) suggests the possible existence of interconnections between routes 1 and 2.…”