Molecular cloning and analysis of the genes encoding the 4-hydroxyphenylacetate hydroxylase from Klebsiella pneumoniae

Abstract

The Klebsiella pneumoniae genes encoding the hydroxylase involved in the meta-cleavage pathway of 4-hydroxyphenylacetic acid (4-HPA) were cloned, and the DNA fragment from the region essential for hydroxylase activity was sequenced. K. pneumoniae 4-HPA hydroxylase was composed of two proteins (HpaA and HpaH) with different molecular masses. HpaA seems to be a flavin-containing hydroxylase with a molecular mass of 58,781 Da. HpaH, with a molecular mass of 18,680 Da, seems to be a "helper" protein required for productive hydroxylation of the substrate. The hpa genes were expressed and the hydroxylase was active in Escherichia coli. Comparison of the enzyme with other monooxygenases indicates that K. pneumoniae 4-HPA hydroxylase is a member of a new family of hydroxylases.

Cloning and Characterization of a 4-Hydroxyphenylacetate 3-Hydroxylase From the Thermophile Geobacillus sp. PA-9

“…These results therefore indicated that formation of the pigment results from the catabolic activity of the cloned 4-HPA hydroxylase and suggests that HpaH might thus recognize other substrates distinct from 4-HPA. Such relaxed substrate specificity is in agreement with results reported for the 4-HPA 3-hydroxylases of E. coli [21] and K. pneumoniae [13,14]. The 4-HPA hydroxylase described in this article is the first from a thermophile species to be analyzed at the genetic level.…”

“…PA-9, designated HpaH, demonstrated homology to the equivalent HpaB and HpaA monooxygenases from E. coli W and K. pneumoniae M5a1, respectively, and to the phenol hydroxylase PheA1 from B. thermoglucosidasius A7. These three proteins are each part of two-component aromatic hydroxylase enzyme systems that are comprised of a monooxygenase and a smaller flavin:NAD(P)H oxidoreductase, and the genes encoding the two components of these enzymes are located in the same operon [10,14,20]. No additional ORF with the potential of encoding an oxidoreductase was found in the intergenic region between the hpaH and hpaI genes in the 2.7-kb genomic DNA fragment cloned from Geobacillus sp.…”

“…Transformants containing 4-HPA hydroxylase genes were identified on LB plates containing ampicillin as brown colonies caused by intracellular accumulation of 3,4-DHPA [13,14].…”

“…4-HPA 3-hydroxylase activity was determined by measuring the liberation of 3,4-DHPA from 4-HPA (Sigma-Aldrich) in 50 mM Tris-HCl buffer (pH 9) at 50°C, according to the method of Anrow [2]. accession number CAA86048) [20] and Klebsiella pneumoniae M5a1 (encoded by hpaA; accession number Q48440) [14]. Furthermore, HpaH shares 31% and 29% sequence identity, respectively, with the phenol 2-hydroxylases of B. thermoglucosidasius A7 (encoded by pheA1; accession number AAF66546) [10] and B. thermoleovorans A2 (encoded by pheA; accession number AAC38324) [11].…”

Functional Analysis of the Small Component of the 4-Hydroxyphenylacetate 3-Monooxygenase ofEscherichia coliW: a Prototype of a New Flavin:NAD(P)H Reductase Subfamily

“…In this regard, a coupling protein enhancing the activity of an aromatic monooxygenase had been also described for the 4-HPA hydroxylase from Pseudomonas putida (2,3). However, in the past four years several TC-FDM enzymes whose amino acid sequences have revealed significant similarities with those of the HpaB and HpaC proteins have been reported in different bacteria, suggesting that HpaB and HpaC could be considered as the representative oxygenase and reductase components, respectively, of this new TC-FDM family (13,16,21,36,41).…”