Functional analysis of the small component of the 4-hydroxyphenylacetate 3-monooxygenase of Escherichia coli W: a prototype of a new Flavin:NAD(P)H reductase subfamily

Abstract

Escherichia coli W uses the aromatic compound 4-hydroxyphenylacetate (4-HPA) as a sole source of carbon and energy for growth. The monooxygenase which converts 4-HPA into 3,4-dihydroxyphenylacetate, the first intermediate of the pathway, consists of two components, HpaB (58.7 kDa) and HpaC (18.6 kDa), encoded by the hpaB and hpaC genes, respectively, that form a single transcription unit. Overproduction of the small HpaC component in E. coli K-12 cells has facilitated the purification of the protein, which was revealed to be a homodimer that catalyzes the reduction of free flavins by NADH in preference to NADPH. Subsequently, the reduced flavins diffuse to the large HpaB component or to other electron acceptors such as cytochrome c and ferric ion. Amino acid sequence comparisons revealed that the HpaC reductase could be considered the prototype of a new subfamily of flavin:NAD(P)H reductases. The construction of a fusion protein between the large HpaB oxygenase component and the choline-binding domain of the major autolysin of Streptococcus pneumoniae allowed us to develop a rapid method to efficiently purify this highly unstable enzyme as a chimeric CH-HpaB protein, which exhibited a 4-HPA hydroxylating activity only when it was supplemented with the HpaC reductase. These results suggest the 4-HPA 3-monooxygenase of E. coli W as a representative member of a novel two-component flavin-diffusible monooxygenase (TC-FDM) family. Relevant features on the evolution and structure-function relationships of these TC-FDM proteins are discussed.

FIG. 1

SDS-PAGE analysis of the overproduction and purification of the two components of the 4-HPA 3-monooxygenase from E. coli W. (A) Lane 1, molecular mass markers; lane 2, soluble control extract from E. coli DH1(pUC18); lane 3, soluble crude extract from E. coli TG1(pAJ28); lane 4, purified HpaC reductase. (B) Lane 1, molecular mass markers; lane 2, soluble control extract from E. coli TG1(pUC18); lane 3, soluble crude extract from E. coli TG1(pAJ31); lane 4, purified CH-HpaB protein. The molecular mass marker proteins are indicated in kilodaltons.

FIG. 2

Schematic representation of the construction of the gene encoding the CH-HpaB fusion protein. Abbreviations: Ap^r, ampicillin resistance; Cm^r, chloramphenicol resistance; E, EcoRI; H, HindIII; S, SalI; P_lac, lac promoter; P_lpp, lpp promoter; RBS, ribosome binding site. Amino acids are indicated by their standard single letter codes.

FIG. 3

Multiple sequence alignment of the oxygenase components of several members of the TC-FDM family. Numbers in parentheses indicate the position of the residues in the complete amino acid sequence of the protein. A consensus sequence was deduced for positions where the residues were identical in more than half of the sequences. AF-HpaA1, putative 4-HPA 3-monooxygenase from Archaeoglobus fulgidus (AE001081); AF-HpaA2, putative 4-HPA 3-monooxygenase from A. fulgidus (AE001043); AF-HpaA3, putative 4-HPA 3-monooxygenase from A. fulgidus (AE001032); PA-PvcC putative hydroxylase of pyoverdine chromophore biosynthesis in Pseudomonas aeruginosa (AF002222); PL-HpaB, putative 4-HPA 3-monooxygenase from Pseudomonas luminescens (AF021839); KP-HpaA, 4-HPA 3-monooxygenase from Klebsiella pneumoniae (L41068); EC-HpaB, 4-HPA 3-monooxygenase from E. coli (Z29081); SD-HpaB, putative 4-HPA 3-monooxygenase from S. dublin (AF144422); BT-PheA, phenol hydroxylase from B. thermoleovorans (AF031325); BS-Yoal, putative 4-HPA 3-monooxygenase from Bacillus subtilis (Z99114); BP-HadA; chlorophenol 4-hydroxylase from B. pickettii (D86544); BC-TftD, chlorophenol 4-hydroxylase from B. cepacia (U83405); RE-DszC, dibenzothiophene monooxygenase from R. erythropolis (L37363); PP-MsuC, putative monooxygenase from P. aeruginosa (AF026067).

FIG. 4

Multiple sequence alignment of HpaC-like proteins. A comparison of the amino acid sequences of HpaC and other proteins of the databases that present a significant similarity is shown. Numbers in parentheses indicate the position of the residues in the complete amino acid sequence of the protein. A consensus sequence was deduced for positions where the residues were identical in more than half of the sequences. SC-ActVB, flavin reductase involved in the biosynthesis of actinorhodin in S. coelicolor (X58833); SR-FrnH, putative flavin reductase involved in frenolicin biosynthesis in Streptomyces roseofulvus (AF058302); SV-Orf34, putative flavin reductase involved in granaticin biosynthesis in Streptomyces violaceoruber (AJ011500); SV-VlmR, putative flavin reductase involved in valanimycin biosynthesis in Streptomyces viridifaciens (U93606); SP-SnaC, flavin reductase involved in pristimamycin II_A biosynthesis in Streptomyces pristinaespiralis (P54994); SA-Orf, putative reductase involved in chlortetracycline biosynthesis in Streptomyces aureofaciens (D38215); CH-NtaB, flavin reductase component of the nitrilotriacetate monooxygenase from C. heintzii (U39411); CH-NmoB, flavin reductase component of the nitrilotriacetate monooxygenase from C. heintzii (L49438); RE-Bph61, putative reductase involved in the metabolism of biphenyl derivatives in R. erythropolis (D88018); MT-14c, putative reductase from Mycobacterium tuberculosis (Z92774); PF-StyB, flavin reductase component of the styrene monooxygenase from Pseudomonas fluorescens (Z92524); PY-StyB, flavin reductase component of the styrene monooxygenase from Pseudomonas sp. strain Y2 (AJ000330); PS-StyB flavin reductase component of the styrene monooxygenase from Pseudomonas sp. strain VLB120 (AF031161); MT-11, putative reductase from M. tuberculosis (AL021929); ML-23, putative reductase from Mycobacterium leprae (AL022486); MT-25c, putative reductase from M. tuberculosis (Z84498); RE-DszD, flavin reductase involved in dibenzothiophene desulfurization from R. erythropolis (AF048979); PL-HpaC, HpaC-like reductase from P. luminescens (AF021838); KP-HpaH, putative reductase component of 4-HPA 3-monooxygenase from K. pneumoniae (L41068); EC-HpaC, reductase component of 4-HPA 3-monooxygenase from E. coli (Z29081); EC-F152, putative HpaC-like reductase from E. coli (AE000202); SD-HpaC, putative HpaC reductase from S. dublin (AF144422); BC-TftC, reductase component of chlorophenol 4-hydroxylase from B. cepacia (U83405); AF-HpaCL, putative reductase from A. fulgidus (AE001047); SS-HpaC-1, HpaC-like protein from Synechococcus sp. (L19521); SS-HpaC-2, HpaC-like protein from Synechococcus sp. (D64000); SS-F594, flavoprotein from Synechocystis sp. (D90900); SS-F578 flavoprotein from Synechocystis sp. (M96929); SS-F597 flavoprotein from Synechocystis sp. (D90914); SS-F573, flavoprotein from Synechocystis sp. (D64003).