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The structure of Haemophilus influenzae prephenate dehydrogenase suggests unique features of bifunctional TyrA enzymes.

ABSTRACT: Chorismate mutase/prephenate dehydrogenase from Haemophilus influenzae Rd KW20 is a bifunctional enzyme that catalyzes the rearrangement of chorismate to prephenate and the NAD(P)(+)-dependent oxidative decarboxylation of prephenate to 4-hydroxyphenylpyruvate in tyrosine biosynthesis. The crystal structure of the prephenate dehydrogenase component (HinfPDH) of the TyrA protein from H. influenzae Rd KW20 in complex with the inhibitor tyrosine and cofactor NAD(+) has been determined to 2.0?Å resolution. HinfPDH is a dimeric enzyme, with each monomer consisting of an N-terminal ?/? dinucleotide-binding domain and a C-terminal ?-helical dimerization domain. The structure reveals key active-site residues at the domain interface, including His200, Arg297 and Ser179 that are involved in catalysis and/or ligand binding and are highly conserved in TyrA proteins from all three kingdoms of life. Tyrosine is bound directly at the catalytic site, suggesting that it is a competitive inhibitor of HinfPDH. Comparisons with its structural homologues reveal important differences around the active site, including the absence of an ?-? motif in HinfPDH that is present in other TyrA proteins, such as Synechocystis sp. arogenate dehydrogenase. Residues from this motif are involved in discrimination between NADP(+) and NAD(+). The loop between ?5 and ?6 in the N-terminal domain is much shorter in HinfPDH and an extra helix is present at the C-terminus. Furthermore, HinfPDH adopts a more closed conformation compared with TyrA proteins that do not have tyrosine bound. This conformational change brings the substrate, cofactor and active-site residues into close proximity for catalysis. An ionic network consisting of Arg297 (a key residue for tyrosine binding), a water molecule, Asp206 (from the loop between ?5 and ?6) and Arg365' (from the additional C-terminal helix of the adjacent monomer) is observed that might be involved in gating the active site.

SUBMITTER: Chiu HJ

PROVIDER: S-EPMC2954222 | biostudies-literature | 2010 Oct

REPOSITORIES: biostudies-literature

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The structure of Haemophilus influenzae prephenate dehydrogenase suggests unique features of bifunctional TyrA enzymes.

Chiu Hsiu Ju HJ Abdubek Polat P Astakhova Tamara T Axelrod Herbert L HL Carlton Dennis D Clayton Thomas T Das Debanu D Deller Marc C MC Duan Lian L Feuerhelm Julie J Grant Joanna C JC Grzechnik Anna A Han Gye Won GW Jaroszewski Lukasz L Jin Kevin K KK Klock Heath E HE Knuth Mark W MW Kozbial Piotr P Krishna S Sri SS Kumar Abhinav A Marciano David D McMullan Daniel D Miller Mitchell D MD Morse Andrew T AT Nigoghossian Edward E Okach Linda L Reyes Ron R Tien Henry J HJ Trame Christine B CB van den Bedem Henry H Weekes Dana D Xu Qingping Q Hodgson Keith O KO Wooley John J Elsliger Marc André MA Deacon Ashley M AM Godzik Adam A Lesley Scott A SA Wilson Ian A IA

Acta crystallographica. Section F, Structural biology and crystallization communications 20100731 Pt 10

Chorismate mutase/prephenate dehydrogenase from Haemophilus influenzae Rd KW20 is a bifunctional enzyme that catalyzes the rearrangement of chorismate to prephenate and the NAD(P)(+)-dependent oxidative decarboxylation of prephenate to 4-hydroxyphenylpyruvate in tyrosine biosynthesis. The crystal structure of the prephenate dehydrogenase component (HinfPDH) of the TyrA protein from H. influenzae Rd KW20 in complex with the inhibitor tyrosine and cofactor NAD(+) has been determined to 2.0 Å resol ...[more]

PMID: 20944228

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Project description:BACKGROUND: The incidence of invasive disease caused by encapsulated Haemophilus influenzae type f (Hif) has increased in the post-H. influenzae type b (Hib) vaccine era. We previously annotated the first complete Hif genome from a clinical isolate (KR494) that caused septic shock and necrotizing myositis. Here, the full genome of Hif KR494 was compared to sequenced reference strains Hib 10810, capsule type d (Hid) Rd Kw20, and finally nontypeable H. influenzae 3655. The goal was to identify possible genomic characteristics that may shed light upon the pathogenesis of Hif. RESULTS: The Hif KR494 genome exhibited large regions of synteny with other H. influenzae, but also distinct genome rearrangements. A predicted Hif core genome of 1390 genes was shared with the reference strains, and 6 unique genomic regions comprising half of the 191 unique coding sequences were revealed. The majority of these regions were inserted genetic fragments, most likely derived from the closely-related Haemophilus spp. including H. aegyptius, H. haemolyticus and H. parainfluenzae. Importantly, the KR494 genome possessed several putative virulence genes that were distinct from non-type f strains. These included the sap2 operon, aef3 fimbriae, and genes for kanamycin nucleotidyltranserase, iron-utilization proteins, and putative YadA-like trimeric autotransporters that may increase the bacterial virulence. Furthermore, Hif KR494 lacked a hisABCDEFGH operon for de novo histidine biosynthesis, hmg locus for lipooligosaccharide biosynthesis and biofilm formation, the Haemophilus antibiotic resistance island and a Haemophilus secondary molybdate transport system. We confirmed the histidine auxotrophy and kanamycin resistance in Hif by functional experiments. Moreover, the pattern of unique or missing genes of Hif KR494 was similar in 20 Hif clinical isolates obtained from different years and geographical areas. A cross-species comparison revealed that the Hif genome shared more characteristics with H. aegyptius than Hid and NTHi. CONCLUSIONS: The genomic comparative analyses facilitated identification of genotypic characteristics that may be related to the specific virulence of Hif. In relation to non-type f H. influenzae strains, the Hif genome contains differences in components involved in metabolism and survival that may contribute to its invasiveness.

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The structure of Haemophilus influenzae prephenate dehydrogenase suggests unique features of bifunctional TyrA enzymes.

Publications

The structure of Haemophilus influenzae prephenate dehydrogenase suggests unique features of bifunctional TyrA enzymes.

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