Crystal structure of Haemophilus influenzae 3-isopropylmalate dehydrogenase (LeuB) in complex with the inhibitor O-isobutenyl oxalylhydroxamate

The bacterial periplasmic protein LpoA is an outer membrane lipoprotein and an activator for the cross-linking activity of PBP1A, a bifunctional peptidoglycan synthase. Previous structures of the amino-terminal (N) domain of LpoA showed it to consist entirely of helices and loops, with at least four tetratricopeptide-like repeats. Although the previously determined orthorhombic crystal structure of the N domain of Haemophilus influenzae LpoA showed a typical curved structure with a concave groove, an NMR structure of the same domain from Escherichia coli was relatively flat. Here, a crystal structure of the N domain of E. coli LpoA was determined to a resolution of 2.1 Å and was found to be more similar to the H. influenzae crystal structure than to the E. coli NMR structure. To provide a quantitative description for these comparisons, the various structures were superimposed pairwise by fitting the first half of each structure to its pairwise partner and then calculating the rotation axis that would optimally superimpose the second half. Differences in both the magnitude of the rotation and the direction of the rotation axis were observed between different pairs of structures. A 1.35 Å resolution structure of a monoclinic crystal form of the N domain of H. influenzae LpoA was also determined. In this structure, the subdomains rotate 10° relative to those in the original orthorhombic H. influenzae crystal structure to further narrow the groove between the subdomains. To accommodate this, a bound chloride ion (in place of sulfate) allowed the closer approach of a helix that forms one side of the groove.

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Crystal Structure of VapBC-1 from NontypeableHaemophilus influenzaeand the Effect of PIN Domain Mutations on Survival during Infection

Journal of Bacteriology ◽

10.1128/jb.00026-19 ◽

2019 ◽

Vol 201 (12) ◽

Cited By ~ 2

Author(s):

Ashley L. Molinaro ◽

Maithri M. Kashipathy ◽

Scott Lovell ◽

Kevin P. Battaile ◽

Nathan P. Coussens ◽

...

Keyword(s):

Crystal Structure ◽

Haemophilus Influenzae ◽

Protein Interactions ◽

Ex Vivo ◽

Single Copy ◽

Human Tissues ◽

Type Ii ◽

Wild Type ◽

Content Type ◽

Native Promoter

ABSTRACTToxin-antitoxin (TA) gene pairs have been identified in nearly all bacterial genomes sequenced to date and are thought to facilitate persistence and antibiotic tolerance. TA loci are classified into various types based upon the characteristics of their antitoxins, with those in type II expressing proteic antitoxins. Many toxins from type II modules are ribonucleases that maintain a PilT N-terminal (PIN) domain containing conserved amino acids considered essential for activity. ThevapBC(virulence-associatedprotein) TA system is the largest subfamily in this class and has been linked to pathogenesis of nontypeableHaemophilus influenzae(NTHi). In this study, the crystal structure of the VapBC-1 complex from NTHi was determined to 2.20 Å resolution. Based on this structure, aspartate-to-asparagine and glutamate-to-glutamine mutations of four conserved residues in the PIN domain of the VapC-1 toxin were constructed and the effects of the mutations on protein-protein interactions, growth ofEscherichia coli, and pathogenesisex vivowere tested. Finally, a novel model system was designed and utilized that consists of an NTHi ΔvapBC-1strain complemented inciswith the TA module containing a mutated or wild-type toxin at an ectopic site on the chromosome. This enabled the analysis of the effect of PIN domain toxin mutants in tandem with their wild-type antitoxin under the control of thevapBC-1native promoter and in single copy. This is the first report of a system facilitating the study of TA mutant operons in the background of NTHi during infections of primary human tissuesex vivo.IMPORTANCEHerein the crystal structure of the VapBC-1 complex from nontypeableHaemophilus influenzae(NTHi) is described. Our results show that some of the mutations in the PIN domain of the VapC-1 toxin were associated with decreased toxicity inE. coli, but the mutants retained the ability to homodimerize and to heterodimerize with the wild-type cognate antitoxin, VapB-1. A new system was designed and constructed to quantify the effects of these mutations on NTHi survival during infections of primary human tissuesex vivo. Any mutation to a conserved amino acid in the PIN domain significantly decreased the number of survivors compared to that of the inciswild-type toxin under the same conditions.

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Enhancement of the latent 3-isopropylmalate dehydrogenase activity of promiscuous homoisocitrate dehydrogenase by directed evolution

Biochemical Journal ◽

10.1042/bj20101246 ◽

2010 ◽

Vol 431 (3) ◽

pp. 401-412 ◽

Cited By ~ 9

Author(s):

Yumewo Suzuki ◽

Kuniko Asada ◽

Junichi Miyazaki ◽

Takeo Tomita ◽

Tomohisa Kuzuyama ◽

...

Keyword(s):

Crystal Structure ◽

Amino Acid ◽

Binding Site ◽

Directed Evolution ◽

Substrate Binding ◽

Dna Shuffling ◽

Turnover Number ◽

Substrate Binding Site ◽

Km Value ◽

Isopropylmalate Dehydrogenase

HICDH (homoisocitrate dehydrogenase), which is involved in lysine biosynthesis through α-aminoadipate, is a paralogue of IPMDH [3-IPM (3-isopropylmalate) dehydrogenase], which is involved in leucine biosynthesis. TtHICDH (Thermus thermophilus HICDH) can recognize isocitrate, as well as homoisocitrate, as the substrate, and also shows IPMDH activity, although at a considerably decreased rate. In the present study, the promiscuous TtHICDH was evolved into an enzyme showing distinct IPMDH activity by directed evolution using a DNA-shuffling technique. Through five repeats of DNA shuffling/screening, variants that allowed Escherichia coli C600 (leuB−) to grow on a minimal medium in 2 days were obtained. One of the variants LR5–1, with eight amino acid replacements, was found to possess a 65-fold increased kcat/Km value for 3-IPM, compared with TtHICDH. Introduction of a single back-replacement H15Y change caused a further increase in the kcat/Km value and a partial recovery of the decreased thermotolerance of LR5–1. Site-directed mutagenesis revealed that most of the amino acid replacements found in LR5–1 effectively increased IPMDH activity; replacements around the substrate-binding site contributed to the improved recognition for 3-IPM, and other replacements at sites away from the substrate-binding site enhanced the turnover number for the IPMDH reaction. The crystal structure of LR5–1 was determined at 2.4 Å resolution and revealed that helix α4 was displaced in a manner suitable for recognition of the hydrophobic γ-moiety of 3-IPM. On the basis of the crystal structure, possible reasons for enhancement of the turnover number are discussed.

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