A Common Catalytic Mechanism for Proteins of the HutI Family

Tyagi, Rajiv; Eswaramoorthy, S.; Burley, S.K.; Raushel, Frank M.; Swaminathan, Subramanyam

doi:10.1021/bi800180g

Cited by 10 publications

(17 citation statements)

References 26 publications

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“…Early kinetic studies used IP produced in situ from urocanate in the presence of highly purified urocanase. As a result, the characterization of IPase lagged behind that of the other Hut enzymes, and IPase was the last of the enzymes to have its crystal structure determined (161,170). Although the details of the reaction mechanism and the nature of the active site metal remain somewhat unclear, two facts are well established: the reaction is essential for utilization of histidine as a carbon source (74,148), and IPase is a hydrolase that cleaves the ring to yield formiminoglutamate, in a different reaction from the nonenzymatic cleavage that yields formylisoglutamine (128).…”

Section: Ipasementioning

confidence: 99%

Regulation of the Histidine Utilization (Hut) System in Bacteria

Bender

2012

Microbiol Mol Biol Rev

123

131

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SUMMARY The ability to degrade the amino acid histidine to ammonia, glutamate, and a one-carbon compound (formate or formamide) is a property that is widely distributed among bacteria. The four or five enzymatic steps of the pathway are highly conserved, and the chemistry of the reactions displays several unusual features, including the rearrangement of a portion of the histidase polypeptide chain to yield an unusual imidazole structure at the active site and the use of a tightly bound NAD molecule as an electrophile rather than a redox-active element in urocanase. Given the importance of this amino acid, it is not surprising that the degradation of histidine is tightly regulated. The study of that regulation led to three central paradigms in bacterial regulation: catabolite repression by glucose and other carbon sources, nitrogen regulation and two-component regulators in general, and autoregulation of bacterial regulators. This review focuses on three groups of organisms for which studies are most complete: the enteric bacteria, for which the regulation is best understood; the pseudomonads, for which the chemistry is best characterized; and Bacillus subtilis , for which the regulatory mechanisms are very different from those of the Gram-negative bacteria. The Hut pathway is fundamentally a catabolic pathway that allows cells to use histidine as a source of carbon, energy, and nitrogen, but other roles for the pathway are also considered briefly here.

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Section: Ipasementioning

confidence: 99%

Regulation of the Histidine Utilization (Hut) System in Bacteria

Bender

2012

Microbiol Mol Biol Rev

123

131

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“…The reverse reaction, an intramolecular condensation, is reminiscent of the assumed formation of pseudochelin A. While sequence alignments of MxcM with different HutI homologs did not indicate significant similarities (see Table S1 in the supplemental material), a manual inspection showed that the active site residues of HutI (26,27) are conserved in MxcM (Fig. S1A).…”

Section: Resultsmentioning

confidence: 99%

Engineering Pseudochelin Production in Myxococcus xanthus

Korp

Winand

Sester

et al. 2018

Appl Environ Microbiol

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Myxobacteria utilize the catechol natural products myxochelin A and B in order to maintain their iron homeostasis. Recently, the production of these siderophores was reported from the marine bacterium S2040, along with a new myxochelin derivative named pseudochelin A. The latter features a characteristic imidazoline moiety, which was proposed to originate from an intramolecular condensation reaction of the β-aminoethyl amide group in myxochelin B. To identify the enzyme catalyzing this conversion, we compared the myxochelin regulons of two myxobacterial strains, which solely produce myxochelin A and B, with S2040. This approach revealed a gene exclusive to the myxochelin regulon in S2040, coding for an enzyme of the amidohydrolase superfamily. To prove that this enzyme is indeed responsible for the postulated conversion, the reaction was reconstituted using a hexahistidyl-tagged recombinant protein made in and myxochelin B as substrate. To test the production of pseudochelin A under conditions, the amidohydrolase gene was cloned into the myxobacterial plasmid pZJY156 and placed under the control of a copper-inducible promoter. The resulting vector was introduced into the myxobacterium DSM16526, a native producer of myxochelin A and B. Following the induction with copper, the myxobacterial expression strain was found to synthesize small quantities of pseudochelin A. Replacement of the copper-inducible promoter with the constitutive promoter led to increased production levels in , which facilitated the isolation and subsequent structural verification of the heterologously produced compound. In this study, an enzyme for imidazoline formation in pseudochelin biosynthesis was identified. Evidence for the involvement of this enzyme in the postulated reaction was obtained after reconstitution. Furthermore, the function of this enzyme was also demonstrated by transferring the corresponding gene into the bacterium , which thereby became a producer of pseudochelin A. Aside from clarifying the molecular basis of imidazoline formation in siderophore biosynthesis, we describe the heterologous expression of a gene in a myxobacterium without its chromosomal integration. Due to its metabolic proficiency, represents an interesting alternative to established host systems for the reconstitution and manipulation of biosynthetic pathways. Since the plasmid used in this study is easily adaptable for the expression of other enzymes as well, we expand the conventional expression strategy for myxobacteria, which is based on the integration of biosynthetic genes into the host genome.

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“…About 1 mg of pure protein was obtained from 15 g of cells. Cc2672 was expressed and purified to homogeneity as previously described (21). …”

Section: Methodsmentioning

confidence: 99%

Functional Identification and Structure Determination of Two Novel Prolidases from cog1228 in the Amidohydrolase Superfamily,

Xiang

Patskovsky

et al. 2010

Biochemistry

Self Cite

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Two uncharacterized enzymes from the amidohydrolase superfamily belonging to cog1228 were cloned, expressed and purified to homogeneity. The two proteins, Sgx9260c (gi|44242006) and Sgx9260b (gi|44479596), were derived from environmental DNA samples originating from the Sargasso Sea. The catalytic function and substrate profiles for Sgx9260c and Sgx9260b were determined using a comprehensive library of dipeptides and N-acyl derivative of L-amino acids. Sgx9260c catalyzes the hydrolysis of Gly-L-Pro, L-Ala-L-Pro and N-acyl derivatives of L-Pro. The best substrate identified to date is N-acetyl-L-Pro with a value of kcat/Km of 3 × 105 M−1 s−1. Sgx9260b catalyzes the hydrolysis of L-hydrophobic L-Pro dipeptides and N-acyl derivatives of L-Pro. The best substrate identified to date is N-propionyl-L-Pro with a value of kcat/Km of 1 × 105 M−1 s−1. Three dimensional structures of both proteins were determined by X-ray diffraction methods (PDB codes: 3MKV and 3FEQ). These proteins fold as distorted (β/α)8-barrels with two divalent cations in the active site. The structure of Sgx9260c was also determined as a complex with the N-methyl phosphonate derivative of L-Pro (PDB code: 3N2C). In this structure the phosphonate moiety bridges the binuclear metal center and one oxygen atom interacts with His-140. The α-carboxylate of the inhibitor interacts with Tyr-231. The proline side chain occupies a small substrate binding cavity formed by residues contributed from the loop that follows β-strand 7 within the (β/α)8-barrel. A total of 38 other proteins from cog1228 are predicted to have the same substrate profile based on conservation of the substrate binding residues. The structure of an evolutionarily related protein, Cc2672 from Caulobacter crecentus, was determined as a complex with the N-methyl phosphonate derivative of L-arginine (PDB code: 3MTW).

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A Common Catalytic Mechanism for Proteins of the HutI Family

Cited by 10 publications

References 26 publications

Regulation of the Histidine Utilization (Hut) System in Bacteria

Regulation of the Histidine Utilization (Hut) System in Bacteria

Engineering Pseudochelin Production in Myxococcus xanthus

Functional Identification and Structure Determination of Two Novel Prolidases from cog1228 in the Amidohydrolase Superfamily,

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