Structure of Aminodeoxychorismate Synthase from Stenotrophomonas maltophilia

Abstract

PabB, aminodeoxychorismate synthase, is the chorismic acid binding component of the heterodimeric PabAB complex that converts chorismic acid to 4-amino-4-deoxychorismate, a precursor of p-aminobenzoate and folic acid in microorganisms. The second component, a glutamine amidotransferase subunit, PabA, generates ammonia that is channeled to the PabB active site where it attacks the C4 carbon of a chorismate derived intermediate that is covalently bound, through C2, to an active site lysine residue. The presence of a PIKGT motif was, until recently, believed to be discriminate PabB enzymes from the closely related enzyme anthranilate synthase, which typically contains a PIAGT active site motif and does not form a covalent enzyme-substrate intermediate with chorismate. A subclass of PabB enzymes that employ an alternative mechanism requiring two equivalents of ammonia from glutamine and that feature a noncovalently bound 2-amino-2-deoxyisochorismate intermediate was recently identified. Here we report the 2.25 Å crystal structure of PabB from the emerging pathogen Stenotrophomonas maltophilia. It is the first reported structure of a PabB that features the PIAGT motif. Surprisingly, no dedicated pabA is evident in the genome of S. maltophilia suggesting that another cellular amidotransferase is able to fulfill the role of PabA in this organism. Evaluation of the ammonia-dependent aminodeoxychorismate synthase activity of S. maltophilia PabB alone revealed that it is virtually inactive. However, in the presence of a heterologous PabA surrogate, typical levels of activity were observed using either glutamine or ammonia as the nitrogen source. Additionally, the structure suggests that a key segment of the polypeptide can remodel itself to interact with a nonspecialized or shared amidotransferase partner in vivo. The structure and mass spectral analysis further suggest that S. maltophilia PabB, like Escherichia coli PabB, binds tryptophan in a vestigial regulatory site. The observation that the binding site is unoccupied in the crystal structure, however, suggests the affinity may be low relative to E. coli PabB.

Structure and inhibition of subunit I of the anthranilate synthase complex of Mycobacterium tuberculosis and expression of the active complex

“…The novel AS-I-AS-I dimer interface seen here fits with a pattern of variability within the wider CUE family among those enzymes that require a GAT subunit (Bera et al, 2012;Parsons et al, 2002). For example, the CUE from the folatebiosynthetic pathway in E. coli is a heterodimer involving a GAT subunit that can associate or dissociate under certain conditions (Roux & Walsh, 1992;Rayl et al, 1996).…”

“…Mtb-AS-I are the three other structurally characterized AS-I subunits from AS complexes (Knö chel et al, 1999;Morollo & Eck, 2001;Spraggon et al, 2001), followed by CUEs from the folate (Parsons et al, 2002;Bera et al, 2012), phenazine (Li et al, 2011), enterobactin (Kerbarh et al, 2006) and mycobactin (Harrison et al, 2006;Zwahlen et al, 2007) biosynthetic pathways. Root-mean-square differences (r.m.s.d.s) in C positions range from 1.47 to 2.42 Å , highlighting the conservation of the core fold within this family of proteins.…”

Cloning, Expression, and Characterization of Para-Aminobenzoic Acid (PABA) Synthase from Agaricus bisporus 02, a Thermotolerant Mushroom Strain

“…S2B). The motif was recently believed to allow discrimination of PabB enzymes from the closely related enzyme anthranilate synthase, which typically contains a PIAGT active-site motif [4]. Thus, pabS cDNA of A. bisporus 02 encodes a protein containing putative aminodeoxychorismate synthase.…”

“…In Escherichia coli, there are three enzymes required for the conversion of chorismate to PABA. PabA is an aminase that supplies ammonia from glutamine hydrolysis; PabB is a member of a family of structurally similar chorismate-utilizing enzymes that catalyze the amination of chorismate, yielding 4-amino-4-deoxychorismate (ADC); PabC is a pyridoxal phosphatedependent enzyme that catalyzes the elimination of pyruvate from ADC, forming PABA [4]. In most bacteria, pabA and pabB are isolated genes, but pabA and pabB homologs are found as one fused gene in a number of actinomycetes and all of the eukaryotes analyzed so far [6,8,12,23].…”

Selection markers for transformation of the sequenced reference monokaryon Okayama 7/#130 and homokaryon AmutBmut of Coprinopsis cinerea

“…This missense mutation resulted in a K546E exchange in the PabB domain within the highly conserved ADC synthase component I motif PIKGT. Lysine in the wildtype (wt) covalently binds to the C 2 of chorismate to initiate with the ammonia-group of glutamine the enzymatic formation of ADC ( [28][29][30][31], Fig. 1a).…”