␤-Ketoacyl-ACP synthases catalyze the condensation steps in fatty acid and polyketide synthesis and are targets for the development of novel antibiotics and anti-obesity and anti-cancer agents. The roles of the active site residues in Streptococcus pneumoniae FabF (␤-ketoacyl-ACP synthase II; SpFabF) were investigated to clarify the mechanism for this enzyme superfamily. The nucleophilic cysteine of the active site triad was required for acyl-enzyme formation and the overall condensation activity. functions as a gatekeeper that controls the order of substrate addition. These data assign specific roles for each active site residue and lead to a revised general mechanism for this important class of enzymes.The condensing enzymes play a central role in fatty acid biosynthesis by elongating the growing acyl chain by two carbon atoms to initiate each elongation cycle (1, 2). Somewhat uniquely in biological synthesis, the enzymes create a carbon-carbon bond via a Claisen-like condensation reaction (3). Specifically, they catalyze the condensation of malonyl-acyl carrier protein (ACP) 2 with an acyl-ACP intermediate via a two-step ping-pong kinetic mechanism. In the first step, an acyl chain from either acyl-CoA or acyl-ACP is transferred to an active site cysteine, and the cofactor is released. During the second step, malonyl-ACP binds, and a carbanion is generated on the C2 of malonate concomitant with the release of the C3 carboxyl group (4 -6). The carbanion then attacks the acyl-enzyme intermediate to produce the ␤-ketoacyl-ACP product. In the dissociated, type II synthases, the condensation reaction is carried out by monofunctional enzymes (7), and most bacteria have only a single elongation-condensing enzyme that belongs to the FabF class. In mammals and yeast, the condensing enzyme component, KS, is fused into a multidomain complex referred to as the type I or associated FAS system (8). However, it is clear from primary sequence analysis that the active site of the FAS I condensation module is very similar to the FAS II elongation enzymes (Fig. 1A). The polyketide synthases also contain a condensing enzyme module in which the same signature active site residues can be identified (Fig. 1A).The importance of the elongation condensing enzymes in regulating fatty acid formation (7, 9) and the unique chemistry of the reaction that they catalyze (3) have focused our interest on understanding the specific tasks of each active site residue in catalysis. In addition, these enzymes have emerged as attractive targets for the development of new broadspectrum antibiotics (10 -12) and anti-obesity/anti-cancer drugs (13-17), and there is growing interest in engineering the polyketide synthases to produce novel therapeutic agents (18). These efforts will be facilitated by a complete mechanistic understanding of the active site. At their catalytic cores, the elongation enzymes possess a Cys-His-His triad. These residues have been mutated and are thought to be critical to the overall forward condensation reaction (8, 19 -22), al...

No abstract

A ␤-1,4-xylan hydrolase (xylanase A) produced by Erwinia chrysanthemi D1 isolated from corn was analyzed with respect to its secondary structure and enzymatic function. The pH and temperature optima for the enzyme were found to be pH 6.0 and 35°C, with a secondary structure under those conditions that consists of approximately 10 to 15% ␣-helices. The enzyme was still active at temperatures higher than 40°C and at pHs of up to 9.0. The loss of enzymatic activity at temperatures above 45°C was accompanied by significant loss of secondary structure. The enzyme was most active on xylan substrates with low ratios of xylose to 4-O-methyl-D-glucuronic acid and appears to require two 4-O-methyl-D-glucuronic acid residues for substrate recognition and/or cleavage of a ␤-1,4-xylosidic bond. The enzyme hydrolyzed sweetgum xylan, generating products with a 4-O-methyl-glucuronic acid-substituted xylose residue one position from the nonreducing terminus of the oligoxyloside product. No internal cleavages of the xylan backbone between substituted xylose residues were observed, giving the enzyme a unique mode of action in the hydrolysis compared to all other xylanases that have been described. Given the size of the oligoxyloside products generated by the enzyme during depolymerization of xylan substrates, the function of the enzyme may be to render substrate available for other depolymerizing enzymes instead of producing oligoxylosides for cellular metabolism and may serve to produce elicitors during the initiation of the infectious process.

No abstract

A novel hypersensitive resistance (HR) in Capsicum baccatum var. pendulum against the bacterial spot of pepper pathogen, Xanthomonas gardneri, was introgressed into C. annuum cv. Early Calwonder (ECW) to create the near-isogenic line designated as ECW-70R. A corresponding avirulence gene avrBs7, in X. gardneri elicited a strong HR in ECW-70R. A homolog of avrBs7, avrBs1.1, was found in X. euvesicatoria 85-10, which showed delayed HR on ECW-70R leaves. Genetic analysis confirmed the presence of a single dominant resistance gene, Bs7, corresponding to the two avr genes. Both AvrBs7 and AvrBs1.1 share a consensus protein tyrosine phosphatase (PTP) active site domain and can dephosphorylate para-nitrophenyl phosphate. Mutation of Cys(265) to Ser in the PTP domain and subsequent loss of enzymatic activity and HR activity indicated the importance of the PTP domain in the recognition of the Avr protein by the Bs7 gene transcripts. Superpositioning of AvrBs7 and AvrBs1.1 homology models indicated variation in the geometry of the loops adjacent to the active sites. These predicted structural differences might be responsible for the differences in HR timing due to differential activation of the resistance gene. Mutating the PTP domain of AvrBs1.1 to match that of AvrBs7 failed to activate HR on ECW-70R, indicating the possibility of differential substrate specificities between AvrBs1.1 and AvrBs7.