Amino acid sequence of a peptide containing the active cysteine residue of histidine ammonia-lyase

Abstract

1. Oxidized (polymerized) histidine ammonia-lyase from Pseudomonas testosteroni was activated with dithiothreitol and the reduced disulphide-linked cysteine residues of the native enzyme were carboxymethylated with iodo[(14)C]acetate. 2. The activity of the carboxymethylated enzyme was similar to that of the polymerized form and approx. 15% of that of the fully reduced form. 3. A tryptic digest of the [(14)C]carboxymethylated enzyme contained only one radioactive peptide. 4. The amino acid sequence of this peptide was shown to be Gly-Leu-Leu-Asp-Gly-Ser-Ala-Ile-Asn-Pro-Ser-His-Pro-Asn-Cys- (CH(2)CO(2)H)-Gly-Arg. 5. These findings show that, during polymerization, the disulphide bonds are formed between identical regions of the enzyme, and that the cysteine residue involved is also the one required in the reduced state for full activity of the enzyme.

Sequence analysis of the hutH gene encoding histidine ammonia-lyase in Pseudomonas putida

“…Earlier studies on histidases from P. acidovorans ATCC 11299b by Klee and Gladner (14) and P. testosteroni NCIB 10808 by Hassall and Soutar (11) reported that the native tetramer form of these proteins could be modified by iodo[14C]acetate, from which a labeled tryptic peptide could be isolated and characterized. Whereas only the composition was reported by Klee and Gladner (14), Hassall and Soutar (11) determined its sequence to be Gly-Leu-Leu-Asp-GlySer-Ala-Ile-Asn-Pro-Ser-His-Pro-Asn-CMCys-Gly-Arg.…”

The purification and properties of l-histidine-2-oxoglutarate aminotransferase from Pseudomonas testosteroni

“…The organism was subcultured monthly and stored at 4°C after incubation for 2 days at 30°C. For purification of histidine-2-oxoglutarate aminotransferase, cells were grown in 100-litre batches on histidine-succinate medium as described by Hassall & Soutar (1974) and harvested in 'late-exponential phase. They were stored frozen at -1 8°C until required.…”

“…The cells were thawed and resuspended in 5 vol. of 0.1M-potassium phosphate buffer, pH7.0, at 4°C and then passed three times through a Manton-Gaulin laboratory homogenizer (Hassall & Soutar, 1974) at a pressure of 55MPa (80001bf/in2). After disruption, cell debris was removed by centrifugation at 30000g for 40min.…”

Crystalline l‐Histidine Ammonia‐Lyase of Achromobacter liquidum

“…L-Histidine ammonia-lyase, which converts L-histidine to urocanic acid and ammonia, is widely distributed in bacteria such as Pseudomonas fluorescens [l-51, P. aeruginosa [6], P.putida [7,8], P. testosteroni [9], Aerobacter aerogenes [lo, 111, Bacillus subtilis [12,13], B. cereus [14], Mycobacterium avium [15], Salmonella typhimurium [16], and Vibrio cholerae [17]. The enzyme occurs also in liver [18,19], blood serum [20], and stratum corneum [21] of higher animals and in plants such as spinach and sunflowers [22].…”

“…The enzyme occurs also in liver [18,19], blood serum [20], and stratum corneum [21] of higher animals and in plants such as spinach and sunflowers [22]. However, there have been only three reports on the exhaustive purification of the enzyme [5,9,19]. Moreover, there has been no report on the crystallization of this enzyme.…”