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Dehydroalanine is present in the histidine ammonia-lyase (histidase) from Pseudomonas putida ATCC 12633 as shown by reaction of purified enzyme with K14CN or NaB3H4 and subsequent identification of [14C]aspartate or [3H]alanine, respectively, following acid hydrolysis of the labeled protein. When labeling with cyanide was conducted under denaturing conditions, 4 mol of [14C]cyanide was incorporated per mol of enzyme (Mr 220 000), equivalent to one dehydroalanine residue being modified per subunit in this protein composed of four essentially identical subunits. In native enzyme, inactivation of catalytic activity by cyanide was complete when 1 mol of [14C]cyanide had reacted per mol of histidase, suggesting that modification of any one of the four dehydroalanine residues in the tetrameric enzyme was sufficient to prevent catalysis at all sites. Loss of activity on treatment with cyanide could be blocked by the addition of the competitive inhibitor cysteine or substrate if Mn2+ was also present. Cross-linking of native enzyme with dimethyl suberimidate produced no species larger than tetramer, thereby eliminating the possibility that an aggregation phenomenon might explain why only one-fourth of the dehydroalanyl residues was modified by cyanide during inactivation. A labeled tryptic peptide was isolated from enzyme inactivated with [14C]cyanide. Its composition was different from that of a tryptic peptide previously isolated from other histidases and shown to contain a highly reactive and catalytically important cysteine residue. Such a finding indicates the dehydroalanine group is distinct from the active site cysteine. Treatment of crude extracts with [14C]cyanide and purification of the inactive enzyme yielded labeled protein that release [14C]aspartate on acid hydrolysis.(ABSTRACT TRUNCATED AT 250 WORDS)

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The complete nucleotide sequence of the hutH gene, encoding histidine ammonia-lyase (histidase), in Pseudomonas putida ATCC 12633 has been determined from the appropriate portions of the hut region that had been cloned into Escherichia coli. The resulting DNA sequence revealed an open reading frame of 1,530 base pairs, corresponding to a protein subunit of approxinate molecular weight 53,600, in the location previously identified for the histidase gene by TnlO00 mutagenesis. Translation began at a GTG codon, but direct protein sequencing revealed that the initiating amino acid was removed posttranslationally to provide an N-terminal threonine; 11 additional residues completely agreed with the predicted amino acid sequence. This sequence excluded the possibility that a dehydroalanine unit, the postulated coenzyme for histidase, is attached at the N terminus of histidase subunits. Comparison of the P. putida histidase gene sequence with that of a Bacillus subtiUs region encoding histidase revealed 42% identity at the protein level. Although the hutU (urocanase) and hutH (histidase) genes are induced by urocanate and normally are transcribed as a unit beginning with hutU, analysis of the region immediately upstream of the histidase gene revealed a potential weak promoter that may possibly be used to maintain a basal level of histidase for the generation of inducer (urocanate) when histidine levels are elevated.Histidine ammonia-lyase (histidase; EC 4.3.1.3) from Pseudomonas putida possesses an essential electrophilic center whose properties are consistent with its tentative identification as a dehydroalanine (DHA) unit (5). This conclusion is based on the chromatographic detection of [3H]alanine from acid hydrolysates of histidase that had been inactivated by reduction with NaB3H4 and a similar identification of ['4C]aspartate from acid-hydrolyzed enzyme that had been treated with Na'4CN. Analogous findings have been obtained for the histidase from Pseudomonas acidovorans ATCC 11299b (9, 31). Little is known regarding the possible mechanistic involvement of DHA in the action of the enzyme or the nature of its binding to the protein.We have previously shown (5) that P. putida histidase is a tetramer with identical subunits of molecular weight approximately 55,000 and 4 mol of DHA per mol of tetrameric protein, although total activity is lost upon covalent modification of one of the DHA units. It was also found that DHA residues are present in the native unpurified enzyme, thereby indicating that they do not arise by 1B elimination of a carbohydrate or similar moiety during the purification process (5). Furthermore, Givot and Abeles inactivated rat liver histidase in vivo by modification of its electrophilic center with nitromethane and demonstrated that the products formed were the same as those found with the P. acidovorans enzyme (8).The structural gene for histidase, along with genes for the other enzymes and major control elements of histidine utilization (hut) in P. putida, has been cloned into Escherichia coli...