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

Abstract

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...

The mechanism of action of phenylalanine ammonia-lyase: the role of prosthetic dehydroalanine.

“…3. Although this mechanism includes chemically implausible steps, it has provided the basis for numerous mechanistic investigations for 25 years (1,3,12,(30)(31)(32). While a carbanionic intermediate was postulated, it was not explained how this could be formed.…”

“…In contrast, in the HAL reaction, the proton abstraction is at least partially rate limiting, as revealed by a kinetic deuterium isotope effect of kH/kD = 1.5-2 (34,35). Since the two enzymes exhibit considerable homology (12,19) (Fig. 7).…”

“…The electrophilic property of the prosthetic dehydroalanine has been demonstrated by addition of various nucleophiles, such as nitromethane (7)(8)(9)(10)(11), cyanide (12), and sodium borohydride (3,8). All these reagents caused inactivation and, using radiolabel, incorporation of the radioisotope into the expected product.…”

Purification of histidase from Streptomyces griseus and nucleotide sequence of the hutH structural gene

“…Histidase has been purified from pseudomonads (9,10,23), Bacillus subtilis (29,32), and rat liver (7,44), and the structural genes have been cloned. In bacteria the synthesis of histidase is regulated by induction (8,11,28,32,33,39), carbon catabolite repression (8,31), and nitrogen regulation (36).…”

“…Either genomic DNA from the wild-type strain of S. griseus or DNA from a library of S. griseus genomic DNA prepared in XEMBL4 was hybridized overnight at 37°C with the radiolabeled probe. The membrane was washed four times for 10 (26) was assessed after transformation with plasmid pKK593; this plasmid was constructed by ligating the 2.4-kb BamHI fragment of pKK540 (containing the hutH structural gene and approximately 600 bp of upstream sequence) to BglII-digested pIJ702 (19). Transformation of S. gniseus was conducted as described previously (2).…”