John K. Hunter

THE DEGRADATION OF L-HISTIDINE AND TRANS- AND CIS-UROCANIC ACID BY BACTERIA FROM SKIN AND THE ROLE OF BACTERIAL CIS-UROCANIC ACID ISOMERASE

UV-B radiation suppresses cell-mediated immunity. Histidine forms trans-urocanic acid (trans-UCA) enzymatically in the stratum corneum. Photoisomerization of trans-UCA to cis-urocanic acid (cis-UCA) has been proposed for the initiation of an immunosuppressive process. Many microorganisms described in the literature metabolize histidine and/or trans-UCA. Our enrichment cultures of soil and sewage contain organisms that can degrade cis-UCA. We have tested microorganisms for degradation of cis-UCA, trans-UCA, or L-histidine when they are incorporated at 0.2% in nutrient broth. Six out of 10 selected genera isolated by our clinical microbiology laboratory degrade one or more of the imidazole substrates. We have cultured over 60 aerobic isolates from human skin. Of these, 33 degrade one or more of the three imidazole substrates and 12 degrade cis-UCA. Isolates from BALB/c mice are also active on cis-UCA. We have identified a cis-UCA-degrading bacterium as Micrococcus luteus. Four ATCC strains of M. luteus have been tested and three are active on histidine or trans-UCA; two are active on cis-UCA. Micrococci that degrade cis-UCA contain a new enzyme, cis-UCA isomerase, which converts the substrate to the trans-isomer. This enzyme provides access to the classical L-histidine degradation pathway. We hypothesize that an epidermal microflora that degrades L-histidine, trans-UCA, or cis-UCA influences the concentration of urocanic acids on the skin and, thus, affects immune suppression.

ADVENTITIOUS INTERCONVERSION OF cis‐ AND trans‐UROCANIC ACID BY LABORATORY LIGHT

Urocanic acid (UCA) is a chromophore in the stratum corneum. Ultraviolet radiation (ultraviolet B) has been shown to suppress mammalian cell‐mediated immunity. The photoisomerization of trans‐UCA to cis‐UCA was proposed as the initiator of the suppression process. Cis‐urocanic acid has been demonstrated to suppress immunity by a variety of experiments. Investigators should be aware that laboratory illumination may be capable of interconverting trans‐UCA and cis‐UCA during experimental manipulations. This possible inadvertent contamination of one isomer by the other may influence results. We demonstrated that fluorescent lamps, daylight, sunlight and incandescent lamps were able to bring about isomerization. Window glass and container materials of plastic and clear glass did not filter out effective wavelengths, but three commercial plastic diffusers on fluorescent fixtures prevented the isomerization. Because the molar extinction coefficient (ɛ) for cis‐UCA is less than that of trans‐UCA, we have exposed 0.1 mMtrans‐UCA to ambient light and monitored the change in absorbance. A method is given to calculate the percentage of trans and cis isomers from the absorbance at 277 nm when the initial purity and absorbance are known. Using this procedure, we validated the molar extinction coefficient of cis‐UCA.

PHOTO ACTIVATION OF UROCANASE IN PSEUDOMONAS PUTIDA: FACTORS INFLUENCING ACTIVATION

Abstract— –When washed cells of histidine‐grown Pseudomonas putida were incubated at 8°C in darkness, urocanase activity diminished after several days; most of this lost activity could be restored by treatment of cell extracts with near‐u.v. light. Sunlight and daylight were also effective for activation. Non‐irradiated extract, when added to the active preparation, did not inhibit the enzyme activity. Heated and boiled extracts with or without irradiation did not catalyze the urocanase reaction nor did they change the activity of an activated extract when added to it. Photoactivation of cell extracts did not require oxygen, was not dependent on temperature and was not prevented by dialysis. Urocanase purified by gel electrophoresis was capable of light activation. It is suggested that the photoreceptor is closely associated with urocanase since it is not separated from the enzyme by dialysis or electrophoresis.

The potential role for urocanic acid and sunlight in the immune suppression associated with protein malnutrition

Irradiation of skin by sunlight or ultraviolet B (UVB, 290-320 nm) brings about a downregulation of cell-mediated immunity. An action spectrum for photoimmune suppression in mice indicates that trans-urocanic acid absorbs UV photons and is isomerized to the cis-isomer in the stratum corneum. Cis-urocanic acid is subsequently shown to suppress cellular immunity in mice. When histidine is elevated in a mouse diet, a higher level of urocanic acid is detected in mouse skin. These mice are more susceptible to photoimmune suppression. There is evidence that humans and animals experiencing protein malnutrition have very high levels of urocanic acid and/or histidine. Urocanic acid is formed by deamination of histidine in one enzymatic step. We discuss the protein malnutrition of kwashiorkor patients. They experience suppressed immunity and disturbed histidine metabolism. Here, we present a testable hypothesis: one cause of the immune deficiency observed in humans with protein malnutrition is the photoconversion by UVB of increased levels of trans-urocanic acid in skin to cis-urocanic acid, which suppresses the cellular immune system.

THE SUBSTRATE‐DEPENDENT PHOTOINACTIVATION OF UROCANASE FROM RAT LIVER

Abstract— Rat liver urocanase was readily inactivated by near‐UV light in the presence of the substrate. Irradiation of substrate or enzyme alone was ineffective. The purpose of this study was to examine the conditions which influenced this inactivation and to investigate the mechanism. The urocanate concentration needed for 50% of the maximum inactivation for a 15 min irradiation was 0.09 μM. Temperatures from 0 to 30°C during irradiation had little influence. Inactivation occurred at ‐75°C, which indicated a photochemical reaction. The pH had little influence on inactivation. Photoinactivation was the same in nitrogen and air. Dialysis experiments showed that unbound small molecules were probably not involved. Inactivated enzyme did not inhibit active enzyme. Chelators, reducing agents, and pyridoxal phosphate did not affect the inactivation. Visible light was not effective. An action spectrum was established with the aid of a monochromator. The action spectrum had a peak at 280 nm and a shoulder extending from 300 to 340 nm which rules out flavins. pyridoxal phosphate, a simple protein, and free urocanate as the chromophore. The results suggest that this photochemical process is not photodynamic action. It appears that only substrate and enzyme are needed for this photoinactivation. The enzyme‐substrate complex may be the chromophore.

Light-Driven Periodic Changes in Urocanase Activity of a Heterotrophic Bacterium

SummaryUrocanase activity in Pseudomonas putida cells showed periodic changes corresponding to a light-dark cycle. Apparent interconversion of the active and inactive forms of urocanase was accomplished by photoactivation by nearultraviolet light and by dark thermal inactivation. The purified enzyme exhibited similar behavior. This system suggests a possible molecular basis for an hour-glass timer. Photoinactivation by light of 290 nm alternated with photoactivation at 340 nm also generated regular changes in urocanase activity.